CA3109702A1 - Peptides and compositions for targeted treatment and imaging - Google Patents
Peptides and compositions for targeted treatment and imaging Download PDFInfo
- Publication number
- CA3109702A1 CA3109702A1 CA3109702A CA3109702A CA3109702A1 CA 3109702 A1 CA3109702 A1 CA 3109702A1 CA 3109702 A CA3109702 A CA 3109702A CA 3109702 A CA3109702 A CA 3109702A CA 3109702 A1 CA3109702 A1 CA 3109702A1
- Authority
- CA
- Canada
- Prior art keywords
- trem
- cancer
- inhibitor
- group
- peptide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 243
- 239000000203 mixture Substances 0.000 title claims abstract description 132
- 102000004196 processed proteins & peptides Human genes 0.000 title claims abstract description 82
- 238000011282 treatment Methods 0.000 title claims abstract description 46
- 238000003384 imaging method Methods 0.000 title claims description 81
- 102000018368 Triggering Receptor Expressed on Myeloid Cells-1 Human genes 0.000 claims abstract description 351
- 108010066451 Triggering Receptor Expressed on Myeloid Cells-1 Proteins 0.000 claims abstract description 351
- 238000000034 method Methods 0.000 claims abstract description 154
- 206010028980 Neoplasm Diseases 0.000 claims abstract description 115
- 201000011510 cancer Diseases 0.000 claims abstract description 68
- 210000000066 myeloid cell Anatomy 0.000 claims abstract description 52
- 201000008754 Tenosynovial giant cell tumor Diseases 0.000 claims abstract description 36
- 238000002560 therapeutic procedure Methods 0.000 claims abstract description 32
- 208000035647 diffuse type tenosynovial giant cell tumor Diseases 0.000 claims abstract description 25
- 208000007420 pigmented villonodular synovitis Diseases 0.000 claims abstract description 24
- 230000019491 signal transduction Effects 0.000 claims abstract description 24
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims abstract description 22
- 206010006895 Cachexia Diseases 0.000 claims abstract description 16
- 208000020816 lung neoplasm Diseases 0.000 claims abstract description 15
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims abstract description 14
- 201000002528 pancreatic cancer Diseases 0.000 claims abstract description 14
- 208000008443 pancreatic carcinoma Diseases 0.000 claims abstract description 14
- 230000001404 mediated effect Effects 0.000 claims abstract description 13
- 208000007990 Giant Cell Tumor of Tendon Sheath Diseases 0.000 claims abstract description 11
- 208000002154 non-small cell lung carcinoma Diseases 0.000 claims abstract description 11
- 208000029729 tumor suppressor gene on chromosome 11 Diseases 0.000 claims abstract description 11
- 210000002435 tendon Anatomy 0.000 claims abstract description 8
- 208000007569 Giant Cell Tumors Diseases 0.000 claims abstract description 7
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims abstract description 7
- 201000005202 lung cancer Diseases 0.000 claims abstract description 7
- 230000020411 cell activation Effects 0.000 claims abstract description 4
- 239000003112 inhibitor Substances 0.000 claims description 121
- 239000000523 sample Substances 0.000 claims description 98
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 63
- 230000002401 inhibitory effect Effects 0.000 claims description 52
- -1 lenolidomide Chemical compound 0.000 claims description 50
- 239000003795 chemical substances by application Substances 0.000 claims description 38
- 108010010234 HDL Lipoproteins Proteins 0.000 claims description 34
- 102000015779 HDL Lipoproteins Human genes 0.000 claims description 34
- 230000011664 signaling Effects 0.000 claims description 34
- 210000004027 cell Anatomy 0.000 claims description 31
- 230000001225 therapeutic effect Effects 0.000 claims description 31
- 102100028198 Macrophage colony-stimulating factor 1 receptor Human genes 0.000 claims description 29
- 108010046938 Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 27
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 27
- 241001465754 Metazoa Species 0.000 claims description 26
- 229960001592 paclitaxel Drugs 0.000 claims description 26
- 241000282414 Homo sapiens Species 0.000 claims description 25
- 229930012538 Paclitaxel Natural products 0.000 claims description 25
- RCINICONZNJXQF-MZXODVADSA-N taxol Chemical compound O([C@@H]1[C@@]2(C[C@@H](C(C)=C(C2(C)C)[C@H](C([C@]2(C)[C@@H](O)C[C@H]3OC[C@]3([C@H]21)OC(C)=O)=O)OC(=O)C)OC(=O)[C@H](O)[C@@H](NC(=O)C=1C=CC=CC=1)C=1C=CC=CC=1)O)C(=O)C1=CC=CC=C1 RCINICONZNJXQF-MZXODVADSA-N 0.000 claims description 25
- AOJJSUZBOXZQNB-TZSSRYMLSA-N Doxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-TZSSRYMLSA-N 0.000 claims description 24
- 230000014509 gene expression Effects 0.000 claims description 24
- 150000002632 lipids Chemical group 0.000 claims description 24
- GGGDNPWHMNJRFN-UHFFFAOYSA-N metrizoic acid Chemical compound CC(=O)N(C)C1=C(I)C(NC(C)=O)=C(I)C(C(O)=O)=C1I GGGDNPWHMNJRFN-UHFFFAOYSA-N 0.000 claims description 24
- 239000002246 antineoplastic agent Substances 0.000 claims description 23
- 239000012472 biological sample Substances 0.000 claims description 23
- 230000004614 tumor growth Effects 0.000 claims description 22
- 230000001093 anti-cancer Effects 0.000 claims description 20
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 19
- 239000003534 dna topoisomerase inhibitor Substances 0.000 claims description 19
- FDLYAMZZIXQODN-UHFFFAOYSA-N olaparib Chemical group FC1=CC=C(CC=2C3=CC=CC=C3C(=O)NN=2)C=C1C(=O)N(CC1)CCN1C(=O)C1CC1 FDLYAMZZIXQODN-UHFFFAOYSA-N 0.000 claims description 19
- 229940044693 topoisomerase inhibitor Drugs 0.000 claims description 19
- 102000015696 Interleukins Human genes 0.000 claims description 18
- 108010063738 Interleukins Proteins 0.000 claims description 18
- 238000011224 anti-cancer immunotherapy Methods 0.000 claims description 17
- 229940022399 cancer vaccine Drugs 0.000 claims description 17
- 230000000694 effects Effects 0.000 claims description 17
- 229960005420 etoposide Drugs 0.000 claims description 17
- VJJPUSNTGOMMGY-MRVIYFEKSA-N etoposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 VJJPUSNTGOMMGY-MRVIYFEKSA-N 0.000 claims description 17
- 230000002519 immonomodulatory effect Effects 0.000 claims description 17
- 238000001959 radiotherapy Methods 0.000 claims description 17
- IAKHMKGGTNLKSZ-INIZCTEOSA-N (S)-colchicine Chemical compound C1([C@@H](NC(C)=O)CC2)=CC(=O)C(OC)=CC=C1C1=C2C=C(OC)C(OC)=C1OC IAKHMKGGTNLKSZ-INIZCTEOSA-N 0.000 claims description 14
- 239000012661 PARP inhibitor Substances 0.000 claims description 14
- 229940121906 Poly ADP ribose polymerase inhibitor Drugs 0.000 claims description 14
- 229960000303 topotecan Drugs 0.000 claims description 14
- UCFGDBYHRUNTLO-QHCPKHFHSA-N topotecan Chemical compound C1=C(O)C(CN(C)C)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UCFGDBYHRUNTLO-QHCPKHFHSA-N 0.000 claims description 14
- 229960003048 vinblastine Drugs 0.000 claims description 14
- 208000003174 Brain Neoplasms Diseases 0.000 claims description 13
- 206010006187 Breast cancer Diseases 0.000 claims description 13
- 208000026310 Breast neoplasm Diseases 0.000 claims description 13
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 13
- 230000004913 activation Effects 0.000 claims description 13
- GXJABQQUPOEUTA-RDJZCZTQSA-N bortezomib Chemical group C([C@@H](C(=O)N[C@@H](CC(C)C)B(O)O)NC(=O)C=1N=CC=NC=1)C1=CC=CC=C1 GXJABQQUPOEUTA-RDJZCZTQSA-N 0.000 claims description 13
- 229920001184 polypeptide Polymers 0.000 claims description 13
- BZTDTCNHAFUJOG-UHFFFAOYSA-N 6-carboxyfluorescein Chemical compound C12=CC=C(O)C=C2OC2=CC(O)=CC=C2C11OC(=O)C2=CC=C(C(=O)O)C=C21 BZTDTCNHAFUJOG-UHFFFAOYSA-N 0.000 claims description 12
- AYZRKFOEZQBUEA-UHFFFAOYSA-N CAN-508 Chemical compound NC1=NNC(N)=C1N=NC1=CC=C(O)C=C1 AYZRKFOEZQBUEA-UHFFFAOYSA-N 0.000 claims description 12
- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 claims description 12
- VTLYFUHAOXGGBS-UHFFFAOYSA-N Fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 claims description 12
- 229910052688 Gadolinium Inorganic materials 0.000 claims description 12
- WAEMQWOKJMHJLA-UHFFFAOYSA-N Manganese(2+) Chemical compound [Mn+2] WAEMQWOKJMHJLA-UHFFFAOYSA-N 0.000 claims description 12
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 12
- 229940079156 Proteasome inhibitor Drugs 0.000 claims description 12
- 229940122429 Tubulin inhibitor Drugs 0.000 claims description 12
- 229940100198 alkylating agent Drugs 0.000 claims description 12
- 239000002168 alkylating agent Substances 0.000 claims description 12
- YVPYQUNUQOZFHG-UHFFFAOYSA-N amidotrizoic acid Chemical compound CC(=O)NC1=C(I)C(NC(C)=O)=C(I)C(C(O)=O)=C1I YVPYQUNUQOZFHG-UHFFFAOYSA-N 0.000 claims description 12
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 claims description 12
- 108010021331 carfilzomib Proteins 0.000 claims description 12
- BLMPQMFVWMYDKT-NZTKNTHTSA-N carfilzomib Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CC(C)C)C(=O)[C@]1(C)OC1)NC(=O)CN1CCOCC1)CC1=CC=CC=C1 BLMPQMFVWMYDKT-NZTKNTHTSA-N 0.000 claims description 12
- BFGKITSFLPAWGI-UHFFFAOYSA-N chromium(3+) Chemical compound [Cr+3] BFGKITSFLPAWGI-UHFFFAOYSA-N 0.000 claims description 12
- 208000029742 colonic neoplasm Diseases 0.000 claims description 12
- 229960005423 diatrizoate Drugs 0.000 claims description 12
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 claims description 12
- 229960004359 iodixanol Drugs 0.000 claims description 12
- NBQNWMBBSKPBAY-UHFFFAOYSA-N iodixanol Chemical compound IC=1C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C(I)C=1N(C(=O)C)CC(O)CN(C(C)=O)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NBQNWMBBSKPBAY-UHFFFAOYSA-N 0.000 claims description 12
- 229960001025 iohexol Drugs 0.000 claims description 12
- NTHXOOBQLCIOLC-UHFFFAOYSA-N iohexol Chemical compound OCC(O)CN(C(=O)C)C1=C(I)C(C(=O)NCC(O)CO)=C(I)C(C(=O)NCC(O)CO)=C1I NTHXOOBQLCIOLC-UHFFFAOYSA-N 0.000 claims description 12
- 229960004647 iopamidol Drugs 0.000 claims description 12
- XQZXYNRDCRIARQ-LURJTMIESA-N iopamidol Chemical compound C[C@H](O)C(=O)NC1=C(I)C(C(=O)NC(CO)CO)=C(I)C(C(=O)NC(CO)CO)=C1I XQZXYNRDCRIARQ-LURJTMIESA-N 0.000 claims description 12
- 229940029407 ioxaglate Drugs 0.000 claims description 12
- TYYBFXNZMFNZJT-UHFFFAOYSA-N ioxaglic acid Chemical compound CNC(=O)C1=C(I)C(N(C)C(C)=O)=C(I)C(C(=O)NCC(=O)NC=2C(=C(C(=O)NCCO)C(I)=C(C(O)=O)C=2I)I)=C1I TYYBFXNZMFNZJT-UHFFFAOYSA-N 0.000 claims description 12
- MMIPFLVOWGHZQD-UHFFFAOYSA-N manganese(3+) Chemical compound [Mn+3] MMIPFLVOWGHZQD-UHFFFAOYSA-N 0.000 claims description 12
- 229960004712 metrizoic acid Drugs 0.000 claims description 12
- 229960002378 oftasceine Drugs 0.000 claims description 12
- 239000003207 proteasome inhibitor Substances 0.000 claims description 12
- DOSGOCSVHPUUIA-UHFFFAOYSA-N samarium(3+) Chemical compound [Sm+3] DOSGOCSVHPUUIA-UHFFFAOYSA-N 0.000 claims description 12
- 229950011257 veliparib Drugs 0.000 claims description 12
- JNAHVYVRKWKWKQ-CYBMUJFWSA-N veliparib Chemical compound N=1C2=CC=CC(C(N)=O)=C2NC=1[C@@]1(C)CCCN1 JNAHVYVRKWKWKQ-CYBMUJFWSA-N 0.000 claims description 12
- 101000777293 Homo sapiens Serine/threonine-protein kinase Chk1 Proteins 0.000 claims description 11
- 102100031081 Serine/threonine-protein kinase Chk1 Human genes 0.000 claims description 11
- 229960004679 doxorubicin Drugs 0.000 claims description 11
- 206010017758 gastric cancer Diseases 0.000 claims description 11
- KTUFNOKKBVMGRW-UHFFFAOYSA-N imatinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 KTUFNOKKBVMGRW-UHFFFAOYSA-N 0.000 claims description 11
- 210000002307 prostate Anatomy 0.000 claims description 11
- 230000004044 response Effects 0.000 claims description 11
- 201000011549 stomach cancer Diseases 0.000 claims description 11
- ZDZOTLJHXYCWBA-VCVYQWHSSA-N N-debenzoyl-N-(tert-butoxycarbonyl)-10-deacetyltaxol Chemical compound O([C@H]1[C@H]2[C@@](C([C@H](O)C3=C(C)[C@@H](OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)C=4C=CC=CC=4)C[C@]1(O)C3(C)C)=O)(C)[C@@H](O)C[C@H]1OC[C@]12OC(=O)C)C(=O)C1=CC=CC=C1 ZDZOTLJHXYCWBA-VCVYQWHSSA-N 0.000 claims description 10
- 125000000539 amino acid group Chemical group 0.000 claims description 10
- 206010009944 Colon cancer Diseases 0.000 claims description 9
- GHASVSINZRGABV-UHFFFAOYSA-N Fluorouracil Chemical compound FC1=CNC(=O)NC1=O GHASVSINZRGABV-UHFFFAOYSA-N 0.000 claims description 9
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 9
- QEFRNWWLZKMPFJ-UHFFFAOYSA-N L-methionine sulphoxide Natural products CS(=O)CCC(N)C(O)=O QEFRNWWLZKMPFJ-UHFFFAOYSA-N 0.000 claims description 9
- 239000005517 L01XE01 - Imatinib Substances 0.000 claims description 9
- 206010038389 Renal cancer Diseases 0.000 claims description 9
- 230000000903 blocking effect Effects 0.000 claims description 9
- 229960002949 fluorouracil Drugs 0.000 claims description 9
- 201000010982 kidney cancer Diseases 0.000 claims description 9
- NRUKOCRGYNPUPR-QBPJDGROSA-N teniposide Chemical compound COC1=C(O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@@H](OC[C@H]4O3)C=3SC=CC=3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 NRUKOCRGYNPUPR-QBPJDGROSA-N 0.000 claims description 9
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 claims description 8
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 claims description 8
- 102100025136 Macrosialin Human genes 0.000 claims description 8
- JXLYSJRDGCGARV-WWYNWVTFSA-N Vinblastine Natural products O=C(O[C@H]1[C@](O)(C(=O)OC)[C@@H]2N(C)c3c(cc(c(OC)c3)[C@]3(C(=O)OC)c4[nH]c5c(c4CCN4C[C@](O)(CC)C[C@H](C3)C4)cccc5)[C@@]32[C@H]2[C@@]1(CC)C=CCN2CC3)C JXLYSJRDGCGARV-WWYNWVTFSA-N 0.000 claims description 8
- 229960004630 chlorambucil Drugs 0.000 claims description 8
- JCKYGMPEJWAADB-UHFFFAOYSA-N chlorambucil Chemical compound OC(=O)CCCC1=CC=C(N(CCCl)CCCl)C=C1 JCKYGMPEJWAADB-UHFFFAOYSA-N 0.000 claims description 8
- 229960004397 cyclophosphamide Drugs 0.000 claims description 8
- 239000003085 diluting agent Substances 0.000 claims description 8
- SDUQYLNIPVEERB-QPPQHZFASA-N gemcitabine Chemical compound O=C1N=C(N)C=CN1[C@H]1C(F)(F)[C@H](O)[C@@H](CO)O1 SDUQYLNIPVEERB-QPPQHZFASA-N 0.000 claims description 8
- 229960005277 gemcitabine Drugs 0.000 claims description 8
- 208000005017 glioblastoma Diseases 0.000 claims description 8
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 8
- 238000001356 surgical procedure Methods 0.000 claims description 8
- 229960001278 teniposide Drugs 0.000 claims description 8
- JXLYSJRDGCGARV-XQKSVPLYSA-N vincaleukoblastine Chemical compound C([C@@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-XQKSVPLYSA-N 0.000 claims description 8
- AOJJSUZBOXZQNB-VTZDEGQISA-N 4'-epidoxorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@@H](O)[C@H](C)O1 AOJJSUZBOXZQNB-VTZDEGQISA-N 0.000 claims description 7
- IDPUKCWIGUEADI-UHFFFAOYSA-N 5-[bis(2-chloroethyl)amino]uracil Chemical compound ClCCN(CCCl)C1=CNC(=O)NC1=O IDPUKCWIGUEADI-UHFFFAOYSA-N 0.000 claims description 7
- 108010006654 Bleomycin Proteins 0.000 claims description 7
- COVZYZSDYWQREU-UHFFFAOYSA-N Busulfan Chemical compound CS(=O)(=O)OCCCCOS(C)(=O)=O COVZYZSDYWQREU-UHFFFAOYSA-N 0.000 claims description 7
- 229940126192 CSF1R kinase inhibitor Drugs 0.000 claims description 7
- DLGOEMSEDOSKAD-UHFFFAOYSA-N Carmustine Chemical compound ClCCNC(=O)N(N=O)CCCl DLGOEMSEDOSKAD-UHFFFAOYSA-N 0.000 claims description 7
- HTIJFSOGRVMCQR-UHFFFAOYSA-N Epirubicin Natural products COc1cccc2C(=O)c3c(O)c4CC(O)(CC(OC5CC(N)C(=O)C(C)O5)c4c(O)c3C(=O)c12)C(=O)CO HTIJFSOGRVMCQR-UHFFFAOYSA-N 0.000 claims description 7
- 108010050904 Interferons Proteins 0.000 claims description 7
- 102000014150 Interferons Human genes 0.000 claims description 7
- 108090001005 Interleukin-6 Proteins 0.000 claims description 7
- 102000004889 Interleukin-6 Human genes 0.000 claims description 7
- GQYIWUVLTXOXAJ-UHFFFAOYSA-N Lomustine Chemical compound ClCCN(N=O)C(=O)NC1CCCCC1 GQYIWUVLTXOXAJ-UHFFFAOYSA-N 0.000 claims description 7
- 229960000548 alemtuzumab Drugs 0.000 claims description 7
- 229960001561 bleomycin Drugs 0.000 claims description 7
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 claims description 7
- 229960001467 bortezomib Drugs 0.000 claims description 7
- 229960002092 busulfan Drugs 0.000 claims description 7
- 229960004562 carboplatin Drugs 0.000 claims description 7
- 229960005243 carmustine Drugs 0.000 claims description 7
- DQLATGHUWYMOKM-UHFFFAOYSA-L cisplatin Chemical compound N[Pt](N)(Cl)Cl DQLATGHUWYMOKM-UHFFFAOYSA-L 0.000 claims description 7
- 229960004316 cisplatin Drugs 0.000 claims description 7
- 229960001338 colchicine Drugs 0.000 claims description 7
- 229960003668 docetaxel Drugs 0.000 claims description 7
- OFDNQWIFNXBECV-VFSYNPLYSA-N dolastatin 10 Chemical compound CC(C)[C@H](N(C)C)C(=O)N[C@@H](C(C)C)C(=O)N(C)[C@@H]([C@@H](C)CC)[C@H](OC)CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C=1SC=CN=1)CC1=CC=CC=C1 OFDNQWIFNXBECV-VFSYNPLYSA-N 0.000 claims description 7
- 229960001904 epirubicin Drugs 0.000 claims description 7
- 229960002411 imatinib Drugs 0.000 claims description 7
- 229960004768 irinotecan Drugs 0.000 claims description 7
- UWKQSNNFCGGAFS-XIFFEERXSA-N irinotecan Chemical compound C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 UWKQSNNFCGGAFS-XIFFEERXSA-N 0.000 claims description 7
- 229960002247 lomustine Drugs 0.000 claims description 7
- 229960001924 melphalan Drugs 0.000 claims description 7
- SGDBTWWWUNNDEQ-LBPRGKRZSA-N melphalan Chemical compound OC(=O)[C@@H](N)CC1=CC=C(N(CCCl)CCCl)C=C1 SGDBTWWWUNNDEQ-LBPRGKRZSA-N 0.000 claims description 7
- 229960003301 nivolumab Drugs 0.000 claims description 7
- 229960000572 olaparib Drugs 0.000 claims description 7
- 229960001756 oxaliplatin Drugs 0.000 claims description 7
- DWAFYCQODLXJNR-BNTLRKBRSA-L oxaliplatin Chemical compound O1C(=O)C(=O)O[Pt]11N[C@@H]2CCCC[C@H]2N1 DWAFYCQODLXJNR-BNTLRKBRSA-L 0.000 claims description 7
- 229960002621 pembrolizumab Drugs 0.000 claims description 7
- CPTBDICYNRMXFX-UHFFFAOYSA-N procarbazine Chemical compound CNNCC1=CC=C(C(=O)NC(C)C)C=C1 CPTBDICYNRMXFX-UHFFFAOYSA-N 0.000 claims description 7
- 229960000624 procarbazine Drugs 0.000 claims description 7
- 108090000623 proteins and genes Proteins 0.000 claims description 7
- 229960004641 rituximab Drugs 0.000 claims description 7
- HMABYWSNWIZPAG-UHFFFAOYSA-N rucaparib Chemical compound C1=CC(CNC)=CC=C1C(N1)=C2CCNC(=O)C3=C2C1=CC(F)=C3 HMABYWSNWIZPAG-UHFFFAOYSA-N 0.000 claims description 7
- 229960001052 streptozocin Drugs 0.000 claims description 7
- ZSJLQEPLLKMAKR-GKHCUFPYSA-N streptozocin Chemical compound O=NN(C)C(=O)N[C@H]1[C@@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O ZSJLQEPLLKMAKR-GKHCUFPYSA-N 0.000 claims description 7
- 229960001055 uracil mustard Drugs 0.000 claims description 7
- LSXOBYNBRKOTIQ-RQUBOUMQSA-N (3s,10r,13e,16s)-10-[(3-chloro-4-methoxyphenyl)methyl]-6,6-dimethyl-3-(2-methylpropyl)-16-[(1s)-1-[(2r,3r)-3-phenyloxiran-2-yl]ethyl]-1,4-dioxa-8,11-diazacyclohexadec-13-ene-2,5,9,12-tetrone Chemical compound C1=C(Cl)C(OC)=CC=C1C[C@@H]1C(=O)NCC(C)(C)C(=O)O[C@@H](CC(C)C)C(=O)O[C@H]([C@H](C)[C@@H]2[C@H](O2)C=2C=CC=CC=2)C/C=C/C(=O)N1 LSXOBYNBRKOTIQ-RQUBOUMQSA-N 0.000 claims description 6
- FDKXTQMXEQVLRF-ZHACJKMWSA-N (E)-dacarbazine Chemical compound CN(C)\N=N\c1[nH]cnc1C(N)=O FDKXTQMXEQVLRF-ZHACJKMWSA-N 0.000 claims description 6
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 claims description 6
- MDOJTZQKHMAPBK-UHFFFAOYSA-N 4-iodo-3-nitrobenzamide Chemical compound NC(=O)C1=CC=C(I)C([N+]([O-])=O)=C1 MDOJTZQKHMAPBK-UHFFFAOYSA-N 0.000 claims description 6
- 229940124957 Cervarix Drugs 0.000 claims description 6
- 229930188224 Cryptophycin Natural products 0.000 claims description 6
- 229940124087 DNA topoisomerase II inhibitor Drugs 0.000 claims description 6
- 229940124897 Gardasil Drugs 0.000 claims description 6
- 101000777277 Homo sapiens Serine/threonine-protein kinase Chk2 Proteins 0.000 claims description 6
- QEFRNWWLZKMPFJ-ZXPFJRLXSA-N L-methionine (R)-S-oxide Chemical compound C[S@@](=O)CC[C@H]([NH3+])C([O-])=O QEFRNWWLZKMPFJ-ZXPFJRLXSA-N 0.000 claims description 6
- 239000005536 L01XE08 - Nilotinib Substances 0.000 claims description 6
- 102100028123 Macrophage colony-stimulating factor 1 Human genes 0.000 claims description 6
- OWPCHSCAPHNHAV-UHFFFAOYSA-N Rhizoxin Natural products C1C(O)C2(C)OC2C=CC(C)C(OC(=O)C2)CC2CC2OC2C(=O)OC1C(C)C(OC)C(C)=CC=CC(C)=CC1=COC(C)=N1 OWPCHSCAPHNHAV-UHFFFAOYSA-N 0.000 claims description 6
- 190014017285 Satraplatin Chemical compound 0.000 claims description 6
- 102100031075 Serine/threonine-protein kinase Chk2 Human genes 0.000 claims description 6
- 239000000317 Topoisomerase II Inhibitor Substances 0.000 claims description 6
- 190014017283 Triplatin tetranitrate Chemical compound 0.000 claims description 6
- 229960001220 amsacrine Drugs 0.000 claims description 6
- XCPGHVQEEXUHNC-UHFFFAOYSA-N amsacrine Chemical compound COC1=CC(NS(C)(=O)=O)=CC=C1NC1=C(C=CC=C2)C2=NC2=CC=CC=C12 XCPGHVQEEXUHNC-UHFFFAOYSA-N 0.000 claims description 6
- 229960002438 carfilzomib Drugs 0.000 claims description 6
- 108010006226 cryptophycin Proteins 0.000 claims description 6
- PSNOPSMXOBPNNV-VVCTWANISA-N cryptophycin 1 Chemical compound C1=C(Cl)C(OC)=CC=C1C[C@@H]1C(=O)NC[C@@H](C)C(=O)O[C@@H](CC(C)C)C(=O)O[C@H]([C@H](C)[C@@H]2[C@H](O2)C=2C=CC=CC=2)C/C=C/C(=O)N1 PSNOPSMXOBPNNV-VVCTWANISA-N 0.000 claims description 6
- 108010083340 cryptophycin 52 Proteins 0.000 claims description 6
- YFGZFQNBPSCWPN-UHFFFAOYSA-N cryptophycin 52 Natural products C1=CC(OC)=CC=C1CC1C(=O)NCC(C)C(=O)OC(CC(C)C)C(=O)OC(C(C)C2C(O2)C=2C=CC=CC=2)CC=CC(=O)N1 YFGZFQNBPSCWPN-UHFFFAOYSA-N 0.000 claims description 6
- PSNOPSMXOBPNNV-UHFFFAOYSA-N cryptophycin-327 Natural products C1=C(Cl)C(OC)=CC=C1CC1C(=O)NCC(C)C(=O)OC(CC(C)C)C(=O)OC(C(C)C2C(O2)C=2C=CC=CC=2)CC=CC(=O)N1 PSNOPSMXOBPNNV-UHFFFAOYSA-N 0.000 claims description 6
- 229960003901 dacarbazine Drugs 0.000 claims description 6
- 229930013356 epothilone Natural products 0.000 claims description 6
- 150000003883 epothilone derivatives Chemical class 0.000 claims description 6
- 229940079322 interferon Drugs 0.000 claims description 6
- 229940000764 kyprolis Drugs 0.000 claims description 6
- ISGGVCWFTPTHIX-UHFFFAOYSA-N n'-(2-hydroxy-3-piperidin-1-ylpropoxy)pyridine-3-carboximidamide;dihydrochloride Chemical compound Cl.Cl.C1CCCCN1CC(O)CONC(=N)C1=CC=CN=C1 ISGGVCWFTPTHIX-UHFFFAOYSA-N 0.000 claims description 6
- 229950007221 nedaplatin Drugs 0.000 claims description 6
- HHZIURLSWUIHRB-UHFFFAOYSA-N nilotinib Chemical compound C1=NC(C)=CN1C1=CC(NC(=O)C=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)=CC(C(F)(F)F)=C1 HHZIURLSWUIHRB-UHFFFAOYSA-N 0.000 claims description 6
- 229960001346 nilotinib Drugs 0.000 claims description 6
- BWKDAMBGCPRVPI-ZQRPHVBESA-N ortataxel Chemical compound O([C@@H]1[C@]23OC(=O)O[C@H]2[C@@H](C(=C([C@@H](OC(C)=O)C(=O)[C@]2(C)[C@@H](O)C[C@H]4OC[C@]4([C@H]21)OC(C)=O)C3(C)C)C)OC(=O)[C@H](O)[C@@H](NC(=O)OC(C)(C)C)CC(C)C)C(=O)C1=CC=CC=C1 BWKDAMBGCPRVPI-ZQRPHVBESA-N 0.000 claims description 6
- 229960000688 pomalidomide Drugs 0.000 claims description 6
- UVSMNLNDYGZFPF-UHFFFAOYSA-N pomalidomide Chemical compound O=C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O UVSMNLNDYGZFPF-UHFFFAOYSA-N 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 229940034080 provenge Drugs 0.000 claims description 6
- OWPCHSCAPHNHAV-LMONGJCWSA-N rhizoxin Chemical compound C/C([C@H](OC)[C@@H](C)[C@@H]1C[C@H](O)[C@]2(C)O[C@@H]2/C=C/[C@@H](C)[C@]2([H])OC(=O)C[C@@](C2)(C[C@@H]2O[C@H]2C(=O)O1)[H])=C\C=C\C(\C)=C\C1=COC(C)=N1 OWPCHSCAPHNHAV-LMONGJCWSA-N 0.000 claims description 6
- 229960005399 satraplatin Drugs 0.000 claims description 6
- 229960000714 sipuleucel-t Drugs 0.000 claims description 6
- 125000002456 taxol group Chemical group 0.000 claims description 6
- 229960003433 thalidomide Drugs 0.000 claims description 6
- PKVRCIRHQMSYJX-AIFWHQITSA-N trabectedin Chemical compound C([C@@]1(C(OC2)=O)NCCC3=C1C=C(C(=C3)O)OC)S[C@@H]1C3=C(OC(C)=O)C(C)=C4OCOC4=C3[C@H]2N2[C@@H](O)[C@H](CC=3C4=C(O)C(OC)=C(C)C=3)N(C)[C@H]4[C@@H]21 PKVRCIRHQMSYJX-AIFWHQITSA-N 0.000 claims description 6
- 229960000977 trabectedin Drugs 0.000 claims description 6
- 229950002860 triplatin tetranitrate Drugs 0.000 claims description 6
- 229940099039 velcade Drugs 0.000 claims description 6
- JXLYSJRDGCGARV-CFWMRBGOSA-N vinblastine Chemical compound C([C@H](C[C@]1(C(=O)OC)C=2C(=CC3=C([C@]45[C@H]([C@@]([C@H](OC(C)=O)[C@]6(CC)C=CCN([C@H]56)CC4)(O)C(=O)OC)N3C)C=2)OC)C[C@@](C2)(O)CC)N2CCC2=C1NC1=CC=CC=C21 JXLYSJRDGCGARV-CFWMRBGOSA-N 0.000 claims description 6
- 108010012934 Albumin-Bound Paclitaxel Proteins 0.000 claims description 5
- 206010005003 Bladder cancer Diseases 0.000 claims description 5
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 5
- 108010069514 Cyclic Peptides Proteins 0.000 claims description 5
- 102000001189 Cyclic Peptides Human genes 0.000 claims description 5
- 150000008574 D-amino acids Chemical class 0.000 claims description 5
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 claims description 5
- 239000005551 L01XE03 - Erlotinib Substances 0.000 claims description 5
- 208000034578 Multiple myelomas Diseases 0.000 claims description 5
- 206010033128 Ovarian cancer Diseases 0.000 claims description 5
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 5
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 5
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 claims description 5
- 229940028652 abraxane Drugs 0.000 claims description 5
- AAKJLRGGTJKAMG-UHFFFAOYSA-N erlotinib Chemical compound C=12C=C(OCCOC)C(OCCOC)=CC2=NC=NC=1NC1=CC=CC(C#C)=C1 AAKJLRGGTJKAMG-UHFFFAOYSA-N 0.000 claims description 5
- 239000012634 fragment Substances 0.000 claims description 5
- 208000032839 leukemia Diseases 0.000 claims description 5
- 201000007270 liver cancer Diseases 0.000 claims description 5
- 208000014018 liver neoplasm Diseases 0.000 claims description 5
- 201000008968 osteosarcoma Diseases 0.000 claims description 5
- JGWRKYUXBBNENE-UHFFFAOYSA-N pexidartinib Chemical group C1=NC(C(F)(F)F)=CC=C1CNC(N=C1)=CC=C1CC1=CNC2=NC=C(Cl)C=C12 JGWRKYUXBBNENE-UHFFFAOYSA-N 0.000 claims description 5
- 201000000849 skin cancer Diseases 0.000 claims description 5
- 229940121358 tyrosine kinase inhibitor Drugs 0.000 claims description 5
- 239000005483 tyrosine kinase inhibitor Substances 0.000 claims description 5
- 150000004917 tyrosine kinase inhibitor derivatives Chemical class 0.000 claims description 5
- 201000005112 urinary bladder cancer Diseases 0.000 claims description 5
- 206010008342 Cervix carcinoma Diseases 0.000 claims description 4
- 206010014733 Endometrial cancer Diseases 0.000 claims description 4
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 4
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 4
- 206010017993 Gastrointestinal neoplasms Diseases 0.000 claims description 4
- 208000032612 Glial tumor Diseases 0.000 claims description 4
- 201000010915 Glioblastoma multiforme Diseases 0.000 claims description 4
- 206010018338 Glioma Diseases 0.000 claims description 4
- 239000002147 L01XE04 - Sunitinib Substances 0.000 claims description 4
- 206010029260 Neuroblastoma Diseases 0.000 claims description 4
- 201000004404 Neurofibroma Diseases 0.000 claims description 4
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 4
- 206010060862 Prostate cancer Diseases 0.000 claims description 4
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 4
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 claims description 4
- 201000010881 cervical cancer Diseases 0.000 claims description 4
- 229950000521 entrectinib Drugs 0.000 claims description 4
- 201000004101 esophageal cancer Diseases 0.000 claims description 4
- 201000010536 head and neck cancer Diseases 0.000 claims description 4
- 208000014829 head and neck neoplasm Diseases 0.000 claims description 4
- VHOGYURTWQBHIL-UHFFFAOYSA-N leflunomide Chemical compound O1N=CC(C(=O)NC=2C=CC(=CC=2)C(F)(F)F)=C1C VHOGYURTWQBHIL-UHFFFAOYSA-N 0.000 claims description 4
- 201000001441 melanoma Diseases 0.000 claims description 4
- LBWFXVZLPYTWQI-IPOVEDGCSA-N n-[2-(diethylamino)ethyl]-5-[(z)-(5-fluoro-2-oxo-1h-indol-3-ylidene)methyl]-2,4-dimethyl-1h-pyrrole-3-carboxamide;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C LBWFXVZLPYTWQI-IPOVEDGCSA-N 0.000 claims description 4
- HAYYBYPASCDWEQ-UHFFFAOYSA-N n-[5-[(3,5-difluorophenyl)methyl]-1h-indazol-3-yl]-4-(4-methylpiperazin-1-yl)-2-(oxan-4-ylamino)benzamide Chemical compound C1CN(C)CCN1C(C=C1NC2CCOCC2)=CC=C1C(=O)NC(C1=C2)=NNC1=CC=C2CC1=CC(F)=CC(F)=C1 HAYYBYPASCDWEQ-UHFFFAOYSA-N 0.000 claims description 4
- WUWDLXZGHZSWQZ-WQLSENKSSA-N semaxanib Chemical compound N1C(C)=CC(C)=C1\C=C/1C2=CC=CC=C2NC\1=O WUWDLXZGHZSWQZ-WQLSENKSSA-N 0.000 claims description 4
- 229940034785 sutent Drugs 0.000 claims description 4
- 201000002510 thyroid cancer Diseases 0.000 claims description 4
- 108050003188 Disks large-associated protein 1 Proteins 0.000 claims description 3
- 102100033499 Interleukin-34 Human genes 0.000 claims description 3
- 101710181549 Interleukin-34 Proteins 0.000 claims description 3
- 201000007455 central nervous system cancer Diseases 0.000 claims description 3
- 229960005395 cetuximab Drugs 0.000 claims description 3
- NVLDXKCJZRQSDJ-UHFFFAOYSA-L cyclohexane-1,2-diamine;2-(1,2-dihydroxyethyl)-3-hydroxy-5-oxo-2h-furan-4-olate;platinum(2+) Chemical compound [Pt+2].NC1CCCCC1N.OCC(O)C1OC(=O)C([O-])=C1O.OCC(O)C1OC(=O)C([O-])=C1O NVLDXKCJZRQSDJ-UHFFFAOYSA-L 0.000 claims description 3
- 229960001433 erlotinib Drugs 0.000 claims description 3
- XGALLCVXEZPNRQ-UHFFFAOYSA-N gefitinib Chemical compound C=12C=C(OCCCN3CCOCC3)C(OC)=CC2=NC=NC=1NC1=CC=C(F)C(Cl)=C1 XGALLCVXEZPNRQ-UHFFFAOYSA-N 0.000 claims description 3
- 229940080856 gleevec Drugs 0.000 claims description 3
- BCFGMOOMADDAQU-UHFFFAOYSA-N lapatinib Chemical compound O1C(CNCCS(=O)(=O)C)=CC=C1C1=CC=C(N=CN=C2NC=3C=C(Cl)C(OCC=4C=C(F)C=CC=4)=CC=3)C2=C1 BCFGMOOMADDAQU-UHFFFAOYSA-N 0.000 claims description 3
- 239000003550 marker Substances 0.000 claims description 3
- 229960000575 trastuzumab Drugs 0.000 claims description 3
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 claims description 2
- 229960003008 blinatumomab Drugs 0.000 claims description 2
- OMZCMEYTWSXEPZ-UHFFFAOYSA-N canertinib Chemical compound C1=C(Cl)C(F)=CC=C1NC1=NC=NC2=CC(OCCCN3CCOCC3)=C(NC(=O)C=C)C=C12 OMZCMEYTWSXEPZ-UHFFFAOYSA-N 0.000 claims description 2
- 238000002659 cell therapy Methods 0.000 claims description 2
- HWGQMRYQVZSGDQ-HZPDHXFCSA-N chembl3137320 Chemical compound CN1N=CN=C1[C@H]([C@H](N1)C=2C=CC(F)=CC=2)C2=NNC(=O)C3=C2C1=CC(F)=C3 HWGQMRYQVZSGDQ-HZPDHXFCSA-N 0.000 claims description 2
- 229960002204 daratumumab Drugs 0.000 claims description 2
- 238000010894 electron beam technology Methods 0.000 claims description 2
- 229960004137 elotuzumab Drugs 0.000 claims description 2
- 238000010884 ion-beam technique Methods 0.000 claims description 2
- 229960000681 leflunomide Drugs 0.000 claims description 2
- 238000011418 maintenance treatment Methods 0.000 claims description 2
- 229950001457 pexidartinib Drugs 0.000 claims description 2
- 230000002285 radioactive effect Effects 0.000 claims description 2
- 238000002271 resection Methods 0.000 claims description 2
- 229950003647 semaxanib Drugs 0.000 claims description 2
- IVDHYUQIDRJSTI-UHFFFAOYSA-N sorafenib tosylate Chemical compound [H+].CC1=CC=C(S([O-])(=O)=O)C=C1.C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 IVDHYUQIDRJSTI-UHFFFAOYSA-N 0.000 claims description 2
- 229950004550 talazoparib Drugs 0.000 claims description 2
- 229940120982 tarceva Drugs 0.000 claims description 2
- 229950000578 vatalanib Drugs 0.000 claims description 2
- YCOYDOIWSSHVCK-UHFFFAOYSA-N vatalanib Chemical compound C1=CC(Cl)=CC=C1NC(C1=CC=CC=C11)=NN=C1CC1=CC=NC=C1 YCOYDOIWSSHVCK-UHFFFAOYSA-N 0.000 claims description 2
- LLDWLPRYLVPDTG-UHFFFAOYSA-N vatalanib succinate Chemical compound OC(=O)CCC(O)=O.C1=CC(Cl)=CC=C1NC(C1=CC=CC=C11)=NN=C1CC1=CC=NC=C1 LLDWLPRYLVPDTG-UHFFFAOYSA-N 0.000 claims description 2
- 101710150918 Macrophage colony-stimulating factor 1 receptor Proteins 0.000 claims 7
- 238000003745 diagnosis Methods 0.000 claims 4
- 239000000969 carrier Substances 0.000 claims 3
- 102100031250 Disks large-associated protein 1 Human genes 0.000 claims 2
- 190000008236 Carboplatin Chemical compound 0.000 claims 1
- 190000005734 Nedaplatin Chemical compound 0.000 claims 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 claims 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 claims 1
- 239000003937 drug carrier Substances 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 77
- 201000010099 disease Diseases 0.000 abstract description 71
- 206010039710 Scleroderma Diseases 0.000 abstract description 39
- 230000002265 prevention Effects 0.000 abstract description 15
- 238000001514 detection method Methods 0.000 abstract description 13
- 208000011580 syndromic disease Diseases 0.000 abstract description 12
- 210000002865 immune cell Anatomy 0.000 abstract description 11
- 238000002648 combination therapy Methods 0.000 abstract description 9
- 230000028993 immune response Effects 0.000 abstract description 6
- 208000004434 Calcinosis Diseases 0.000 abstract description 5
- 208000012322 Raynaud phenomenon Diseases 0.000 abstract description 5
- 208000009056 telangiectasis Diseases 0.000 abstract description 5
- 208000030644 Esophageal Motility disease Diseases 0.000 abstract description 4
- 206010043189 Telangiectasia Diseases 0.000 abstract description 4
- 201000011191 dyskinesia of esophagus Diseases 0.000 abstract description 4
- 230000006041 cell recruitment Effects 0.000 abstract description 3
- 238000011269 treatment regimen Methods 0.000 abstract description 3
- 239000002245 particle Substances 0.000 description 73
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 58
- 229940024606 amino acid Drugs 0.000 description 56
- 235000001014 amino acid Nutrition 0.000 description 56
- 150000001413 amino acids Chemical class 0.000 description 56
- 102100038717 TYRO protein tyrosine kinase-binding protein Human genes 0.000 description 52
- 108010028921 Lipopeptides Proteins 0.000 description 51
- 101710187864 TYRO protein tyrosine kinase-binding protein Proteins 0.000 description 50
- 102000004895 Lipoproteins Human genes 0.000 description 46
- 108090001030 Lipoproteins Proteins 0.000 description 46
- 150000001875 compounds Chemical class 0.000 description 43
- 210000002540 macrophage Anatomy 0.000 description 43
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Chemical compound CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 42
- 208000024891 symptom Diseases 0.000 description 41
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 32
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 32
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 30
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 26
- 101800002011 Amphipathic peptide Proteins 0.000 description 23
- 102000007651 Macrophage Colony-Stimulating Factor Human genes 0.000 description 22
- 241000699670 Mus sp. Species 0.000 description 22
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 21
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 21
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 21
- 230000003993 interaction Effects 0.000 description 21
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 21
- 229940124597 therapeutic agent Drugs 0.000 description 21
- 239000004474 valine Substances 0.000 description 21
- 125000002924 primary amino group Chemical class [H]N([H])* 0.000 description 20
- 239000003981 vehicle Substances 0.000 description 20
- 239000004475 Arginine Substances 0.000 description 19
- 208000022559 Inflammatory bowel disease Diseases 0.000 description 19
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 19
- 241001111421 Pannus Species 0.000 description 18
- 210000004072 lung Anatomy 0.000 description 18
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 17
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 17
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 17
- 235000009582 asparagine Nutrition 0.000 description 17
- 229960001230 asparagine Drugs 0.000 description 17
- 239000003814 drug Substances 0.000 description 17
- 239000002158 endotoxin Substances 0.000 description 17
- 230000002757 inflammatory effect Effects 0.000 description 17
- 229920006008 lipopolysaccharide Polymers 0.000 description 17
- 102000005962 receptors Human genes 0.000 description 17
- 108020003175 receptors Proteins 0.000 description 17
- 208000030507 AIDS Diseases 0.000 description 16
- 102000005666 Apolipoprotein A-I Human genes 0.000 description 16
- 108010059886 Apolipoprotein A-I Proteins 0.000 description 16
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 16
- 108091008874 T cell receptors Proteins 0.000 description 16
- 102000016266 T-Cell Antigen Receptors Human genes 0.000 description 16
- 230000006870 function Effects 0.000 description 16
- 235000013922 glutamic acid Nutrition 0.000 description 16
- 239000004220 glutamic acid Substances 0.000 description 16
- 125000000291 glutamic acid group Chemical group N[C@@H](CCC(O)=O)C(=O)* 0.000 description 16
- 101000795107 Homo sapiens Triggering receptor expressed on myeloid cells 1 Proteins 0.000 description 15
- 239000004472 Lysine Substances 0.000 description 15
- 201000011152 Pemphigus Diseases 0.000 description 15
- 201000004681 Psoriasis Diseases 0.000 description 15
- 230000001363 autoimmune Effects 0.000 description 15
- 201000001976 pemphigus vulgaris Diseases 0.000 description 15
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 14
- 125000000174 L-prolyl group Chemical group [H]N1C([H])([H])C([H])([H])C([H])([H])[C@@]1([H])C(*)=O 0.000 description 14
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 14
- 230000000172 allergic effect Effects 0.000 description 14
- 208000010668 atopic eczema Diseases 0.000 description 14
- 208000011231 Crohn disease Diseases 0.000 description 13
- 239000004471 Glycine Substances 0.000 description 13
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 13
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 13
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 13
- 125000003630 glycyl group Chemical group [H]N([H])C([H])([H])C(*)=O 0.000 description 13
- 201000006417 multiple sclerosis Diseases 0.000 description 13
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 13
- 206010039073 rheumatoid arthritis Diseases 0.000 description 13
- 102000014452 scavenger receptors Human genes 0.000 description 13
- 201000000596 systemic lupus erythematosus Diseases 0.000 description 13
- 102000007592 Apolipoproteins Human genes 0.000 description 12
- 108010071619 Apolipoproteins Proteins 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 12
- 101000916644 Homo sapiens Macrophage colony-stimulating factor 1 receptor Proteins 0.000 description 12
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 12
- 235000019441 ethanol Nutrition 0.000 description 12
- 229930182817 methionine Natural products 0.000 description 12
- 229960004452 methionine Drugs 0.000 description 12
- 201000001320 Atherosclerosis Diseases 0.000 description 11
- 206010042953 Systemic sclerosis Diseases 0.000 description 11
- 102000054961 human TREM1 Human genes 0.000 description 11
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Substances [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 10
- 108010058398 Macrophage Colony-Stimulating Factor Receptor Proteins 0.000 description 10
- 241000699666 Mus <mouse, genus> Species 0.000 description 10
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 10
- CTSPAMFJBXKSOY-UHFFFAOYSA-N ellipticine Chemical compound N1=CC=C2C(C)=C(NC=3C4=CC=CC=3)C4=C(C)C2=C1 CTSPAMFJBXKSOY-UHFFFAOYSA-N 0.000 description 10
- 210000002216 heart Anatomy 0.000 description 10
- 230000002829 reductive effect Effects 0.000 description 10
- 230000004083 survival effect Effects 0.000 description 10
- 210000001519 tissue Anatomy 0.000 description 10
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 10
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 10
- 125000001493 tyrosinyl group Chemical group [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 10
- 208000023275 Autoimmune disease Diseases 0.000 description 9
- 208000017442 Retinal disease Diseases 0.000 description 9
- 206010038923 Retinopathy Diseases 0.000 description 9
- 206010040047 Sepsis Diseases 0.000 description 9
- 201000009594 Systemic Scleroderma Diseases 0.000 description 9
- 210000004556 brain Anatomy 0.000 description 9
- 230000012202 endocytosis Effects 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 9
- 239000003446 ligand Substances 0.000 description 9
- 239000002105 nanoparticle Substances 0.000 description 9
- 210000000056 organ Anatomy 0.000 description 9
- 108010078070 scavenger receptors Proteins 0.000 description 9
- 210000002784 stomach Anatomy 0.000 description 9
- 230000008685 targeting Effects 0.000 description 9
- 208000001395 Acute radiation syndrome Diseases 0.000 description 8
- 208000024827 Alzheimer disease Diseases 0.000 description 8
- 206010002412 Angiocentric lymphomas Diseases 0.000 description 8
- 206010012689 Diabetic retinopathy Diseases 0.000 description 8
- 206010014568 Empyema Diseases 0.000 description 8
- 208000015872 Gaucher disease Diseases 0.000 description 8
- 206010018691 Granuloma Diseases 0.000 description 8
- 208000032456 Hemorrhagic Shock Diseases 0.000 description 8
- 208000023105 Huntington disease Diseases 0.000 description 8
- 208000029523 Interstitial Lung disease Diseases 0.000 description 8
- 206010022680 Intestinal ischaemia Diseases 0.000 description 8
- 208000011200 Kawasaki disease Diseases 0.000 description 8
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 8
- 125000000998 L-alanino group Chemical group [H]N([*])[C@](C([H])([H])[H])([H])C(=O)O[H] 0.000 description 8
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 8
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 8
- 208000019693 Lung disease Diseases 0.000 description 8
- 208000004535 Mesenteric Ischemia Diseases 0.000 description 8
- NWIBSHFKIJFRCO-WUDYKRTCSA-N Mytomycin Chemical compound C1N2C(C(C(C)=C(N)C3=O)=O)=C3[C@@H](COC(N)=O)[C@@]2(OC)[C@@H]2[C@H]1N2 NWIBSHFKIJFRCO-WUDYKRTCSA-N 0.000 description 8
- 208000018737 Parkinson disease Diseases 0.000 description 8
- 206010068142 Radiation sickness syndrome Diseases 0.000 description 8
- 206010038933 Retinopathy of prematurity Diseases 0.000 description 8
- 206010049771 Shock haemorrhagic Diseases 0.000 description 8
- 208000021386 Sjogren Syndrome Diseases 0.000 description 8
- 208000006011 Stroke Diseases 0.000 description 8
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 8
- 239000004473 Threonine Substances 0.000 description 8
- 206010047115 Vasculitis Diseases 0.000 description 8
- 230000035508 accumulation Effects 0.000 description 8
- 238000009825 accumulation Methods 0.000 description 8
- 230000001154 acute effect Effects 0.000 description 8
- 235000004279 alanine Nutrition 0.000 description 8
- 208000026935 allergic disease Diseases 0.000 description 8
- 208000006673 asthma Diseases 0.000 description 8
- 208000022362 bacterial infectious disease Diseases 0.000 description 8
- 210000000481 breast Anatomy 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 210000001072 colon Anatomy 0.000 description 8
- 229960003957 dexamethasone Drugs 0.000 description 8
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 8
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 8
- 208000019622 heart disease Diseases 0.000 description 8
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 8
- 208000006116 lymphomatoid granulomatosis Diseases 0.000 description 8
- 208000001725 mucocutaneous lymph node syndrome Diseases 0.000 description 8
- 208000030613 peripheral artery disease Diseases 0.000 description 8
- 208000037803 restenosis Diseases 0.000 description 8
- 201000000306 sarcoidosis Diseases 0.000 description 8
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 8
- 201000008827 tuberculosis Diseases 0.000 description 8
- 206010001889 Alveolitis Diseases 0.000 description 7
- 206010002556 Ankylosing Spondylitis Diseases 0.000 description 7
- 208000008439 Biliary Liver Cirrhosis Diseases 0.000 description 7
- 208000033222 Biliary cirrhosis primary Diseases 0.000 description 7
- 206010008909 Chronic Hepatitis Diseases 0.000 description 7
- 208000015943 Coeliac disease Diseases 0.000 description 7
- 206010012438 Dermatitis atopic Diseases 0.000 description 7
- 206010012442 Dermatitis contact Diseases 0.000 description 7
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 7
- 208000001204 Hashimoto Disease Diseases 0.000 description 7
- 208000030836 Hashimoto thyroiditis Diseases 0.000 description 7
- 206010019755 Hepatitis chronic active Diseases 0.000 description 7
- 208000009889 Herpes Simplex Diseases 0.000 description 7
- 208000007514 Herpes zoster Diseases 0.000 description 7
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 7
- 201000002481 Myositis Diseases 0.000 description 7
- 208000031845 Pernicious anaemia Diseases 0.000 description 7
- 208000012654 Primary biliary cholangitis Diseases 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 206010053613 Type IV hypersensitivity reaction Diseases 0.000 description 7
- 206010003246 arthritis Diseases 0.000 description 7
- 201000008937 atopic dermatitis Diseases 0.000 description 7
- 230000008499 blood brain barrier function Effects 0.000 description 7
- 210000001218 blood-brain barrier Anatomy 0.000 description 7
- 230000005800 cardiovascular problem Effects 0.000 description 7
- 208000010247 contact dermatitis Diseases 0.000 description 7
- 201000001155 extrinsic allergic alveolitis Diseases 0.000 description 7
- 208000022098 hypersensitivity pneumonitis Diseases 0.000 description 7
- 230000004968 inflammatory condition Effects 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 208000008494 pericarditis Diseases 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 7
- 230000026731 phosphorylation Effects 0.000 description 7
- 238000006366 phosphorylation reaction Methods 0.000 description 7
- 230000002062 proliferating effect Effects 0.000 description 7
- 230000000241 respiratory effect Effects 0.000 description 7
- 230000005808 skin problem Effects 0.000 description 7
- 239000011780 sodium chloride Substances 0.000 description 7
- 230000005951 type IV hypersensitivity Effects 0.000 description 7
- 208000027930 type IV hypersensitivity disease Diseases 0.000 description 7
- PORPENFLTBBHSG-MGBGTMOVSA-N 1,2-dihexadecanoyl-sn-glycerol-3-phosphate Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(O)=O)OC(=O)CCCCCCCCCCCCCCC PORPENFLTBBHSG-MGBGTMOVSA-N 0.000 description 6
- TZCPCKNHXULUIY-RGULYWFUSA-N 1,2-distearoyl-sn-glycero-3-phosphoserine Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC[C@H](COP(O)(=O)OC[C@H](N)C(O)=O)OC(=O)CCCCCCCCCCCCCCCCC TZCPCKNHXULUIY-RGULYWFUSA-N 0.000 description 6
- 208000022309 Alcoholic Liver disease Diseases 0.000 description 6
- IYMAXBFPHPZYIK-BQBZGAKWSA-N Arg-Gly-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O IYMAXBFPHPZYIK-BQBZGAKWSA-N 0.000 description 6
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 6
- 102000004127 Cytokines Human genes 0.000 description 6
- 108090000695 Cytokines Proteins 0.000 description 6
- 229940123780 DNA topoisomerase I inhibitor Drugs 0.000 description 6
- JZNWSCPGTDBMEW-UHFFFAOYSA-N Glycerophosphorylethanolamin Natural products NCCOP(O)(=O)OCC(O)CO JZNWSCPGTDBMEW-UHFFFAOYSA-N 0.000 description 6
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 description 6
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 description 6
- 239000000365 Topoisomerase I Inhibitor Substances 0.000 description 6
- ATBOMIWRCZXYSZ-XZBBILGWSA-N [1-[2,3-dihydroxypropoxy(hydroxy)phosphoryl]oxy-3-hexadecanoyloxypropan-2-yl] (9e,12e)-octadeca-9,12-dienoate Chemical compound CCCCCCCCCCCCCCCC(=O)OCC(COP(O)(=O)OCC(O)CO)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC ATBOMIWRCZXYSZ-XZBBILGWSA-N 0.000 description 6
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 6
- 230000003510 anti-fibrotic effect Effects 0.000 description 6
- VSRXQHXAPYXROS-UHFFFAOYSA-N azanide;cyclobutane-1,1-dicarboxylic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OC(=O)C1(C(O)=O)CCC1 VSRXQHXAPYXROS-UHFFFAOYSA-N 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 210000004155 blood-retinal barrier Anatomy 0.000 description 6
- 230000004378 blood-retinal barrier Effects 0.000 description 6
- 235000012000 cholesterol Nutrition 0.000 description 6
- 230000004087 circulation Effects 0.000 description 6
- ZGSPNIOCEDOHGS-UHFFFAOYSA-L disodium [3-[2,3-di(octadeca-9,12-dienoyloxy)propoxy-oxidophosphoryl]oxy-2-hydroxypropyl] 2,3-di(octadeca-9,12-dienoyloxy)propyl phosphate Chemical compound [Na+].[Na+].CCCCCC=CCC=CCCCCCCCC(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COP([O-])(=O)OCC(O)COP([O-])(=O)OCC(OC(=O)CCCCCCCC=CCC=CCCCCC)COC(=O)CCCCCCCC=CCC=CCCCCC ZGSPNIOCEDOHGS-UHFFFAOYSA-L 0.000 description 6
- 208000035475 disorder Diseases 0.000 description 6
- 208000027866 inflammatory disease Diseases 0.000 description 6
- 238000002595 magnetic resonance imaging Methods 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 150000008104 phosphatidylethanolamines Chemical class 0.000 description 6
- 150000003905 phosphatidylinositols Chemical class 0.000 description 6
- 150000003904 phospholipids Chemical class 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 238000002600 positron emission tomography Methods 0.000 description 6
- 238000001338 self-assembly Methods 0.000 description 6
- NRHMKIHPTBHXPF-TUJRSCDTSA-M sodium cholate Chemical compound [Na+].C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC([O-])=O)C)[C@@]2(C)[C@@H](O)C1 NRHMKIHPTBHXPF-TUJRSCDTSA-M 0.000 description 6
- 210000004981 tumor-associated macrophage Anatomy 0.000 description 6
- ZIXGXMMUKPLXBB-UHFFFAOYSA-N Guatambuinine Natural products N1C2=CC=CC=C2C2=C1C(C)=C1C=CN=C(C)C1=C2 ZIXGXMMUKPLXBB-UHFFFAOYSA-N 0.000 description 5
- 241000725303 Human immunodeficiency virus Species 0.000 description 5
- SUYXJDLXGFPMCQ-INIZCTEOSA-N SJ000287331 Natural products CC1=c2cnccc2=C(C)C2=Nc3ccccc3[C@H]12 SUYXJDLXGFPMCQ-INIZCTEOSA-N 0.000 description 5
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 5
- 230000009471 action Effects 0.000 description 5
- KLNFSAOEKUDMFA-UHFFFAOYSA-N azanide;2-hydroxyacetic acid;platinum(2+) Chemical compound [NH2-].[NH2-].[Pt+2].OCC(O)=O KLNFSAOEKUDMFA-UHFFFAOYSA-N 0.000 description 5
- 210000000170 cell membrane Anatomy 0.000 description 5
- 230000003176 fibrotic effect Effects 0.000 description 5
- 239000012216 imaging agent Substances 0.000 description 5
- 102000027596 immune receptors Human genes 0.000 description 5
- 108091008915 immune receptors Proteins 0.000 description 5
- 229940100601 interleukin-6 Drugs 0.000 description 5
- 229960005386 ipilimumab Drugs 0.000 description 5
- 230000037361 pathway Effects 0.000 description 5
- 235000018102 proteins Nutrition 0.000 description 5
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 5
- 229940043267 rhodamine b Drugs 0.000 description 5
- 125000003607 serino group Chemical group [H]N([H])[C@]([H])(C(=O)[*])C(O[H])([H])[H] 0.000 description 5
- 238000002626 targeted therapy Methods 0.000 description 5
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 4
- 108010082126 Alanine transaminase Proteins 0.000 description 4
- 101710155857 C-C motif chemokine 2 Proteins 0.000 description 4
- 102000000018 Chemokine CCL2 Human genes 0.000 description 4
- 101000733802 Homo sapiens Apolipoprotein A-I Proteins 0.000 description 4
- 201000003088 Limited Scleroderma Diseases 0.000 description 4
- 208000024140 Limited cutaneous systemic sclerosis Diseases 0.000 description 4
- 102100034184 Macrophage scavenger receptor types I and II Human genes 0.000 description 4
- 101710134306 Macrophage scavenger receptor types I and II Proteins 0.000 description 4
- 102000004278 Receptor Protein-Tyrosine Kinases Human genes 0.000 description 4
- 108090000873 Receptor Protein-Tyrosine Kinases Proteins 0.000 description 4
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 4
- 230000003110 anti-inflammatory effect Effects 0.000 description 4
- 210000005100 blood-tumour barrier Anatomy 0.000 description 4
- 210000003169 central nervous system Anatomy 0.000 description 4
- 238000002591 computed tomography Methods 0.000 description 4
- SDZRWUKZFQQKKV-JHADDHBZSA-N cytochalasin D Chemical compound C([C@H]1[C@@H]2[C@@H](C([C@@H](O)[C@H]\3[C@]2([C@@H](/C=C/[C@@](C)(O)C(=O)[C@@H](C)C/C=C/3)OC(C)=O)C(=O)N1)=C)C)C1=CC=CC=C1 SDZRWUKZFQQKKV-JHADDHBZSA-N 0.000 description 4
- 238000002405 diagnostic procedure Methods 0.000 description 4
- 102000051062 human APOA1 Human genes 0.000 description 4
- 230000005965 immune activity Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000002427 irreversible effect Effects 0.000 description 4
- 210000001865 kupffer cell Anatomy 0.000 description 4
- 108091005485 macrophage scavenger receptors Proteins 0.000 description 4
- 230000009456 molecular mechanism Effects 0.000 description 4
- 230000007935 neutral effect Effects 0.000 description 4
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 4
- 230000005855 radiation Effects 0.000 description 4
- 229940044551 receptor antagonist Drugs 0.000 description 4
- 239000002464 receptor antagonist Substances 0.000 description 4
- 238000010186 staining Methods 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- 238000005199 ultracentrifugation Methods 0.000 description 4
- 229960000653 valrubicin Drugs 0.000 description 4
- ZOCKGBMQLCSHFP-KQRAQHLDSA-N valrubicin Chemical compound O([C@H]1C[C@](CC2=C(O)C=3C(=O)C4=CC=CC(OC)=C4C(=O)C=3C(O)=C21)(O)C(=O)COC(=O)CCCC)[C@H]1C[C@H](NC(=O)C(F)(F)F)[C@H](O)[C@H](C)O1 ZOCKGBMQLCSHFP-KQRAQHLDSA-N 0.000 description 4
- HVCOBJNICQPDBP-UHFFFAOYSA-N 3-[3-[3,5-dihydroxy-6-methyl-4-(3,4,5-trihydroxy-6-methyloxan-2-yl)oxyoxan-2-yl]oxydecanoyloxy]decanoic acid;hydrate Chemical compound O.OC1C(OC(CC(=O)OC(CCCCCCC)CC(O)=O)CCCCCCC)OC(C)C(O)C1OC1C(O)C(O)C(O)C(C)O1 HVCOBJNICQPDBP-UHFFFAOYSA-N 0.000 description 3
- BWGNESOTFCXPMA-UHFFFAOYSA-N Dihydrogen disulfide Chemical compound SS BWGNESOTFCXPMA-UHFFFAOYSA-N 0.000 description 3
- ZPLQIPFOCGIIHV-UHFFFAOYSA-N Gimeracil Chemical compound OC1=CC(=O)C(Cl)=CN1 ZPLQIPFOCGIIHV-UHFFFAOYSA-N 0.000 description 3
- 229930186217 Glycolipid Natural products 0.000 description 3
- 150000008575 L-amino acids Chemical class 0.000 description 3
- QEFRNWWLZKMPFJ-YGVKFDHGSA-N L-methionine (R)-S-oxide group Chemical group N[C@@H](CCS(=O)C)C(=O)O QEFRNWWLZKMPFJ-YGVKFDHGSA-N 0.000 description 3
- 239000002202 Polyethylene glycol Substances 0.000 description 3
- IAPCTXZQXAVYNG-UHFFFAOYSA-M Potassium 2,6-dihydroxytriazinecarboxylate Chemical compound [K+].[O-]C(=O)C1=NC(=O)NC(=O)N1 IAPCTXZQXAVYNG-UHFFFAOYSA-M 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 229940121363 anti-inflammatory agent Drugs 0.000 description 3
- 239000002260 anti-inflammatory agent Substances 0.000 description 3
- 230000000840 anti-viral effect Effects 0.000 description 3
- 239000003963 antioxidant agent Substances 0.000 description 3
- 235000006708 antioxidants Nutrition 0.000 description 3
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 229940125692 cardiovascular agent Drugs 0.000 description 3
- 239000002327 cardiovascular agent Substances 0.000 description 3
- 239000002872 contrast media Substances 0.000 description 3
- 230000016396 cytokine production Effects 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000001086 cytosolic effect Effects 0.000 description 3
- 150000001982 diacylglycerols Chemical class 0.000 description 3
- 239000000539 dimer Substances 0.000 description 3
- 230000007783 downstream signaling Effects 0.000 description 3
- 239000011363 dried mixture Substances 0.000 description 3
- 238000001035 drying Methods 0.000 description 3
- 229950009822 gimeracil Drugs 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 230000008595 infiltration Effects 0.000 description 3
- 238000001764 infiltration Methods 0.000 description 3
- 210000003734 kidney Anatomy 0.000 description 3
- 229940043355 kinase inhibitor Drugs 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 208000019423 liver disease Diseases 0.000 description 3
- 239000006166 lysate Substances 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 229950000193 oteracil Drugs 0.000 description 3
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 3
- 210000002381 plasma Anatomy 0.000 description 3
- 239000003909 protein kinase inhibitor Substances 0.000 description 3
- 210000003491 skin Anatomy 0.000 description 3
- 150000003408 sphingolipids Chemical class 0.000 description 3
- 230000001629 suppression Effects 0.000 description 3
- 229940063683 taxotere Drugs 0.000 description 3
- 229960001674 tegafur Drugs 0.000 description 3
- WFWLQNSHRPWKFK-ZCFIWIBFSA-N tegafur Chemical compound O=C1NC(=O)C(F)=CN1[C@@H]1OCCC1 WFWLQNSHRPWKFK-ZCFIWIBFSA-N 0.000 description 3
- IMCGHZIGRANKHV-AJNGGQMLSA-N tert-butyl (3s,5s)-2-oxo-5-[(2s,4s)-5-oxo-4-propan-2-yloxolan-2-yl]-3-propan-2-ylpyrrolidine-1-carboxylate Chemical compound O1C(=O)[C@H](C(C)C)C[C@H]1[C@H]1N(C(=O)OC(C)(C)C)C(=O)[C@H](C(C)C)C1 IMCGHZIGRANKHV-AJNGGQMLSA-N 0.000 description 3
- WYWHKKSPHMUBEB-UHFFFAOYSA-N tioguanine Chemical compound N1C(N)=NC(=S)C2=C1N=CN2 WYWHKKSPHMUBEB-UHFFFAOYSA-N 0.000 description 3
- 150000003626 triacylglycerols Chemical class 0.000 description 3
- DCXXMTOCNZCJGO-UHFFFAOYSA-N tristearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(OC(=O)CCCCCCCCCCCCCCCCC)COC(=O)CCCCCCCCCCCCCCCCC DCXXMTOCNZCJGO-UHFFFAOYSA-N 0.000 description 3
- STQGQHZAVUOBTE-UHFFFAOYSA-N 7-Cyan-hept-2t-en-4,6-diinsaeure Natural products C1=2C(O)=C3C(=O)C=4C(OC)=CC=CC=4C(=O)C3=C(O)C=2CC(O)(C(C)=O)CC1OC1CC(N)C(O)C(C)O1 STQGQHZAVUOBTE-UHFFFAOYSA-N 0.000 description 2
- 108010024976 Asparaginase Proteins 0.000 description 2
- 102000015790 Asparaginase Human genes 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- 201000003066 Diffuse Scleroderma Diseases 0.000 description 2
- 208000009386 Experimental Arthritis Diseases 0.000 description 2
- 229920000855 Fucoidan Polymers 0.000 description 2
- 101000809875 Homo sapiens TYRO protein tyrosine kinase-binding protein Proteins 0.000 description 2
- 108010003272 Hyaluronate lyase Proteins 0.000 description 2
- 102000001974 Hyaluronidases Human genes 0.000 description 2
- 108010078049 Interferon alpha-2 Proteins 0.000 description 2
- 108050003558 Interleukin-17 Proteins 0.000 description 2
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 2
- 208000002193 Pain Diseases 0.000 description 2
- 201000001263 Psoriatic Arthritis Diseases 0.000 description 2
- 208000036824 Psoriatic arthropathy Diseases 0.000 description 2
- 108091005487 SCARB1 Proteins 0.000 description 2
- NKANXQFJJICGDU-QPLCGJKRSA-N Tamoxifen Chemical compound C=1C=CC=CC=1C(/CC)=C(C=1C=CC(OCCN(C)C)=CC=1)/C1=CC=CC=C1 NKANXQFJJICGDU-QPLCGJKRSA-N 0.000 description 2
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 2
- SHGAZHPCJJPHSC-YCNIQYBTSA-N all-trans-retinoic acid Chemical compound OC(=O)\C=C(/C)\C=C\C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-YCNIQYBTSA-N 0.000 description 2
- 230000000259 anti-tumor effect Effects 0.000 description 2
- 238000003782 apoptosis assay Methods 0.000 description 2
- 229960003272 asparaginase Drugs 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-M asparaginate Chemical compound [O-]C(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-M 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- KVUAALJSMIVURS-ZEDZUCNESA-L calcium folinate Chemical compound [Ca+2].C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC([O-])=O)C([O-])=O)C=C1 KVUAALJSMIVURS-ZEDZUCNESA-L 0.000 description 2
- 238000002512 chemotherapy Methods 0.000 description 2
- 230000008045 co-localization Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 229960000640 dactinomycin Drugs 0.000 description 2
- 229960000975 daunorubicin Drugs 0.000 description 2
- STQGQHZAVUOBTE-VGBVRHCVSA-N daunorubicin Chemical compound O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(C)=O)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 STQGQHZAVUOBTE-VGBVRHCVSA-N 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 230000009144 enzymatic modification Effects 0.000 description 2
- PJZDLZXMGBOJRF-CXOZILEQSA-L folfirinox Chemical compound [Pt+4].[O-]C(=O)C([O-])=O.[NH-][C@H]1CCCC[C@@H]1[NH-].FC1=CNC(=O)NC1=O.C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1.C1=C2C(CC)=C3CN(C(C4=C([C@@](C(=O)OC4)(O)CC)C=4)=O)C=4C3=NC2=CC=C1OC(=O)N(CC1)CCC1N1CCCCC1 PJZDLZXMGBOJRF-CXOZILEQSA-L 0.000 description 2
- 230000030279 gene silencing Effects 0.000 description 2
- 210000004394 hip joint Anatomy 0.000 description 2
- 229960002773 hyaluronidase Drugs 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 210000001503 joint Anatomy 0.000 description 2
- 210000000629 knee joint Anatomy 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- GLVAUDGFNGKCSF-UHFFFAOYSA-N mercaptopurine Chemical compound S=C1NC=NC2=C1NC=N2 GLVAUDGFNGKCSF-UHFFFAOYSA-N 0.000 description 2
- 229960000485 methotrexate Drugs 0.000 description 2
- 229960004857 mitomycin Drugs 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 210000000440 neutrophil Anatomy 0.000 description 2
- 206010053219 non-alcoholic steatohepatitis Diseases 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 229940075993 receptor modulator Drugs 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000036962 time dependent Effects 0.000 description 2
- 229960004528 vincristine Drugs 0.000 description 2
- OGWKCGZFUXNPDA-XQKSVPLYSA-N vincristine Chemical compound C([N@]1C[C@@H](C[C@]2(C(=O)OC)C=3C(=CC4=C([C@]56[C@H]([C@@]([C@H](OC(C)=O)[C@]7(CC)C=CCN([C@H]67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)C[C@@](C1)(O)CC)CC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-XQKSVPLYSA-N 0.000 description 2
- OGWKCGZFUXNPDA-UHFFFAOYSA-N vincristine Natural products C1C(CC)(O)CC(CC2(C(=O)OC)C=3C(=CC4=C(C56C(C(C(OC(C)=O)C7(CC)C=CCN(C67)CC5)(O)C(=O)OC)N4C=O)C=3)OC)CN1CCC1=C2NC2=CC=CC=C12 OGWKCGZFUXNPDA-UHFFFAOYSA-N 0.000 description 2
- XRASPMIURGNCCH-UHFFFAOYSA-N zoledronic acid Chemical compound OP(=O)(O)C(P(O)(O)=O)(O)CN1C=CN=C1 XRASPMIURGNCCH-UHFFFAOYSA-N 0.000 description 2
- 229960004276 zoledronic acid Drugs 0.000 description 2
- BMKDZUISNHGIBY-ZETCQYMHSA-N (+)-dexrazoxane Chemical compound C([C@H](C)N1CC(=O)NC(=O)C1)N1CC(=O)NC(=O)C1 BMKDZUISNHGIBY-ZETCQYMHSA-N 0.000 description 1
- WDJHHCAKBRKCLW-IBGZPJMESA-N (2S)-2-[[2-[3-(6-carbamimidoyl-1H-benzimidazol-2-yl)-4-hydroxy-5-(2-hydroxy-5-sulfamoylphenyl)phenyl]acetyl]amino]butanedioic acid Chemical compound N=1C2=CC(C(=N)N)=CC=C2NC=1C(C=1O)=CC(CC(=O)N[C@@H](CC(O)=O)C(O)=O)=CC=1C1=CC(S(N)(=O)=O)=CC=C1O WDJHHCAKBRKCLW-IBGZPJMESA-N 0.000 description 1
- WZJRQXZSYQYFJV-QAXVQDKQSA-N (2s)-6-amino-2-[[(2s)-1-[(2s,3s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s,3r)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-1-[(2s)-2-[[(2s)-2-[[(2s)-2-amino-4-carboxybutanoyl]amino]propanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]pyrrolidine-2-car Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCCN)C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O)[C@@H](C)O)C1=CC=CC=C1 WZJRQXZSYQYFJV-QAXVQDKQSA-N 0.000 description 1
- PSVUJBVBCOISSP-SPFKKGSWSA-N (2s,3r,4s,5s,6r)-2-bis(2-chloroethylamino)phosphoryloxy-6-(hydroxymethyl)oxane-3,4,5-triol Chemical compound OC[C@H]1O[C@@H](OP(=O)(NCCCl)NCCCl)[C@H](O)[C@@H](O)[C@@H]1O PSVUJBVBCOISSP-SPFKKGSWSA-N 0.000 description 1
- FPVKHBSQESCIEP-UHFFFAOYSA-N (8S)-3-(2-deoxy-beta-D-erythro-pentofuranosyl)-3,6,7,8-tetrahydroimidazo[4,5-d][1,3]diazepin-8-ol Natural products C1C(O)C(CO)OC1N1C(NC=NCC2O)=C2N=C1 FPVKHBSQESCIEP-UHFFFAOYSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- DEVSOMFAQLZNKR-RJRFIUFISA-N (z)-3-[3-[3,5-bis(trifluoromethyl)phenyl]-1,2,4-triazol-1-yl]-n'-pyrazin-2-ylprop-2-enehydrazide Chemical compound FC(F)(F)C1=CC(C(F)(F)F)=CC(C2=NN(\C=C/C(=O)NNC=3N=CC=NC=3)C=N2)=C1 DEVSOMFAQLZNKR-RJRFIUFISA-N 0.000 description 1
- XGQXULJHBWKUJY-LYIKAWCPSA-N (z)-but-2-enedioic acid;n-[2-(diethylamino)ethyl]-5-[(z)-(5-fluoro-2-oxo-1h-indol-3-ylidene)methyl]-2,4-dimethyl-1h-pyrrole-3-carboxamide Chemical compound OC(=O)\C=C/C(O)=O.CCN(CC)CCNC(=O)C1=C(C)NC(\C=C/2C3=CC(F)=CC=C3NC\2=O)=C1C XGQXULJHBWKUJY-LYIKAWCPSA-N 0.000 description 1
- HJTAZXHBEBIQQX-UHFFFAOYSA-N 1,5-bis(chloromethyl)naphthalene Chemical compound C1=CC=C2C(CCl)=CC=CC2=C1CCl HJTAZXHBEBIQQX-UHFFFAOYSA-N 0.000 description 1
- VSNHCAURESNICA-NJFSPNSNSA-N 1-oxidanylurea Chemical compound N[14C](=O)NO VSNHCAURESNICA-NJFSPNSNSA-N 0.000 description 1
- 108010058566 130-nm albumin-bound paclitaxel Proteins 0.000 description 1
- QXLQZLBNPTZMRK-UHFFFAOYSA-N 2-[(dimethylamino)methyl]-1-(2,4-dimethylphenyl)prop-2-en-1-one Chemical compound CN(C)CC(=C)C(=O)C1=CC=C(C)C=C1C QXLQZLBNPTZMRK-UHFFFAOYSA-N 0.000 description 1
- SNKUSVNHTCUELQ-UHFFFAOYSA-N 2-[[4-amino-2-[[2-[[2-[2-[[2-[[2-[[2-[[2-amino-3-(4-hydroxyphenyl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]acetyl]amino]propanoylamino]-3-carboxypropanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-4-methylpentanoic aci Chemical compound CC(C)CC(C(O)=O)NC(=O)C(CC(N)=O)NC(=O)C(CC(C)C)NC(=O)C(CC(O)=O)NC(=O)C(C)NC(=O)CNC(=O)C(CO)NC(=O)C(CC(C)C)NC(=O)C(N)CC1=CC=C(O)C=C1 SNKUSVNHTCUELQ-UHFFFAOYSA-N 0.000 description 1
- UZFPOOOQHWICKY-UHFFFAOYSA-N 3-[13-[1-[1-[8,12-bis(2-carboxyethyl)-17-(1-hydroxyethyl)-3,7,13,18-tetramethyl-21,24-dihydroporphyrin-2-yl]ethoxy]ethyl]-18-(2-carboxyethyl)-8-(1-hydroxyethyl)-3,7,12,17-tetramethyl-22,23-dihydroporphyrin-2-yl]propanoic acid Chemical compound N1C(C=C2C(=C(CCC(O)=O)C(C=C3C(=C(C)C(C=C4N5)=N3)CCC(O)=O)=N2)C)=C(C)C(C(C)O)=C1C=C5C(C)=C4C(C)OC(C)C1=C(N2)C=C(N3)C(C)=C(C(O)C)C3=CC(C(C)=C3CCC(O)=O)=NC3=CC(C(CCC(O)=O)=C3C)=NC3=CC2=C1C UZFPOOOQHWICKY-UHFFFAOYSA-N 0.000 description 1
- VIJCCFFEBCOOIE-JOCHJYFZSA-N 3-[[(3r)-1-cyclohexyl-5-(3,3-dimethyl-2-oxobutyl)-4-oxo-2,3-dihydro-1,5-benzodiazepin-3-yl]carbamoylamino]benzoic acid Chemical compound N([C@@H]1CN(C2=CC=CC=C2N(C1=O)CC(=O)C(C)(C)C)C1CCCCC1)C(=O)NC1=CC=CC(C(O)=O)=C1 VIJCCFFEBCOOIE-JOCHJYFZSA-N 0.000 description 1
- 108010082808 4-1BB Ligand Proteins 0.000 description 1
- UWXSAYUXVSFDBQ-CYBMUJFWSA-N 4-n-[3-chloro-4-(1,3-thiazol-2-ylmethoxy)phenyl]-6-n-[(4r)-4-methyl-4,5-dihydro-1,3-oxazol-2-yl]quinazoline-4,6-diamine Chemical compound C[C@@H]1COC(NC=2C=C3C(NC=4C=C(Cl)C(OCC=5SC=CN=5)=CC=4)=NC=NC3=CC=2)=N1 UWXSAYUXVSFDBQ-CYBMUJFWSA-N 0.000 description 1
- NMUSYJAQQFHJEW-KVTDHHQDSA-N 5-azacytidine Chemical compound O=C1N=C(N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NMUSYJAQQFHJEW-KVTDHHQDSA-N 0.000 description 1
- ZYRLHJIMTROTBO-UHFFFAOYSA-N 6,8-bis(benzylsulfanyl)octanoic acid Chemical compound C=1C=CC=CC=1CSC(CCCCC(=O)O)CCSCC1=CC=CC=C1 ZYRLHJIMTROTBO-UHFFFAOYSA-N 0.000 description 1
- VVIAGPKUTFNRDU-UHFFFAOYSA-N 6S-folinic acid Natural products C1NC=2NC(N)=NC(=O)C=2N(C=O)C1CNC1=CC=C(C(=O)NC(CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-UHFFFAOYSA-N 0.000 description 1
- SHGAZHPCJJPHSC-ZVCIMWCZSA-N 9-cis-retinoic acid Chemical compound OC(=O)/C=C(\C)/C=C/C=C(/C)\C=C\C1=C(C)CCCC1(C)C SHGAZHPCJJPHSC-ZVCIMWCZSA-N 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 108020004306 Alpha-ketoglutarate dehydrogenase Proteins 0.000 description 1
- 102000006589 Alpha-ketoglutarate dehydrogenase Human genes 0.000 description 1
- 229930183010 Amphotericin Natural products 0.000 description 1
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 description 1
- BFYIZQONLCFLEV-DAELLWKTSA-N Aromasine Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(C(CC4)=O)[C@@H]4[C@@H]3CC(=C)C2=C1 BFYIZQONLCFLEV-DAELLWKTSA-N 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 description 1
- MLDQJTXFUGDVEO-UHFFFAOYSA-N BAY-43-9006 Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=CC(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 MLDQJTXFUGDVEO-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 238000011740 C57BL/6 mouse Methods 0.000 description 1
- 108010077333 CAP1-6D Proteins 0.000 description 1
- 102100031168 CCN family member 2 Human genes 0.000 description 1
- 239000012275 CTLA-4 inhibitor Substances 0.000 description 1
- 229940045513 CTLA4 antagonist Drugs 0.000 description 1
- 101100289888 Caenorhabditis elegans lys-5 gene Proteins 0.000 description 1
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UORFTKCHSA-N Capecitabine Chemical compound C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1[C@H]1[C@H](O)[C@H](O)[C@@H](C)O1 GAGWJHPBXLXJQN-UORFTKCHSA-N 0.000 description 1
- GAGWJHPBXLXJQN-UHFFFAOYSA-N Capecitabine Natural products C1=C(F)C(NC(=O)OCCCCC)=NC(=O)N1C1C(O)C(O)C(C)O1 GAGWJHPBXLXJQN-UHFFFAOYSA-N 0.000 description 1
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108090000397 Caspase 3 Proteins 0.000 description 1
- 102100029855 Caspase-3 Human genes 0.000 description 1
- 102100026550 Caspase-9 Human genes 0.000 description 1
- 108090000566 Caspase-9 Proteins 0.000 description 1
- JWBOIMRXGHLCPP-UHFFFAOYSA-N Chloditan Chemical compound C=1C=CC=C(Cl)C=1C(C(Cl)Cl)C1=CC=C(Cl)C=C1 JWBOIMRXGHLCPP-UHFFFAOYSA-N 0.000 description 1
- 101800001982 Cholecystokinin Proteins 0.000 description 1
- 102100025841 Cholecystokinin Human genes 0.000 description 1
- PTOAARAWEBMLNO-KVQBGUIXSA-N Cladribine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@H]1C[C@H](O)[C@@H](CO)O1 PTOAARAWEBMLNO-KVQBGUIXSA-N 0.000 description 1
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 1
- 108090000404 Cyclin G1 Proteins 0.000 description 1
- 102000004012 Cyclin G1 Human genes 0.000 description 1
- 102100024458 Cyclin-dependent kinase inhibitor 2A Human genes 0.000 description 1
- 101710154003 Cyclin-dependent kinase inhibitor 2A Proteins 0.000 description 1
- PMATZTZNYRCHOR-CGLBZJNRSA-N Cyclosporin A Chemical compound CC[C@@H]1NC(=O)[C@H]([C@H](O)[C@H](C)C\C=C\C)N(C)C(=O)[C@H](C(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](CC(C)C)N(C)C(=O)[C@@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](CC(C)C)N(C)C(=O)CN(C)C1=O PMATZTZNYRCHOR-CGLBZJNRSA-N 0.000 description 1
- 108010036949 Cyclosporine Proteins 0.000 description 1
- UHDGCWIWMRVCDJ-CCXZUQQUSA-N Cytarabine Chemical compound O=C1N=C(N)C=CN1[C@H]1[C@@H](O)[C@H](O)[C@@H](CO)O1 UHDGCWIWMRVCDJ-CCXZUQQUSA-N 0.000 description 1
- 108010019673 Darbepoetin alfa Proteins 0.000 description 1
- 102100039694 Death-associated protein 1 Human genes 0.000 description 1
- 229940124186 Dehydrogenase inhibitor Drugs 0.000 description 1
- 102100024746 Dihydrofolate reductase Human genes 0.000 description 1
- 229940123171 Dihydropyrimidine dehydrogenase inhibitor Drugs 0.000 description 1
- OFDNQWIFNXBECV-UHFFFAOYSA-N Dolastatin 10 Natural products CC(C)C(N(C)C)C(=O)NC(C(C)C)C(=O)N(C)C(C(C)CC)C(OC)CC(=O)N1CCCC1C(OC)C(C)C(=O)NC(C=1SC=CN=1)CC1=CC=CC=C1 OFDNQWIFNXBECV-UHFFFAOYSA-N 0.000 description 1
- MWWSFMDVAYGXBV-RUELKSSGSA-N Doxorubicin hydrochloride Chemical compound Cl.O([C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1C[C@H](N)[C@H](O)[C@H](C)O1 MWWSFMDVAYGXBV-RUELKSSGSA-N 0.000 description 1
- 102100023266 Dual specificity mitogen-activated protein kinase kinase 2 Human genes 0.000 description 1
- 101710146529 Dual specificity mitogen-activated protein kinase kinase 2 Proteins 0.000 description 1
- 101150029707 ERBB2 gene Proteins 0.000 description 1
- 101150082819 ERBB3 gene Proteins 0.000 description 1
- MBYXEBXZARTUSS-QLWBXOBMSA-N Emetamine Natural products O(C)c1c(OC)cc2c(c(C[C@@H]3[C@H](CC)CN4[C@H](c5c(cc(OC)c(OC)c5)CC4)C3)ncc2)c1 MBYXEBXZARTUSS-QLWBXOBMSA-N 0.000 description 1
- 229940123818 Epidermal growth factor antagonist Drugs 0.000 description 1
- 108010074604 Epoetin Alfa Proteins 0.000 description 1
- 101150004694 Erbb4 gene Proteins 0.000 description 1
- 108010041356 Estrogen Receptor beta Proteins 0.000 description 1
- 102100029951 Estrogen receptor beta Human genes 0.000 description 1
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 1
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 206010016654 Fibrosis Diseases 0.000 description 1
- 108010029961 Filgrastim Proteins 0.000 description 1
- VWUXBMIQPBEWFH-WCCTWKNTSA-N Fulvestrant Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3[C@H](CCCCCCCCCS(=O)CCCC(F)(F)C(F)(F)F)CC2=C1 VWUXBMIQPBEWFH-WCCTWKNTSA-N 0.000 description 1
- 229940123570 Fyn tyrosine kinase inhibitor Drugs 0.000 description 1
- 102000030902 Galactosyltransferase Human genes 0.000 description 1
- 108060003306 Galactosyltransferase Proteins 0.000 description 1
- 102100031351 Galectin-9 Human genes 0.000 description 1
- 101100229077 Gallus gallus GAL9 gene Proteins 0.000 description 1
- 108010069236 Goserelin Proteins 0.000 description 1
- 102100039619 Granulocyte colony-stimulating factor Human genes 0.000 description 1
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 description 1
- 102000003693 Hedgehog Proteins Human genes 0.000 description 1
- 108090000031 Hedgehog Proteins Proteins 0.000 description 1
- 102100022623 Hepatocyte growth factor receptor Human genes 0.000 description 1
- 101710184069 Hepatocyte growth factor receptor Proteins 0.000 description 1
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 description 1
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101100520189 Homo sapiens PKN3 gene Proteins 0.000 description 1
- 101001117317 Homo sapiens Programmed cell death 1 ligand 1 Proteins 0.000 description 1
- 101100207072 Homo sapiens TNFSF9 gene Proteins 0.000 description 1
- 101000604583 Homo sapiens Tyrosine-protein kinase SYK Proteins 0.000 description 1
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 description 1
- 229960005545 IMM-101 Drugs 0.000 description 1
- XDXDZDZNSLXDNA-TZNDIEGXSA-N Idarubicin Chemical compound C1[C@H](N)[C@H](O)[C@H](C)O[C@H]1O[C@@H]1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2C[C@@](O)(C(C)=O)C1 XDXDZDZNSLXDNA-TZNDIEGXSA-N 0.000 description 1
- XDXDZDZNSLXDNA-UHFFFAOYSA-N Idarubicin Natural products C1C(N)C(O)C(C)OC1OC1C2=C(O)C(C(=O)C3=CC=CC=C3C3=O)=C3C(O)=C2CC(O)(C(C)=O)C1 XDXDZDZNSLXDNA-UHFFFAOYSA-N 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 229940076838 Immune checkpoint inhibitor Drugs 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- 102100030694 Interleukin-11 Human genes 0.000 description 1
- 102000013691 Interleukin-17 Human genes 0.000 description 1
- 108010002350 Interleukin-2 Proteins 0.000 description 1
- 102000000588 Interleukin-2 Human genes 0.000 description 1
- 102100033627 Killer cell immunoglobulin-like receptor 3DL1 Human genes 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 239000005411 L01XE02 - Gefitinib Substances 0.000 description 1
- 239000005511 L01XE05 - Sorafenib Substances 0.000 description 1
- 239000002136 L01XE07 - Lapatinib Substances 0.000 description 1
- 239000002138 L01XE21 - Regorafenib Substances 0.000 description 1
- 239000002139 L01XE22 - Masitinib Substances 0.000 description 1
- 239000002177 L01XE27 - Ibrutinib Substances 0.000 description 1
- 229940125563 LAG3 inhibitor Drugs 0.000 description 1
- IVRXNBXKWIJUQB-UHFFFAOYSA-N LY-2157299 Chemical compound CC1=CC=CC(C=2C(=C3CCCN3N=2)C=2C3=CC(=CC=C3N=CC=2)C(N)=O)=N1 IVRXNBXKWIJUQB-UHFFFAOYSA-N 0.000 description 1
- 229940122145 LYN tyrosine kinase inhibitor Drugs 0.000 description 1
- 108010000817 Leuprolide Proteins 0.000 description 1
- HLFSDGLLUJUHTE-SNVBAGLBSA-N Levamisole Chemical compound C1([C@H]2CN3CCSC3=N2)=CC=CC=C1 HLFSDGLLUJUHTE-SNVBAGLBSA-N 0.000 description 1
- 206010067125 Liver injury Diseases 0.000 description 1
- 208000000185 Localized scleroderma Diseases 0.000 description 1
- 102000002576 MAP Kinase Kinase 1 Human genes 0.000 description 1
- 108010068342 MAP Kinase Kinase 1 Proteins 0.000 description 1
- 101710127797 Macrophage colony-stimulating factor 1 Proteins 0.000 description 1
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 description 1
- XOGTZOOQQBDUSI-UHFFFAOYSA-M Mesna Chemical compound [Na+].[O-]S(=O)(=O)CCS XOGTZOOQQBDUSI-UHFFFAOYSA-M 0.000 description 1
- 102000003735 Mesothelin Human genes 0.000 description 1
- 108090000015 Mesothelin Proteins 0.000 description 1
- QXKHYNVANLEOEG-UHFFFAOYSA-N Methoxsalen Chemical compound C1=CC(=O)OC2=C1C=C1C=COC1=C2OC QXKHYNVANLEOEG-UHFFFAOYSA-N 0.000 description 1
- 229930192392 Mitomycin Natural products 0.000 description 1
- 102000007298 Mucin-1 Human genes 0.000 description 1
- 108010008707 Mucin-1 Proteins 0.000 description 1
- 102000015728 Mucins Human genes 0.000 description 1
- 108010063954 Mucins Proteins 0.000 description 1
- HRNLUBSXIHFDHP-UHFFFAOYSA-N N-(2-aminophenyl)-4-[[[4-(3-pyridinyl)-2-pyrimidinyl]amino]methyl]benzamide Chemical compound NC1=CC=CC=C1NC(=O)C(C=C1)=CC=C1CNC1=NC=CC(C=2C=NC=CC=2)=N1 HRNLUBSXIHFDHP-UHFFFAOYSA-N 0.000 description 1
- 108091008877 NK cell receptors Proteins 0.000 description 1
- 102000010648 Natural Killer Cell Receptors Human genes 0.000 description 1
- 229940088705 Neurokinin receptor agonist Drugs 0.000 description 1
- 102000001756 Notch2 Receptor Human genes 0.000 description 1
- 108010029751 Notch2 Receptor Proteins 0.000 description 1
- 102000001760 Notch3 Receptor Human genes 0.000 description 1
- 108010029756 Notch3 Receptor Proteins 0.000 description 1
- 229940122515 Orotate phosphoribosyltransferase inhibitor Drugs 0.000 description 1
- 239000012270 PD-1 inhibitor Substances 0.000 description 1
- 239000012668 PD-1-inhibitor Substances 0.000 description 1
- 239000012272 PD-L2 inhibitor Substances 0.000 description 1
- 101150083673 PKN3 gene Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- KMSKQZKKOZQFFG-HSUXVGOQSA-N Pirarubicin Chemical compound O([C@H]1[C@@H](N)C[C@@H](O[C@H]1C)O[C@H]1C[C@@](O)(CC=2C(O)=C3C(=O)C=4C=CC=C(C=4C(=O)C3=C(O)C=21)OC)C(=O)CO)[C@H]1CCCCO1 KMSKQZKKOZQFFG-HSUXVGOQSA-N 0.000 description 1
- 229940124090 Platelet-derived growth factor (PDGF) receptor antagonist Drugs 0.000 description 1
- 229940119220 Poly ADP ribose polymerase 1 inhibitor Drugs 0.000 description 1
- 229940122482 Poly ADP ribose polymerase 2 inhibitor Drugs 0.000 description 1
- 229940118469 Poly ADP ribose polymerase 3 inhibitor Drugs 0.000 description 1
- 108010050808 Procollagen Proteins 0.000 description 1
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 description 1
- 206010064911 Pulmonary arterial hypertension Diseases 0.000 description 1
- 208000007135 Retinal Neovascularization Diseases 0.000 description 1
- AUVVAXYIELKVAI-UHFFFAOYSA-N SJ000285215 Natural products N1CCC2=CC(OC)=C(OC)C=C2C1CC1CC2C3=CC(OC)=C(OC)C=C3CCN2CC1CC AUVVAXYIELKVAI-UHFFFAOYSA-N 0.000 description 1
- 229940123612 Secretin agonist Drugs 0.000 description 1
- 229940122055 Serine protease inhibitor Drugs 0.000 description 1
- 101710102218 Serine protease inhibitor Proteins 0.000 description 1
- 102000013380 Smoothened Receptor Human genes 0.000 description 1
- 108010090739 Smoothened Receptor Proteins 0.000 description 1
- 208000018359 Systemic autoimmune disease Diseases 0.000 description 1
- 101150041736 TNFSF9 gene Proteins 0.000 description 1
- NAVMQTYZDKMPEU-UHFFFAOYSA-N Targretin Chemical compound CC1=CC(C(CCC2(C)C)(C)C)=C2C=C1C(=C)C1=CC=C(C(O)=O)C=C1 NAVMQTYZDKMPEU-UHFFFAOYSA-N 0.000 description 1
- 229940123237 Taxane Drugs 0.000 description 1
- 108010017842 Telomerase Proteins 0.000 description 1
- BPEGJWRSRHCHSN-UHFFFAOYSA-N Temozolomide Chemical compound O=C1N(C)N=NC2=C(C(N)=O)N=CN21 BPEGJWRSRHCHSN-UHFFFAOYSA-N 0.000 description 1
- FOCVUCIESVLUNU-UHFFFAOYSA-N Thiotepa Chemical compound C1CN1P(N1CC1)(=S)N1CC1 FOCVUCIESVLUNU-UHFFFAOYSA-N 0.000 description 1
- 102000006601 Thymidine Kinase Human genes 0.000 description 1
- 108020004440 Thymidine kinase Proteins 0.000 description 1
- 229940122149 Thymidylate synthase inhibitor Drugs 0.000 description 1
- 102000004060 Transforming Growth Factor-beta Type II Receptor Human genes 0.000 description 1
- 108010082684 Transforming Growth Factor-beta Type II Receptor Proteins 0.000 description 1
- 102100024598 Tumor necrosis factor ligand superfamily member 10 Human genes 0.000 description 1
- 101710097160 Tumor necrosis factor ligand superfamily member 10 Proteins 0.000 description 1
- 102100032101 Tumor necrosis factor ligand superfamily member 9 Human genes 0.000 description 1
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 description 1
- 102100038183 Tyrosine-protein kinase SYK Human genes 0.000 description 1
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 description 1
- 229940122803 Vinca alkaloid Drugs 0.000 description 1
- 102100022748 Wilms tumor protein Human genes 0.000 description 1
- 101710127857 Wilms tumor protein Proteins 0.000 description 1
- PCWZKQSKUXXDDJ-UHFFFAOYSA-N Xanthotoxin Natural products COCc1c2OC(=O)C=Cc2cc3ccoc13 PCWZKQSKUXXDDJ-UHFFFAOYSA-N 0.000 description 1
- 108010023617 abarelix Proteins 0.000 description 1
- 229960002184 abarelix Drugs 0.000 description 1
- AIWRTTMUVOZGPW-HSPKUQOVSA-N abarelix Chemical compound C([C@@H](C(=O)N[C@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCNC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N[C@H](C)C(N)=O)N(C)C(=O)[C@H](CO)NC(=O)[C@@H](CC=1C=NC=CC=1)NC(=O)[C@@H](CC=1C=CC(Cl)=CC=1)NC(=O)[C@@H](CC=1C=C2C=CC=CC2=CC=1)NC(C)=O)C1=CC=C(O)C=C1 AIWRTTMUVOZGPW-HSPKUQOVSA-N 0.000 description 1
- 229940009456 adriamycin Drugs 0.000 description 1
- 229960001686 afatinib Drugs 0.000 description 1
- ULXXDDBFHOBEHA-CWDCEQMOSA-N afatinib Chemical compound N1=CN=C2C=C(O[C@@H]3COCC3)C(NC(=O)/C=C/CN(C)C)=CC2=C1NC1=CC=C(F)C(Cl)=C1 ULXXDDBFHOBEHA-CWDCEQMOSA-N 0.000 description 1
- 229950003067 aglatimagene besadenovec Drugs 0.000 description 1
- 101150045355 akt1 gene Proteins 0.000 description 1
- 229960005310 aldesleukin Drugs 0.000 description 1
- 108700025316 aldesleukin Proteins 0.000 description 1
- 229960001445 alitretinoin Drugs 0.000 description 1
- 230000002152 alkylating effect Effects 0.000 description 1
- 229960003459 allopurinol Drugs 0.000 description 1
- OFCNXPDARWKPPY-UHFFFAOYSA-N allopurinol Chemical compound OC1=NC=NC2=C1C=NN2 OFCNXPDARWKPPY-UHFFFAOYSA-N 0.000 description 1
- 229960000473 altretamine Drugs 0.000 description 1
- 229960001097 amifostine Drugs 0.000 description 1
- JKOQGQFVAUAYPM-UHFFFAOYSA-N amifostine Chemical compound NCCCNCCSP(O)(O)=O JKOQGQFVAUAYPM-UHFFFAOYSA-N 0.000 description 1
- 229940009444 amphotericin Drugs 0.000 description 1
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 description 1
- 229960002932 anastrozole Drugs 0.000 description 1
- YBBLVLTVTVSKRW-UHFFFAOYSA-N anastrozole Chemical compound N#CC(C)(C)C1=CC(C(C)(C#N)C)=CC(CN2N=CN=C2)=C1 YBBLVLTVTVSKRW-UHFFFAOYSA-N 0.000 description 1
- 229940045799 anthracyclines and related substance Drugs 0.000 description 1
- 238000011394 anticancer treatment Methods 0.000 description 1
- GOLCXWYRSKYTSP-UHFFFAOYSA-N arsenic trioxide Inorganic materials O1[As]2O[As]1O2 GOLCXWYRSKYTSP-UHFFFAOYSA-N 0.000 description 1
- 229960002594 arsenic trioxide Drugs 0.000 description 1
- 229960003852 atezolizumab Drugs 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229940120638 avastin Drugs 0.000 description 1
- 229950002916 avelumab Drugs 0.000 description 1
- 229960002756 azacitidine Drugs 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 229950007843 bavituximab Drugs 0.000 description 1
- 229960002938 bexarotene Drugs 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 208000029028 brain injury Diseases 0.000 description 1
- 229960001169 brivudine Drugs 0.000 description 1
- 235000008207 calcium folinate Nutrition 0.000 description 1
- 239000011687 calcium folinate Substances 0.000 description 1
- IVFYLRMMHVYGJH-PVPPCFLZSA-N calusterone Chemical compound C1C[C@]2(C)[C@](O)(C)CC[C@H]2[C@@H]2[C@@H](C)CC3=CC(=O)CC[C@]3(C)[C@H]21 IVFYLRMMHVYGJH-PVPPCFLZSA-N 0.000 description 1
- 229950009823 calusterone Drugs 0.000 description 1
- 229940127093 camptothecin Drugs 0.000 description 1
- VSJKWCGYPAHWDS-FQEVSTJZSA-N camptothecin Chemical compound C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-FQEVSTJZSA-N 0.000 description 1
- 230000004611 cancer cell death Effects 0.000 description 1
- 229960004117 capecitabine Drugs 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 229960000590 celecoxib Drugs 0.000 description 1
- RZEKVGVHFLEQIL-UHFFFAOYSA-N celecoxib Chemical compound C1=CC(C)=CC=C1C1=CC(C(F)(F)F)=NN1C1=CC=C(S(N)(=O)=O)C=C1 RZEKVGVHFLEQIL-UHFFFAOYSA-N 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 1
- 229940107137 cholecystokinin Drugs 0.000 description 1
- 239000003743 cholecystokinin B receptor antagonist Substances 0.000 description 1
- 229960001265 ciclosporin Drugs 0.000 description 1
- 229960002436 cladribine Drugs 0.000 description 1
- 229960000928 clofarabine Drugs 0.000 description 1
- WDDPHFBMKLOVOX-AYQXTPAHSA-N clofarabine Chemical compound C1=NC=2C(N)=NC(Cl)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1F WDDPHFBMKLOVOX-AYQXTPAHSA-N 0.000 description 1
- 208000018631 connective tissue disease Diseases 0.000 description 1
- 229930182912 cyclosporin Natural products 0.000 description 1
- 229960000684 cytarabine Drugs 0.000 description 1
- 229960005029 darbepoetin alfa Drugs 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 229940121548 devimistat Drugs 0.000 description 1
- 229960000605 dexrazoxane Drugs 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 108020001096 dihydrofolate reductase Proteins 0.000 description 1
- VSJKWCGYPAHWDS-UHFFFAOYSA-N dl-camptothecin Natural products C1=CC=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)C5(O)CC)C4=NC2=C1 VSJKWCGYPAHWDS-UHFFFAOYSA-N 0.000 description 1
- 108010045524 dolastatin 10 Proteins 0.000 description 1
- 229960002918 doxorubicin hydrochloride Drugs 0.000 description 1
- NOTIQUSPUUHHEH-UXOVVSIBSA-N dromostanolone propionate Chemical compound C([C@@H]1CC2)C(=O)[C@H](C)C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H](OC(=O)CC)[C@@]2(C)CC1 NOTIQUSPUUHHEH-UXOVVSIBSA-N 0.000 description 1
- 229950004683 drostanolone propionate Drugs 0.000 description 1
- 229950009791 durvalumab Drugs 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- AUVVAXYIELKVAI-CKBKHPSWSA-N emetine Chemical compound N1CCC2=CC(OC)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@@H]1CC AUVVAXYIELKVAI-CKBKHPSWSA-N 0.000 description 1
- 229960002694 emetine Drugs 0.000 description 1
- AUVVAXYIELKVAI-UWBTVBNJSA-N emetine Natural products N1CCC2=CC(OC)=C(OC)C=C2[C@H]1C[C@H]1C[C@H]2C3=CC(OC)=C(OC)C=C3CCN2C[C@H]1CC AUVVAXYIELKVAI-UWBTVBNJSA-N 0.000 description 1
- 229960003388 epoetin alfa Drugs 0.000 description 1
- 229960001842 estramustine Drugs 0.000 description 1
- FRPJXPJMRWBBIH-RBRWEJTLSA-N estramustine Chemical compound ClCCN(CCCl)C(=O)OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 FRPJXPJMRWBBIH-RBRWEJTLSA-N 0.000 description 1
- CYCFEEXTLQGJEL-XEOXDSMQSA-N ethyl 4-[(2s)-3-[3-[(e)-(hydroxyhydrazinylidene)methyl]phenyl]-2-[[2,4,6-tri(propan-2-yl)phenyl]sulfonylamino]propanoyl]piperazine-1-carboxylate Chemical compound C1CN(C(=O)OCC)CCN1C(=O)[C@@H](NS(=O)(=O)C=1C(=CC(=CC=1C(C)C)C(C)C)C(C)C)CC1=CC=CC(\C=N\NO)=C1 CYCFEEXTLQGJEL-XEOXDSMQSA-N 0.000 description 1
- 229960000752 etoposide phosphate Drugs 0.000 description 1
- LIQODXNTTZAGID-OCBXBXKTSA-N etoposide phosphate Chemical compound COC1=C(OP(O)(O)=O)C(OC)=CC([C@@H]2C3=CC=4OCOC=4C=C3[C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H]4O[C@H](C)OC[C@H]4O3)O)[C@@H]3[C@@H]2C(OC3)=O)=C1 LIQODXNTTZAGID-OCBXBXKTSA-N 0.000 description 1
- 229960000255 exemestane Drugs 0.000 description 1
- 230000004761 fibrosis Effects 0.000 description 1
- 229960004177 filgrastim Drugs 0.000 description 1
- 229960000961 floxuridine Drugs 0.000 description 1
- ODKNJVUHOIMIIZ-RRKCRQDMSA-N floxuridine Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(F)=C1 ODKNJVUHOIMIIZ-RRKCRQDMSA-N 0.000 description 1
- 229960000390 fludarabine Drugs 0.000 description 1
- GIUYCYHIANZCFB-FJFJXFQQSA-N fludarabine phosphate Chemical compound C1=NC=2C(N)=NC(F)=NC=2N1[C@@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O GIUYCYHIANZCFB-FJFJXFQQSA-N 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- IJJVMEJXYNJXOJ-UHFFFAOYSA-N fluquinconazole Chemical compound C=1C=C(Cl)C=C(Cl)C=1N1C(=O)C2=CC(F)=CC=C2N=C1N1C=NC=N1 IJJVMEJXYNJXOJ-UHFFFAOYSA-N 0.000 description 1
- 235000008191 folinic acid Nutrition 0.000 description 1
- 239000011672 folinic acid Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 229960002258 fulvestrant Drugs 0.000 description 1
- 229950000456 galunisertib Drugs 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 229960002584 gefitinib Drugs 0.000 description 1
- 229960000578 gemtuzumab Drugs 0.000 description 1
- 229940045109 genistein Drugs 0.000 description 1
- 235000006539 genistein Nutrition 0.000 description 1
- TZBJGXHYKVUXJN-UHFFFAOYSA-N genistein Natural products C1=CC(O)=CC=C1C1=COC2=CC(O)=CC(O)=C2C1=O TZBJGXHYKVUXJN-UHFFFAOYSA-N 0.000 description 1
- ZCOLJUOHXJRHDI-CMWLGVBASA-N genistein 7-O-beta-D-glucoside Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=C2C(=O)C(C=3C=CC(O)=CC=3)=COC2=C1 ZCOLJUOHXJRHDI-CMWLGVBASA-N 0.000 description 1
- 229950011595 glufosfamide Drugs 0.000 description 1
- 229960003690 goserelin acetate Drugs 0.000 description 1
- 210000004247 hand Anatomy 0.000 description 1
- ODZBBRURCPAEIQ-PIXDULNESA-N helpin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(=O)NC(=O)C(\C=C\Br)=C1 ODZBBRURCPAEIQ-PIXDULNESA-N 0.000 description 1
- 231100000234 hepatic damage Toxicity 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 208000006454 hepatitis Diseases 0.000 description 1
- UUVWYPNAQBNQJQ-UHFFFAOYSA-N hexamethylmelamine Chemical compound CN(C)C1=NC(N(C)C)=NC(N(C)C)=N1 UUVWYPNAQBNQJQ-UHFFFAOYSA-N 0.000 description 1
- 229940121372 histone deacetylase inhibitor Drugs 0.000 description 1
- 239000003276 histone deacetylase inhibitor Substances 0.000 description 1
- 229960003911 histrelin acetate Drugs 0.000 description 1
- BKEMVGVBBDMHKL-VYFXDUNUSA-N histrelin acetate Chemical compound CC(O)=O.CC(O)=O.CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC(N=C1)=CN1CC1=CC=CC=C1 BKEMVGVBBDMHKL-VYFXDUNUSA-N 0.000 description 1
- 229960001507 ibrutinib Drugs 0.000 description 1
- XYFPWWZEPKGCCK-GOSISDBHSA-N ibrutinib Chemical compound C1=2C(N)=NC=NC=2N([C@H]2CN(CCC2)C(=O)C=C)N=C1C(C=C1)=CC=C1OC1=CC=CC=C1 XYFPWWZEPKGCCK-GOSISDBHSA-N 0.000 description 1
- 229960000908 idarubicin Drugs 0.000 description 1
- 229960001101 ifosfamide Drugs 0.000 description 1
- HOMGKSMUEGBAAB-UHFFFAOYSA-N ifosfamide Chemical compound ClCCNP1(=O)OCCCN1CCCl HOMGKSMUEGBAAB-UHFFFAOYSA-N 0.000 description 1
- 229960003685 imatinib mesylate Drugs 0.000 description 1
- YLMAHDNUQAMNNX-UHFFFAOYSA-N imatinib methanesulfonate Chemical compound CS(O)(=O)=O.C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3N=C(C=CN=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 YLMAHDNUQAMNNX-UHFFFAOYSA-N 0.000 description 1
- 239000012274 immune-checkpoint protein inhibitor Substances 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 238000012744 immunostaining Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 229960003521 interferon alfa-2a Drugs 0.000 description 1
- 229960003507 interferon alfa-2b Drugs 0.000 description 1
- 229940047124 interferons Drugs 0.000 description 1
- 230000002601 intratumoral effect Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 229950009645 istiratumab Drugs 0.000 description 1
- 210000003127 knee Anatomy 0.000 description 1
- 229960004891 lapatinib Drugs 0.000 description 1
- 229960004942 lenalidomide Drugs 0.000 description 1
- GOTYRUGSSMKFNF-UHFFFAOYSA-N lenalidomide Chemical compound C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O GOTYRUGSSMKFNF-UHFFFAOYSA-N 0.000 description 1
- 229960003881 letrozole Drugs 0.000 description 1
- HPJKCIUCZWXJDR-UHFFFAOYSA-N letrozole Chemical compound C1=CC(C#N)=CC=C1C(N1N=CN=C1)C1=CC=C(C#N)C=C1 HPJKCIUCZWXJDR-UHFFFAOYSA-N 0.000 description 1
- 229960001691 leucovorin Drugs 0.000 description 1
- GFIJNRVAKGFPGQ-LIJARHBVSA-N leuprolide Chemical compound CCNC(=O)[C@@H]1CCCN1C(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC=1N=CNC=1)NC(=O)[C@H]1NC(=O)CC1)CC1=CC=C(O)C=C1 GFIJNRVAKGFPGQ-LIJARHBVSA-N 0.000 description 1
- 229960004338 leuprorelin Drugs 0.000 description 1
- 229960001614 levamisole Drugs 0.000 description 1
- 125000003473 lipid group Chemical group 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000008818 liver damage Effects 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 229940124302 mTOR inhibitor Drugs 0.000 description 1
- 239000003628 mammalian target of rapamycin inhibitor Substances 0.000 description 1
- 229960004655 masitinib Drugs 0.000 description 1
- WJEOLQLKVOPQFV-UHFFFAOYSA-N masitinib Chemical compound C1CN(C)CCN1CC1=CC=C(C(=O)NC=2C=C(NC=3SC=C(N=3)C=3C=NC=CC=3)C(C)=CC=2)C=C1 WJEOLQLKVOPQFV-UHFFFAOYSA-N 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229960004296 megestrol acetate Drugs 0.000 description 1
- RQZAXGRLVPAYTJ-GQFGMJRRSA-N megestrol acetate Chemical compound C1=C(C)C2=CC(=O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@@](C(C)=O)(OC(=O)C)[C@@]1(C)CC2 RQZAXGRLVPAYTJ-GQFGMJRRSA-N 0.000 description 1
- 229960000901 mepacrine Drugs 0.000 description 1
- 229960001428 mercaptopurine Drugs 0.000 description 1
- 229960004635 mesna Drugs 0.000 description 1
- 229960004469 methoxsalen Drugs 0.000 description 1
- VQJHOPSWBGJHQS-UHFFFAOYSA-N metoprine, methodichlorophen Chemical compound CC1=NC(N)=NC(N)=C1C1=CC=C(Cl)C(Cl)=C1 VQJHOPSWBGJHQS-UHFFFAOYSA-N 0.000 description 1
- 229960000282 metronidazole Drugs 0.000 description 1
- VAOCPAMSLUNLGC-UHFFFAOYSA-N metronidazole Chemical compound CC1=NC=C([N+]([O-])=O)N1CCO VAOCPAMSLUNLGC-UHFFFAOYSA-N 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- CFCUWKMKBJTWLW-BKHRDMLASA-N mithramycin Chemical compound O([C@@H]1C[C@@H](O[C@H](C)[C@H]1O)OC=1C=C2C=C3C[C@H]([C@@H](C(=O)C3=C(O)C2=C(O)C=1C)O[C@@H]1O[C@H](C)[C@@H](O)[C@H](O[C@@H]2O[C@H](C)[C@H](O)[C@H](O[C@@H]3O[C@H](C)[C@@H](O)[C@@](C)(O)C3)C2)C1)[C@H](OC)C(=O)[C@@H](O)[C@@H](C)O)[C@H]1C[C@@H](O)[C@H](O)[C@@H](C)O1 CFCUWKMKBJTWLW-BKHRDMLASA-N 0.000 description 1
- 229960000350 mitotane Drugs 0.000 description 1
- 229960001156 mitoxantrone Drugs 0.000 description 1
- KKZJGLLVHKMTCM-UHFFFAOYSA-N mitoxantrone Chemical compound O=C1C2=C(O)C=CC(O)=C2C(=O)C2=C1C(NCCNCCO)=CC=C2NCCNCCO KKZJGLLVHKMTCM-UHFFFAOYSA-N 0.000 description 1
- 229950007812 mocetinostat Drugs 0.000 description 1
- VOWOEBADKMXUBU-UHFFFAOYSA-J molecular oxygen;tetrachlorite;hydrate Chemical compound O.O=O.[O-]Cl=O.[O-]Cl=O.[O-]Cl=O.[O-]Cl=O VOWOEBADKMXUBU-UHFFFAOYSA-J 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- BLCLNMBMMGCOAS-UHFFFAOYSA-N n-[1-[[1-[[1-[[1-[[1-[[1-[[1-[2-[(carbamoylamino)carbamoyl]pyrrolidin-1-yl]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-[(2-methylpropan-2-yl)oxy]-1-oxopropan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amin Chemical compound C1CCC(C(=O)NNC(N)=O)N1C(=O)C(CCCN=C(N)N)NC(=O)C(CC(C)C)NC(=O)C(COC(C)(C)C)NC(=O)C(NC(=O)C(CO)NC(=O)C(CC=1C2=CC=CC=C2NC=1)NC(=O)C(CC=1NC=NC=1)NC(=O)C1NC(=O)CC1)CC1=CC=C(O)C=C1 BLCLNMBMMGCOAS-UHFFFAOYSA-N 0.000 description 1
- RDSACQWTXKSHJT-NSHDSACASA-N n-[3,4-difluoro-2-(2-fluoro-4-iodoanilino)-6-methoxyphenyl]-1-[(2s)-2,3-dihydroxypropyl]cyclopropane-1-sulfonamide Chemical compound C1CC1(C[C@H](O)CO)S(=O)(=O)NC=1C(OC)=CC(F)=C(F)C=1NC1=CC=C(I)C=C1F RDSACQWTXKSHJT-NSHDSACASA-N 0.000 description 1
- HUFOZJXAKZVRNJ-UHFFFAOYSA-N n-[3-[[2-[4-(4-acetylpiperazin-1-yl)-2-methoxyanilino]-5-(trifluoromethyl)pyrimidin-4-yl]amino]phenyl]prop-2-enamide Chemical compound COC1=CC(N2CCN(CC2)C(C)=O)=CC=C1NC(N=1)=NC=C(C(F)(F)F)C=1NC1=CC=CC(NC(=O)C=C)=C1 HUFOZJXAKZVRNJ-UHFFFAOYSA-N 0.000 description 1
- 229960004719 nandrolone Drugs 0.000 description 1
- NPAGDVCDWIYMMC-IZPLOLCNSA-N nandrolone Chemical compound O=C1CC[C@@H]2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 NPAGDVCDWIYMMC-IZPLOLCNSA-N 0.000 description 1
- 229950003237 nastorazepide Drugs 0.000 description 1
- 229960000801 nelarabine Drugs 0.000 description 1
- IXOXBSCIXZEQEQ-UHTZMRCNSA-N nelarabine Chemical compound C1=NC=2C(OC)=NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@@H]1O IXOXBSCIXZEQEQ-UHTZMRCNSA-N 0.000 description 1
- 230000004770 neurodegeneration Effects 0.000 description 1
- 208000015122 neurodegenerative disease Diseases 0.000 description 1
- 229950010203 nimotuzumab Drugs 0.000 description 1
- 229960002450 ofatumumab Drugs 0.000 description 1
- 238000006384 oligomerization reaction Methods 0.000 description 1
- 238000011275 oncology therapy Methods 0.000 description 1
- 229960001840 oprelvekin Drugs 0.000 description 1
- 108010046821 oprelvekin Proteins 0.000 description 1
- 229950007283 oregovomab Drugs 0.000 description 1
- 229960003278 osimertinib Drugs 0.000 description 1
- DUYJMQONPNNFPI-UHFFFAOYSA-N osimertinib Chemical compound COC1=CC(N(C)CCN(C)C)=C(NC(=O)C=C)C=C1NC1=NC=CC(C=2C3=CC=CC=C3N(C)C=2)=N1 DUYJMQONPNNFPI-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229960002404 palifermin Drugs 0.000 description 1
- 229940046231 pamidronate Drugs 0.000 description 1
- WRUUGTRCQOWXEG-UHFFFAOYSA-N pamidronate Chemical compound NCCC(O)(P(O)(O)=O)P(O)(O)=O WRUUGTRCQOWXEG-UHFFFAOYSA-N 0.000 description 1
- 229960001972 panitumumab Drugs 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229940121655 pd-1 inhibitor Drugs 0.000 description 1
- 229940121654 pd-l2 inhibitor Drugs 0.000 description 1
- HQQSBEDKMRHYME-UHFFFAOYSA-N pefloxacin mesylate Chemical compound [H+].CS([O-])(=O)=O.C1=C2N(CC)C=C(C(O)=O)C(=O)C2=CC(F)=C1N1CCN(C)CC1 HQQSBEDKMRHYME-UHFFFAOYSA-N 0.000 description 1
- 229960001218 pegademase Drugs 0.000 description 1
- 108010027841 pegademase bovine Proteins 0.000 description 1
- 229960001744 pegaspargase Drugs 0.000 description 1
- 108010001564 pegaspargase Proteins 0.000 description 1
- 229960001373 pegfilgrastim Drugs 0.000 description 1
- 108010044644 pegfilgrastim Proteins 0.000 description 1
- 229960005547 pelareorep Drugs 0.000 description 1
- 229960005079 pemetrexed Drugs 0.000 description 1
- QOFFJEBXNKRSPX-ZDUSSCGKSA-N pemetrexed Chemical compound C1=N[C]2NC(N)=NC(=O)C2=C1CCC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QOFFJEBXNKRSPX-ZDUSSCGKSA-N 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 229960002340 pentostatin Drugs 0.000 description 1
- FPVKHBSQESCIEP-JQCXWYLXSA-N pentostatin Chemical compound C1[C@H](O)[C@@H](CO)O[C@H]1N1C(N=CNC[C@H]2O)=C2N=C1 FPVKHBSQESCIEP-JQCXWYLXSA-N 0.000 description 1
- 229960002087 pertuzumab Drugs 0.000 description 1
- 229960000952 pipobroman Drugs 0.000 description 1
- NJBFOOCLYDNZJN-UHFFFAOYSA-N pipobroman Chemical compound BrCCC(=O)N1CCN(C(=O)CCBr)CC1 NJBFOOCLYDNZJN-UHFFFAOYSA-N 0.000 description 1
- 229960001221 pirarubicin Drugs 0.000 description 1
- 150000003057 platinum Chemical class 0.000 description 1
- 229960003171 plicamycin Drugs 0.000 description 1
- 229960004293 porfimer sodium Drugs 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229940121649 protein inhibitor Drugs 0.000 description 1
- 239000012268 protein inhibitor Substances 0.000 description 1
- 229940076155 protein modulator Drugs 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- GPKJTRJOBQGKQK-UHFFFAOYSA-N quinacrine Chemical compound C1=C(OC)C=C2C(NC(C)CCCN(CC)CC)=C(C=CC(Cl)=C3)C3=NC2=C1 GPKJTRJOBQGKQK-UHFFFAOYSA-N 0.000 description 1
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 1
- 102000016914 ras Proteins Human genes 0.000 description 1
- 108010014186 ras Proteins Proteins 0.000 description 1
- 229960000424 rasburicase Drugs 0.000 description 1
- 108010084837 rasburicase Proteins 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 229950008933 refametinib Drugs 0.000 description 1
- 229960004836 regorafenib Drugs 0.000 description 1
- FNHKPVJBJVTLMP-UHFFFAOYSA-N regorafenib Chemical compound C1=NC(C(=O)NC)=CC(OC=2C=C(F)C(NC(=O)NC=3C=C(C(Cl)=CC=3)C(F)(F)F)=CC=2)=C1 FNHKPVJBJVTLMP-UHFFFAOYSA-N 0.000 description 1
- 210000001525 retina Anatomy 0.000 description 1
- 230000002207 retinal effect Effects 0.000 description 1
- 229950009855 rociletinib Drugs 0.000 description 1
- 229950004707 rucaparib Drugs 0.000 description 1
- 229960002530 sargramostim Drugs 0.000 description 1
- 108010038379 sargramostim Proteins 0.000 description 1
- 229950010613 selinexor Drugs 0.000 description 1
- CYOHGALHFOKKQC-UHFFFAOYSA-N selumetinib Chemical compound OCCONC(=O)C=1C=C2N(C)C=NC2=C(F)C=1NC1=CC=C(Br)C=C1Cl CYOHGALHFOKKQC-UHFFFAOYSA-N 0.000 description 1
- 229950010746 selumetinib Drugs 0.000 description 1
- 239000003001 serine protease inhibitor Substances 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 1
- 229960002930 sirolimus Drugs 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
- 210000002460 smooth muscle Anatomy 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 229940083542 sodium Drugs 0.000 description 1
- MIXCUJKCXRNYFM-UHFFFAOYSA-M sodium;diiodomethanesulfonate;n-propyl-n-[2-(2,4,6-trichlorophenoxy)ethyl]imidazole-1-carboxamide Chemical compound [Na+].[O-]S(=O)(=O)C(I)I.C1=CN=CN1C(=O)N(CCC)CCOC1=C(Cl)C=C(Cl)C=C1Cl MIXCUJKCXRNYFM-UHFFFAOYSA-M 0.000 description 1
- 229960005325 sonidegib Drugs 0.000 description 1
- VZZJRYRQSPEMTK-CALCHBBNSA-N sonidegib Chemical compound C1[C@@H](C)O[C@@H](C)CN1C(N=C1)=CC=C1NC(=O)C1=CC=CC(C=2C=CC(OC(F)(F)F)=CC=2)=C1C VZZJRYRQSPEMTK-CALCHBBNSA-N 0.000 description 1
- 229960003787 sorafenib Drugs 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 210000001179 synovial fluid Anatomy 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000002466 tachykinin receptor agonist Substances 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229940033134 talc Drugs 0.000 description 1
- 229960001603 tamoxifen Drugs 0.000 description 1
- 229950007435 tarextumab Drugs 0.000 description 1
- DKPFODGZWDEEBT-QFIAKTPHSA-N taxane Chemical class C([C@]1(C)CCC[C@@H](C)[C@H]1C1)C[C@H]2[C@H](C)CC[C@@H]1C2(C)C DKPFODGZWDEEBT-QFIAKTPHSA-N 0.000 description 1
- 229960004964 temozolomide Drugs 0.000 description 1
- 229950003547 tertomotide Drugs 0.000 description 1
- 229960005353 testolactone Drugs 0.000 description 1
- BPEWUONYVDABNZ-DZBHQSCQSA-N testolactone Chemical compound O=C1C=C[C@]2(C)[C@H]3CC[C@](C)(OC(=O)CC4)[C@@H]4[C@@H]3CCC2=C1 BPEWUONYVDABNZ-DZBHQSCQSA-N 0.000 description 1
- 229960001196 thiotepa Drugs 0.000 description 1
- 239000003734 thymidylate synthase inhibitor Substances 0.000 description 1
- 229960003087 tioguanine Drugs 0.000 description 1
- 229960005026 toremifene Drugs 0.000 description 1
- XFCLJVABOIYOMF-QPLCGJKRSA-N toremifene Chemical compound C1=CC(OCCN(C)C)=CC=C1C(\C=1C=CC=CC=1)=C(\CCCl)C1=CC=CC=C1 XFCLJVABOIYOMF-QPLCGJKRSA-N 0.000 description 1
- 229960005267 tositumomab Drugs 0.000 description 1
- FNCMIJWGZNHSBF-UHFFFAOYSA-N trabedersen Chemical compound CC1=CN(C2CC(O)C(COP(=O)(S)OC3CC(OC3COP(=O)(S)OC4CC(OC4COP(=O)(S)OC5CC(OC5COP(=O)(S)OC6CC(OC6COP(=O)(S)OC7CC(OC7COP(=O)(S)OC8CC(OC8COP(=O)(S)OC9CC(OC9COP(=O)(S)OC%10CC(OC%10COP(=O)(S)OC%11CC(OC%11COP(=O)(S)OC%12CC(OC%12COP(=O)(S)OC%13CC(OC%13COP(=O)(S)OC%14CC(OC%14COP(=O)(S)OC%15CC(OC%15CO)N%16C=CC(=NC%16=O)N)n%17cnc%18C(=O)NC(=Nc%17%18)N)n%19cnc%20C(=O)NC(=Nc%19%20)N)N%21C=CC(=NC%21=O)N)n%22cnc%23c(N)ncnc%22%23)N%24C=C(C)C(=O)NC%24=O)n%25cnc%26C(=O)NC(=Nc%25%26)N)N%27C=C(C)C(=O)NC%27=O)N%28C=CC(=NC%28=O)N)N%29C=C(C)C(=O)NC%29=O)n%30cnc%31c(N)ncnc%30%31)N%32C=C(C)C(=O)NC%32=O)N%33C=C(C)C(=O)NC%33=O)O2)C(=O)NC1=O.CC%34=CN(C%35CC(OP(=O)(S)OCC%36OC(CC%36OP(=O)(S)OCC%37OC(CC%37OP(=O)(S)OCC%38OC(CC%38O)n%39cnc%40c(N)ncnc%39%40)N%41C=C(C)C(=O)NC%41=O)n%42cnc%43C(=O)NC(=Nc%42%43)N)C(COP(=O)S)O%35)C(=O)NC%34=O FNCMIJWGZNHSBF-UHFFFAOYSA-N 0.000 description 1
- 229950002824 trabedersen Drugs 0.000 description 1
- 229960004066 trametinib Drugs 0.000 description 1
- LIRYPHYGHXZJBZ-UHFFFAOYSA-N trametinib Chemical compound CC(=O)NC1=CC=CC(N2C(N(C3CC3)C(=O)C3=C(NC=4C(=CC(I)=CC=4)F)N(C)C(=O)C(C)=C32)=O)=C1 LIRYPHYGHXZJBZ-UHFFFAOYSA-N 0.000 description 1
- 229960001727 tretinoin Drugs 0.000 description 1
- 229960001099 trimetrexate Drugs 0.000 description 1
- NOYPYLRCIDNJJB-UHFFFAOYSA-N trimetrexate Chemical compound COC1=C(OC)C(OC)=CC(NCC=2C(=C3C(N)=NC(N)=NC3=CC=2)C)=C1 NOYPYLRCIDNJJB-UHFFFAOYSA-N 0.000 description 1
- 229950008529 upamostat Drugs 0.000 description 1
- 210000002700 urine Anatomy 0.000 description 1
- 229950006605 varlitinib Drugs 0.000 description 1
- 229960002066 vinorelbine Drugs 0.000 description 1
- GBABOYUKABKIAF-GHYRFKGUSA-N vinorelbine Chemical compound C1N(CC=2C3=CC=CC=C3NC=22)CC(CC)=C[C@H]1C[C@]2(C(=O)OC)C1=CC([C@]23[C@H]([C@]([C@H](OC(C)=O)[C@]4(CC)C=CCN([C@H]34)CC2)(O)C(=O)OC)N2C)=C2C=C1OC GBABOYUKABKIAF-GHYRFKGUSA-N 0.000 description 1
- 229950003990 yttrium (90y) clivatuzumab tetraxetan Drugs 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
- A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/08—Peptides having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
- A61K38/10—Peptides having 12 to 20 amino acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
- A61K38/1774—Immunoglobulin superfamily (e.g. CD2, CD4, CD8, ICAM molecules, B7 molecules, Fc-receptors, MHC-molecules)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/0005—Vertebrate antigens
- A61K39/0011—Cancer antigens
- A61K39/001102—Receptors, cell surface antigens or cell surface determinants
- A61K39/001111—Immunoglobulin superfamily
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
- A61K2039/55511—Organic adjuvants
- A61K2039/55516—Proteins; Peptides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Epidemiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- Gastroenterology & Hepatology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Hematology (AREA)
- Diabetes (AREA)
- Ophthalmology & Optometry (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Physical Education & Sports Medicine (AREA)
- Oncology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-1/DAP-12 signaling pathway. The compositions, including peptides and peptide variants, modulate TREM- 1 -mediated immunological response as standalone and combination-therapy treatment regimen. Further, methods are provided for predicting the efficacy of TREM-l modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
Description
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
PEPTIDES AND COMPOSITIONS FOR TARGETED TREATMENT AND IMAGING
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No.
62/717,929, filed August 13, 2018, U.S. Provisional Patent Application No. 62/751,303, filed October 26, 2018, U.S. Provisional Patent Application No. 62/836823, filed April 22, 2019, U.S. Provisional Patent Application No. 62/843,835, filed May 06, 2019, and to U.S. Provisional Patent Application No. 62/875287 filed July 17, 2019, each of which are incorporated herein by reference in their entireties and for all purposes.
FIELD OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-signaling pathway. The compositions, including peptides and peptide variants, modulate TREM-1-mediated immunological response as standalone and combination-therapy treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, .. pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
BACKGROUND OF THE INVENTION
Administration of therapeutic peptides often causes activation of nontarget cells and leads to undesired side effects and increases risk of undesired immunogenic effects. Limitations generally attributed to therapeutic peptides are: a short half-life in the circulation because of their rapid degradation by proteolytic enzymes of the digestive system and blood plasma; rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance); poor ability to cross physiological barriers, such as the blood-brain barrier.
Because of therapeutic peptides having general hydrophilicity; high conformational flexibility, and use resulting sometimes in a lack of selectivity involving interactions with different receptors/targets (poor specific biodistribution), described in part in Vlieghe, et al. Drug Discov Today 2010, 15:40-56.
Consequently, there is need for more effective formulations of therapeutic peptides to improve their targeted delivery, prolonged circulatory half-life, biocompatibility and therapeutic efficiency.
SUMMARY OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-signaling pathway. The compositions, including peptides and peptide variants, modulate TREM-1-mediated immunological response as standalone and combination-therapy treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
The present disclosure describes novel amphipathic trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating diseases related to activated immune cells. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In some embodiments, each trifunctional peptide is capable of at least three functions:
1) mediating the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of interest and/or sites of
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
PEPTIDES AND COMPOSITIONS FOR TARGETED TREATMENT AND IMAGING
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No.
62/717,929, filed August 13, 2018, U.S. Provisional Patent Application No. 62/751,303, filed October 26, 2018, U.S. Provisional Patent Application No. 62/836823, filed April 22, 2019, U.S. Provisional Patent Application No. 62/843,835, filed May 06, 2019, and to U.S. Provisional Patent Application No. 62/875287 filed July 17, 2019, each of which are incorporated herein by reference in their entireties and for all purposes.
FIELD OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-signaling pathway. The compositions, including peptides and peptide variants, modulate TREM-1-mediated immunological response as standalone and combination-therapy treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, .. pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
BACKGROUND OF THE INVENTION
Administration of therapeutic peptides often causes activation of nontarget cells and leads to undesired side effects and increases risk of undesired immunogenic effects. Limitations generally attributed to therapeutic peptides are: a short half-life in the circulation because of their rapid degradation by proteolytic enzymes of the digestive system and blood plasma; rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance); poor ability to cross physiological barriers, such as the blood-brain barrier.
Because of therapeutic peptides having general hydrophilicity; high conformational flexibility, and use resulting sometimes in a lack of selectivity involving interactions with different receptors/targets (poor specific biodistribution), described in part in Vlieghe, et al. Drug Discov Today 2010, 15:40-56.
Consequently, there is need for more effective formulations of therapeutic peptides to improve their targeted delivery, prolonged circulatory half-life, biocompatibility and therapeutic efficiency.
SUMMARY OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-signaling pathway. The compositions, including peptides and peptide variants, modulate TREM-1-mediated immunological response as standalone and combination-therapy treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
The present disclosure describes novel amphipathic trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating diseases related to activated immune cells. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In some embodiments, each trifunctional peptide is capable of at least three functions:
1) mediating the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of interest and/or sites of
2 disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and .. targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages. The invention further relates to personalized medical treatments for cancer that involve targeting specific cancers by their tumor environment. The invention further relates to personalized medical treatments for scleroderma (systemic sclerosis, SSc). More specifically, the invention provides for treatment of scleroderma or a related autoimmune or a fibrotic condition by using modulators of the pathway standalone or together with other antifibrotic therapies and the use of such combinations in the treatment of scleroderma.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof, said method comprising administering to said patient a therapeutically effective .. amount of at least one modulator that is effective for modulating the TREM-1/DAP-12 signaling pathway together with a therapeutically amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof. In one embodiment, said method further comprises administering said modulator together with a pharmaceutically acceptable .. excipient, carrier, diluents, or a combination thereof. In some embodiments, said carrier is selected from the group consisting of lipids, proteins or polypeptides, and mixtures thereof. In one embodiment, said method further comprises prior to administering the first dose of said modulator, the subject received a prior therapy selected from the group consising of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof.
However it is not meant to limit such prior therapies. In some embodiments, said cancer recurred or progressed after the prior therapy. In some embodiments, said administration of said modulator to said patient is continued as a long-term maintenance treatment for duration between about two weeks to about five years, preferably said administration is continued for duration of .. up to one year. In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, Sipuleucel-T/Provenge, and the like. In some embodiments,
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof, said method comprising administering to said patient a therapeutically effective .. amount of at least one modulator that is effective for modulating the TREM-1/DAP-12 signaling pathway together with a therapeutically amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof. In one embodiment, said method further comprises administering said modulator together with a pharmaceutically acceptable .. excipient, carrier, diluents, or a combination thereof. In some embodiments, said carrier is selected from the group consisting of lipids, proteins or polypeptides, and mixtures thereof. In one embodiment, said method further comprises prior to administering the first dose of said modulator, the subject received a prior therapy selected from the group consising of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof.
However it is not meant to limit such prior therapies. In some embodiments, said cancer recurred or progressed after the prior therapy. In some embodiments, said administration of said modulator to said patient is continued as a long-term maintenance treatment for duration between about two weeks to about five years, preferably said administration is continued for duration of .. up to one year. In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, Sipuleucel-T/Provenge, and the like. In some embodiments,
3 said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Ofatumumab, Nivolumab, Pembrolizumab, Rituximab, Blinatumomab, Daratumumab, Trastuzumab, Cetuximab, Elotuzumab, adoptive T-cell therapy, T-Vec, Interferon, Interleukin, and a combination thereof In some embodiments, said anticancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anticancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, a PARP
inhibitor, a tyrosine kinase inhibitor, CSF-1/CSF-1R inhibitor, doxorubicin, gemcitabine, entrectinib, epirubicin, vinblastine, etoposide, topotecan, bleomycin, mytomycin c, and the like.
In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, Oxaliplatin, and the like. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Abraxane, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, IDN-5109, and the like. In some embodiments, said topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of Irinotecan, Topotecan, Camptothecins (CPT), and the like.
In some embodiments, said topoisomerase inhibitor is a topoisomerase II
inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, ellipticine, and the like. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib), and the like. In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-641397, and the like. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, Talazoparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231, and the like. In some embodiments, said tyrosine kinase inhibitor is selected from the group consisting of pexidartinib, entrectinib, matinib mesylate (STI571; Gleevec), gefitinib (Iressa), erlotinib (OSI-1774;
Tarceva), lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248), and leflunomide (SU101), and the like. In some
inhibitor, a tyrosine kinase inhibitor, CSF-1/CSF-1R inhibitor, doxorubicin, gemcitabine, entrectinib, epirubicin, vinblastine, etoposide, topotecan, bleomycin, mytomycin c, and the like.
In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, Oxaliplatin, and the like. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Abraxane, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, IDN-5109, and the like. In some embodiments, said topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of Irinotecan, Topotecan, Camptothecins (CPT), and the like.
In some embodiments, said topoisomerase inhibitor is a topoisomerase II
inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, ellipticine, and the like. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib), and the like. In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-641397, and the like. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, Talazoparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231, and the like. In some embodiments, said tyrosine kinase inhibitor is selected from the group consisting of pexidartinib, entrectinib, matinib mesylate (STI571; Gleevec), gefitinib (Iressa), erlotinib (OSI-1774;
Tarceva), lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248), and leflunomide (SU101), and the like. In some
4 embodiments, said CSF-1/CSF-1R inhibitor is selected from the group consisting of CSF-1R
kinase inhibitor, an antibody that binds CSF-1R and is capable of blocking binding of CSF-1 and/or IL-34 to CSF-1R, and the like. In some embodiments, said CSF-1R kinase inhibitor is imatinib, nilotinib or PLX3397. In some embodiments, said radiation therapy is selected from the group consisting of X-rays, ion beams, electron beams, gamma-rays, UV-rays, and decay of a radioactive isotope, or a combination thereof. In some embodiments, said surgery is surgical tumor resection. In some embodiments, said cancer is lung cancer including non-small cell lung cancer, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, central nervous system cancer, stomach cancer, prostate, colon cancer, colorectal cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, stomach cancer, bladder cancer, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis and other cancers in which myeloid cells are involved or recruited and cancer cachexia. In some embodiments, said at least one said modulator comprises a variant peptide sequence that is capable of binding TREM-1/DAP-12 and reducing or blocking TREM-1/DAP-12 activity (signaling and/or activation). In some embodiments, said variant peptide sequence comprises at least one D-amino acid. In some embodiments, said variant peptide sequence is a cyclic peptide. In some embodiments, said variant peptide sequence is derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, or a combination thereof. In some embodiments, said variant peptide sequence comprises LR12 and/or LP17 peptide variants and the like or a combination thereof In some embodiments, said modulator comprises at least one isolated antibody or fragment thereof, that is capable of specifically binding TREM-1/DAP-12 and which is capable of reducing or blocking TREM-1/DAP-12 activity (signaling and/or activation). In one embodiment, said method further comprises a diagnostic method. In one embodiment, said diagnostic method is performed prior to administering the first dose of said modulator to predict response of said patient to a therapy of the method of claim 1. In some embodiments, said diagnostic method comprises isolating a biological sample from said patient and determining in .. said sample the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of CD68-positive cells or a combination thereof, wherein the higher is the expression level of CSF-1, CSF-1R, IL-
kinase inhibitor, an antibody that binds CSF-1R and is capable of blocking binding of CSF-1 and/or IL-34 to CSF-1R, and the like. In some embodiments, said CSF-1R kinase inhibitor is imatinib, nilotinib or PLX3397. In some embodiments, said radiation therapy is selected from the group consisting of X-rays, ion beams, electron beams, gamma-rays, UV-rays, and decay of a radioactive isotope, or a combination thereof. In some embodiments, said surgery is surgical tumor resection. In some embodiments, said cancer is lung cancer including non-small cell lung cancer, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, central nervous system cancer, stomach cancer, prostate, colon cancer, colorectal cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, stomach cancer, bladder cancer, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis and other cancers in which myeloid cells are involved or recruited and cancer cachexia. In some embodiments, said at least one said modulator comprises a variant peptide sequence that is capable of binding TREM-1/DAP-12 and reducing or blocking TREM-1/DAP-12 activity (signaling and/or activation). In some embodiments, said variant peptide sequence comprises at least one D-amino acid. In some embodiments, said variant peptide sequence is a cyclic peptide. In some embodiments, said variant peptide sequence is derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, or a combination thereof. In some embodiments, said variant peptide sequence comprises LR12 and/or LP17 peptide variants and the like or a combination thereof In some embodiments, said modulator comprises at least one isolated antibody or fragment thereof, that is capable of specifically binding TREM-1/DAP-12 and which is capable of reducing or blocking TREM-1/DAP-12 activity (signaling and/or activation). In one embodiment, said method further comprises a diagnostic method. In one embodiment, said diagnostic method is performed prior to administering the first dose of said modulator to predict response of said patient to a therapy of the method of claim 1. In some embodiments, said diagnostic method comprises isolating a biological sample from said patient and determining in .. said sample the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of CD68-positive cells or a combination thereof, wherein the higher is the expression level of CSF-1, CSF-1R, IL-
5
6, TREM-1 or the higher is number of CD68-positive cells or a combination thereof, the better the patient is predicted to respond to a therapy of the method of claim 1. In some embodiments, said method comprises: (a) administering to said patient an amount of at least one said modulator of the method of claim 1 that is capable of binding TREM-1 and is conjugated to at least one imaging probe, or a combination thereof, in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location and amount of the labeled probe corresponds to at least one symptom of the myeloid cell-related cancer condition and correlates with the TREM-1 expression levels and the higher the levels are, the better the patient is predicted to respond to a therapy of the method of claim 1. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99m, cr51 , Ga67, Ga68, cu64, Rb82,m099, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol, or a combination thereof.
The present invention encompasses the discovery that it is possible to combine multiple functions in one amphipathic polypeptide amino acid sequence to confer a variety of properties on the resulting peptide and provides novel peptides and compounds, which are capable of executing at least, three functions: 1) mediation of formation of naturally long half-life lipopeptide/lipoprotein particles (LP) upon interaction with native lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In one embodiment, said peptides and compounds of the present invention are used in combinations thereof The peptides and compounds of the present invention and combinations thereof have a wide variety of uses, particularly in the areas of oncology, transplantology, dermatology, hepatology, ophthalmology, cardiovascular diseases, sepsis, autoimmune diseases, neurodegenerative diseases and other diseases and conditions.
They also are useful in the production of medical devices (for example, medical implants and implantable devices).
In some embodiments, the invention provides a synthetic trifunctional peptide comprising: (a) a first amino acid domain that does not interact with native lipoproteins in isolated form, wherein said first amino acid domain is at least 3 amino acids in length and is capable of treating, preventing and/or detecting an immune-related disease or condition; and (b)
The present invention encompasses the discovery that it is possible to combine multiple functions in one amphipathic polypeptide amino acid sequence to confer a variety of properties on the resulting peptide and provides novel peptides and compounds, which are capable of executing at least, three functions: 1) mediation of formation of naturally long half-life lipopeptide/lipoprotein particles (LP) upon interaction with native lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In one embodiment, said peptides and compounds of the present invention are used in combinations thereof The peptides and compounds of the present invention and combinations thereof have a wide variety of uses, particularly in the areas of oncology, transplantology, dermatology, hepatology, ophthalmology, cardiovascular diseases, sepsis, autoimmune diseases, neurodegenerative diseases and other diseases and conditions.
They also are useful in the production of medical devices (for example, medical implants and implantable devices).
In some embodiments, the invention provides a synthetic trifunctional peptide comprising: (a) a first amino acid domain that does not interact with native lipoproteins in isolated form, wherein said first amino acid domain is at least 3 amino acids in length and is capable of treating, preventing and/or detecting an immune-related disease or condition; and (b)
7 a second amino acid domain that mediates formation of lipopeptide/lipoprotein particles upon interaction of the peptide with native lipoproteins and targets these particles to cells of interest and/or sites of disease or condition, which second amino acid domain is at least 6 amino acids in length and has an amphipathic alpha helical amino acid sequence. In some embodiments, said first amino acid domain comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine. In some embodiments, said first amino acid domain comprises amino acid sequence Met-Trp-Lys-Thr-Pro-Thr-Leu-Lys-Tyr-Phe, wherein Met is methionine, Trp is tryptophan, Lys is lysine, Thr is threonine, Pro is proline, Leu is leucine, Tyr is tyrosine, and Phe is phenylalanine. In some embodiments, said second amino acid domain comprises amino acid sequence Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val, wherein Tyr is tyrosine, Leu is leucine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some embodiments, said second amino acid domain comprises amino acid sequence Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val, wherein Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, and Val is valine. In some embodiments, said first amino acid domain and/or said second amino acid domain are conjugated to at least one imaging probe.
In some embodiments, the invention provides a method of imaging an immune-related disease or condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) a compound of claim 8, wherein the composition has an affinity for immune receptors; b) administering said composition to said patient in a detectably effective amount, c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the immune-related disease or condition.
In some embodiments, the invention provides a method of treating an immune-related disease or condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) the composition of claim 1 capable of modulating immune receptors; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said immune-related disease or condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, psoriatic arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, sepsis, inflammatory lung diseases (e.g., interstitial pneumonitis and asthma), retinopathy (e.g., retinopathy of prematurity and diabetic retinopathy), neurodegenrative diseases (e.g., Alzheimer's, Parkinson's and Huntington's diseases), gastroenterological diseases and conditions (e.g. inflammatory bowel disease, Crohn's disease, celiac disease), Guillain-Barre syndrome, Hashimoto's disease, pernicious anemia, primary biliary cirrhosis, chronic active hepatitis, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, skin problems (e.g. atopic dermatitis, psoriasis, pemphigus vulgaris), cardiovascular problems (e.g. autoimmune pericarditis), allergic diathesis (e.g. delayed type hypersensitivity), contact dermatitis, herpes simplex/zoster, respiratory conditions (e.g. allergic alveolitis), inflammatory conditions (e.g. myositis), ankylosing spondylitis, tissue/organ transplant (e.g., heart/lung transplants) rejection reactions, brain and spinal cord injuries, and other diseases and conditions where immune cells are involved or recruited.
In some embodiments, the invention provides a synthetic trifunctional peptide comprising: (a) a first amino acid domain that does not interact with native lipoproteins in isolated form, which first amino acid domain is at least 3 amino acids in length and is capable of treating, preventing and/or detecting an immune-related disease or condition;
and (b) a second amino acid domain that mediates formation of lipopeptide/lipoprotein particles upon interaction of the peptide with native lipoproteins and targets these particles to cells of interest and/or sites of disease or condition, which second amino acid domain is at least 6 amino acids in length and has an amphipathic alpha helical amino acid sequence. In some embodiments, said first amino acid domain comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine.
In some embodiments, said first amino acid domain comprises amino acid sequence Met-Trp-
In some embodiments, the invention provides a method of imaging an immune-related disease or condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) a compound of claim 8, wherein the composition has an affinity for immune receptors; b) administering said composition to said patient in a detectably effective amount, c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the immune-related disease or condition.
In some embodiments, the invention provides a method of treating an immune-related disease or condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) the composition of claim 1 capable of modulating immune receptors; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said immune-related disease or condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, psoriatic arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, sepsis, inflammatory lung diseases (e.g., interstitial pneumonitis and asthma), retinopathy (e.g., retinopathy of prematurity and diabetic retinopathy), neurodegenrative diseases (e.g., Alzheimer's, Parkinson's and Huntington's diseases), gastroenterological diseases and conditions (e.g. inflammatory bowel disease, Crohn's disease, celiac disease), Guillain-Barre syndrome, Hashimoto's disease, pernicious anemia, primary biliary cirrhosis, chronic active hepatitis, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, skin problems (e.g. atopic dermatitis, psoriasis, pemphigus vulgaris), cardiovascular problems (e.g. autoimmune pericarditis), allergic diathesis (e.g. delayed type hypersensitivity), contact dermatitis, herpes simplex/zoster, respiratory conditions (e.g. allergic alveolitis), inflammatory conditions (e.g. myositis), ankylosing spondylitis, tissue/organ transplant (e.g., heart/lung transplants) rejection reactions, brain and spinal cord injuries, and other diseases and conditions where immune cells are involved or recruited.
In some embodiments, the invention provides a synthetic trifunctional peptide comprising: (a) a first amino acid domain that does not interact with native lipoproteins in isolated form, which first amino acid domain is at least 3 amino acids in length and is capable of treating, preventing and/or detecting an immune-related disease or condition;
and (b) a second amino acid domain that mediates formation of lipopeptide/lipoprotein particles upon interaction of the peptide with native lipoproteins and targets these particles to cells of interest and/or sites of disease or condition, which second amino acid domain is at least 6 amino acids in length and has an amphipathic alpha helical amino acid sequence. In some embodiments, said first amino acid domain comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine.
In some embodiments, said first amino acid domain comprises amino acid sequence Met-Trp-
8 Lys-Thr-Pro-Thr-Leu-Lys-Tyr-Phe, wherein Met is methionine, Trp is tryptophan, Lys is lysine, Thr is threonine, Pro is proline, Leu is leucine, Tyr is tyrosine, and Phe is phenylalanine. In some embodiments, said second amino acid domain comprises amino acid sequence Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val, wherein Tyr is tyrosine, Leu is leucine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some embodiments, said the second amino acid domain comprises amino acid sequence Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val, wherein Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, and Val is valine. In some embodiments, said the first amino acid domain and/or the second amino acid domain are conjugated to at least one imaging probe.
In some embodiments, the invention provides a method of imaging an immune-related disease or condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) a compound of claim 8, wherein the composition has an affinity for immune receptors; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the immune-related disease or condition.
In some embodiments, the invention provides a method of treating an immune-related disease or condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) the composition of claim 1 capable of modulating immune receptors; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said immune-related disease or condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, psoriatic arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease,
In some embodiments, the invention provides a method of imaging an immune-related disease or condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) a compound of claim 8, wherein the composition has an affinity for immune receptors; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the immune-related disease or condition.
In some embodiments, the invention provides a method of treating an immune-related disease or condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) the composition of claim 1 capable of modulating immune receptors; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said immune-related disease or condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, psoriatic arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease,
9 sepsis, inflammatory lung diseases (e.g., interstitial pneumonitis and asthma), retinopathy (e.g., retinopathy of prematurity and diabetic retinopathy), neurodegenrative diseases (e.g., Alzheimer's, Parkinson's and Huntington's diseases), gastroenterological diseases and conditions (e.g. inflammatory bowel disease, Crohn's disease, celiac disease), Guillain-Barre syndrome, Hashimoto's disease, pernicious anemia, primary biliary cirrhosis, chronic active hepatitis, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, skin problems (e.g. atopic dermatitis, psoriasis, pemphigus vulgaris), cardiovascular problems (e.g. autoimmune pericarditis), allergic diathesis (e.g. delayed type hypersensitivity), contact dermatitis, herpes simplex/zoster, respiratory conditions (e.g. allergic alveolitis), inflammatory conditions (e.g. myositis), ankylosing spondylitis, tissue/organ transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where immune cells are involved or recruited.
The present disclosure provides novel peptides and compounds, which are capable of executing three functions: 1) assistance in the self-assembly of naturally long half-life lipopeptide particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In one embodiment, said peptides and compounds of the present invention form synthetic lipopeptide particles upon binding to lipid or lipid mixtures.
In some embodiments, the invention provides a synthetic trifunctional polypeptide comprising at least one peptide domain of 3 to 35 amino acids in length having a C-terminal amino acid and at least one amphipathic domain of 6 to 45 to amino acids in length comprising an amphipathic lipopeptide having an N-terminal amino acid, wherein said first domain's C-terminal amino acid is attached to said second domain's N-terminal amino acid.
In one embodiment, said synthetic trifunctional polypeptide further comprises an imaging agent. In one embodiment, said synthetic trifunctional polypeptide further comprises a therapeutic agent. In one embodiment, said synthetic trifunctional polypeptide further comprises a targeting agent. In one embodiment, said synthetic trifunctional polypeptide further comprises a lipopeptide nanoparticle.
In some embodiments, the invention provides a population of spherical lipopeptide nanoparticles or discoidal lipopeptide nanoparticles comprising a plurality of synthetic trifunctional polypeptides, wherein said synthetic trifunctional polypeptide comprising at least one peptide domain of 3 to 35 amino acids in length having a C-terminal amino acid and at least one amphipathic domain of 6 to 45 to amino acids in length comprising an amphipathic lipopeptide having an N-terminal amino acid, wherein said first domain's C-terminal amino acid is attached to said second domain's N-terminal amino acid.
In some embodiments, the invention provides a method of treating an immune-related disease or condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) a synthetic trifunctional polypeptide comprising at least one peptide domain of 3 to 35 amino acids in length having a C-terminal amino acid and at least one amphipathic domain of 6 to 45 to amino acids in length comprising an amphipathic lipopeptide having an N-terminal amino acid, wherein said first domain's C-terminal amino acid is attached to said second domain's N-terminal amino acid, wherein said trifunctional polypeptide is capable of modulating immune receptors; b) administering said synthetic trifunctional polypeptide to said patient under conditions such that said at least one symptom is reduced.
The invention relates to personalized medical treatments for cancer that involve targeting specific cancers by their tumor environment. More specifically, the invention provides for treatment of various cancers by using inhibitors of the TREM-1/DAP-12 pathway.
These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. In addition, the invention provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on macrophages. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe. In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with cancer using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention provides for a method of treating cancer in an individual in need thereof by administering to the individual an effective amount of an inhibitor of the pathway. In one aspect, the inhibitors are selected from peptide variants and compositions that suppress tumor growth by modulating the TREM-1/DAP-12 signaling pathway. In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to the peptide variants and compositions of the invention. In one embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MRI). In one embodiment, the imaging agent is .. a [64Cu]-containing imaging probe for imaging systems such as a positron emission tomography (PET) imaging systems (and combined PET/computer tomography (CT) and PET/MM
systems).
In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In certain embodiments, the peptide .. variants and compositions of the present invention are incorporated into long half-life synthetic lipopeptide particles (SLP). In certain embodiments, the peptide variants and compositions of the invention may incorporate into lipopeptide particles (LP) in vivo upon administration to the individual. In certain embodiments, the peptides and compositions of the invention can cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention provides for a method for suppressing tumor growth in an individual in need thereof by administering to the individual an amount of a TREM-1 inhibitor that is effective for suppressing tumor growth.
In some embodiments, methods of treating a proliferative disorder involving a synovial joint and/or tendon sheath in a subject are provided, comprising administering to the subject an effective amount of a compound or composition that modulates TREM-1/DAP-12 activity. In some embodiments, the proliferative disorder is selected from pigmented villonodular synovitis (PVNS), giant cell tumor of the tendon sheath (GCTTS), and tenosynovial giant cell tumor (TGCT) such as diffuse type tenosynovial gian cell tumor (dtTGCT). In some embodiments, the disorder is pigmented villonodular synovitis/diffuse type tenosynovial gian cell tumor (PVNS/dtTGCT).
In some embodiments, the PVNS tumor volume is reduced by at least 30% or at least 40% or at least 50% or at least 60% or at least 70% after administration of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses of the compound or composition that modulates TREM-1/DAP-12 activity. In some embodiments, the tumor volume is tumor volume in a single joint. In some embodiments, the single join is selected from a hip joint and a knee joint. In some embodiments, the tumor volume is total tumor volume in all joints affected by PVNS. In some embodiments, the subject experiences one or more than one of the following improvements in symptoms:
(a) a reduction in joint pain, (b) an increase range of motion in a joint, and (c) an increase in functional capacity of a joint, following at least one dose of the compound or composition.
In some embodiments, the compounds or compositions of the present invention are selected peptide variants and compositions (see, e.g., US 9,981,004; US
8,513,185; US
9,815,883; US 9,273,111; US 8,013,116) that modulate the TREM-1/DAP-12 signaling pathway.
In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein complexes and targets these complexes to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to the compounds and compositions of the invention.
In certain embodiments, the compounds and compositions of the present invention are incorporated into long half-life synthetic lipopeptide complexes (LPC). In certain embodiments, the compounds and compositions of the invention may incorporate into natural lipoprotein particles (LP) in vivo upon administration to the individual. See, e.g., US
20110256224 and (Sigalov 2014, Shen and Sigalov 2017, Shen et al. 2017, Rojas et al. 2018, Tornai et al. 2019).
In certain embodiments, the preferred TREM-1 modulatory compounds and compositions are TREM-1 inhibitory peptide sequences such e.g., as GF9 described in (described in (Sigalov 2014, Rojas et al. 2017, Shen and Sigalov 2017, Shen and Sigalov 2017) and disclosed in (US
8,513,185 and US 9,981,004) or LR12 and LP17 (described in Gibot, et al.
Infect Immun 2006, 74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J
Immunol 2007, 37:456-466; Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J
Clin Pharmacol 2018, in press; Zhou, et al. Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et .. al., US 8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US
9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136). In certain embodiments, the preferred TREM-1 modulatory compounds and compositions are antibodies that bind and block TREM-1 such e.g., as those disclosed in US 10,189,902. In some embodiments, combinations of different TREM-1 modulatory compounds and compositions of the invention is used.
In another aspect, the invention provides for a method of predicting the efficacy of TREM-1 targeted therapies in an individual with the proliferative disorder by:
(a) obtaining a biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample; (d) measuring the level of soluble form of the human TREM-1 receptor in the biological sample. See, e.g., US 8,021,836.
In some embodiments, prior to administering the first dose of the compound or composition that modulates the TREM-1/DAP-12 receptor complex signaling, the subject receives a first therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, and joint replacement. In some embodiments, the PVNS
recurred or progressed after the first therapy. In some embodiments, the compound or composition of the present invention is administered prior to a therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, and joint replacement. In some embodiments, the tumor is unresectable. In some embodiments, the subject has not received prior therapy with imatinib, nilotinib or a CSF1/CSF1R inhibitor, while in other embodiments the subject has received prior treatment with imatinib, nilotinib or a CSF1/CSF1R
inhibitor. In some embodiments, the subject has not received prior treatment with a CSF1/CSF1R
inhibitor, while in other embodiments the subject has received prior treatment with a CSF1/CSF1R inhibitor. In some embodiments, the compound or composition that modulates the TREM-1/DAP-12 receptor complex signaling is administered with imatinib, nilotinib, a CSF1/CSF1R
inhibitor, anti-programmed cell death protein 1 (anti-PD1) or anti-programmed cell death ligand 1 (PDL1) antibodies.
In one embodiment the compound or composition of the present invention is provided as a pharmaceutical composition for intravenous administration. In one embodiment, the compound or composition of the present invention is provided as a pharmaceutical composition for oral administration. In one embodiment, the compound is administered once a day. In one embodiment, the compound is administered twice a day. In one embodiment, the method includes administering to the patient one or more additional therapeutic compounds. In one embodiment, the one or more additional therapeutic compound is selected from one or more of a Btk tyrosine kinase inhibitor, an Erbb2 tyrosine kinase receptor inhibitor; an Erbb4 tyrosine kinase receptor inhibitor, an mTOR inhibitor, a thymidylate synthase inhibitor, an EGFR
tyrosine kinase receptor inhibitor, an epidermal growth factor antagonist, a Fyn tyrosine kinase inhibitor, a kit tyrosine kinase inhibitor, a Lyn tyrosine kinase inhibitor, a NK cell receptor modulator, a PDGF receptor antagonist, a PARP inhibitor, a poly ADP ribose polymerase inhibitor, a poly ADP ribose polymerase 1 inhibitor, a poly ADP ribose polymerase 2 inhibitor, a poly ADP ribose polymerase 3 inhibitor, a galactosyltransferase modulator, a dihydropyrimidine dehydrogenase inhibitor, an orotate phosphoribosyltransferase inhibitor, a telomerase modulator, a mucin 1 inhibitor, a mucin inhibitor, a secretin agonist, a TNF related apoptosis inducing ligand modulator, an IL-17 gene stimulator, an interleukin-17E ligand, a neurokinin receptor agonist, a cyclin G1 inhibitor, a checkpoint inhibitor, a PD-1 inhibitor, a PD-Li inhibitor, a CTLA4 inhibitor, a topoisomerase I inhibitor, an Alk-5 protein kinase inhibitor, a connective tissue growth factor ligand inhibitor, a notch-2 receptor antagonist, a notch-3 receptor antagonist, a hyaluronidase stimulator, a MEK-1 protein kinase inhibitor; MEK-2 protein kinase inhibitor, a GM-C SF receptor modulator; TNF alpha ligand modulator, a mesothelin modulator, an asparaginase stimulator, a caspase-3 stimulator; caspase-9 stimulator, a PKN3 gene inhibitor, a hedgehog protein inhibitor; smoothened receptor antagonist, an AKT1 gene inhibitor, a DHFR
inhibitor, a thymidine kinase stimulator, a CD29 modulator, a fibronectin modulator, an interleukin-2 ligand, a serine protease inhibitor, a D4OLG gene stimulator;
TNFSF9 gene stimulator, a 2-oxoglutarate dehydrogenase inhibitor, a TGF-beta type II
receptor antagonist, an Erbb3 tyrosine kinase receptor inhibitor, a cholecystokinin CCK2 receptor antagonist, a Wilms tumor protein modulator, a Ras GTPase modulator, an histone deacetylase inhibitor, a cyclin-dependent kinase 4 inhibitor A modulator, an estrogen receptor beta modulator, a 4-1BB
inhibitor, a 4-1BBL inhibitor, a PD-L2 inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a BTLA
inhibitor, a HVEM inhibitor, aTIM3 inhibitor, a GAL9 inhibitor, a LAG3 inhibitor, a VISTA
inhibitor, a KIR inhibitor, a 2B4 inhibitor, a CD160 inhibitor and a CD66e modulator. In one embodiment, the one or more additional therapeutic compounds is selected from one or more of bavituximab, IMM-101, CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483, TG-01, .. mocetinostat, LOAd-703, CPI-613, upamostat, CRS-207, NovaCaps, trametinib, Atu-027, sonidegib, GRASPA, trabedersen, nastorazepide, Vaccell, oregovomab, istiratumab, refametinib, regorafenib, lapatinib, selumetinib, rucaparib, pelareorep, tarextumab, PEGylated hyaluronidase, varlitinib, aglatimagene besadenovec, GB S-01, GI-4000, WF-10, galunisertib, afatinib, RX-0201, FG-3019, pertuzumab, DCVax-Direct, selinexor, glufosfamide, virulizin, yttrium (90Y) clivatuzumab tetraxetan, brivudine, nimotuzumab, algenpantucel-L, tegafur+gimeracil+oteracil potassium+calcium folinate, olaparib, ibrutinib, pirarubicin, Rh-Apo2L, tertomotide, tegafur+gimeracil+oteracil potassium, tegafur +gimeracil +oteracil potassium, masitinib, Rexin-G, mitomycin, erlotinib, adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons, platinum derivatives, taxane, paclitaxel, vinca alkaloids, vinblastine, anthracyclines, doxorubicin, epipodophyllotoxins, etoposide, cisplatin, rapamycin, methotrexate, actinomycin D, dolastatin 10, colchicine, emetine, trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents, chlorambucil, 5-fluorouracil, campthothecin, metronidazole, Gleevec, Avastin, Vectibix, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, BCG Live, bevacuzimab, fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, camptothecin, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin, dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, dromostanolone propionate, epirubicin, epoetin alfa, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, megestrol acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed di sodium, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, rociletinib, sargramostim, sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronate, zoledronic acid, pembrolizumab, nivolumab, D3I-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, FOLFIRINOX and KY-1003. In one embodiment, the one or more additional therapeutic compound is FOLFIRINOX. In one embodiment, the one or more additional therapeutic compounds are gemcitabine and paclitaxel.
In one embodiment, the one or more additional therapeutic compounds are gemcitabine and nab-paclitaxel.
In some embodiments, the invention provides diagnostic markers to prognose the response to TREM-1 therapy. In some embodiments, the invention provides prognostic markers to prognose the response to TREM-1 therapy. It is not meant to limit the markers to those described herein.
Accordingly, the invention provides for a method of treating cancer in an individual in need thereof by administering to the individual a therapeutically effective amount of at least one modulator which affects myeloid cells by action on the TREM-1/DAP-12 signaling pathway together with a therapeutically effective amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof. The subject of the present invention includes any human subject who has been diagnosed with, has symptoms of, or is at risk of developing a cancer or a pre- or post-cancerous condition.
The invention relates to personalized combination-therapy treatments for cancer that involve targeting specific cancers by their tumor environment. More specifically, the invention provides a method for treating various cancers by using modulators of the TREM-pathway together with other cancer therapies and the use of such combinations in the treatment of cancer. In certain embodiments, these modulators may possess the antitumor activity. In some embodiments, these modulators may not possess the antitumor activity. In one embodiment, these modulators include peptide variants and compositions that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation). In one embodiment these peptide variants and compositions modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on monocytes, macrophages and neutrophils. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on tumor-associated macrophages. In one embodiment, the invention provides a method for predicting the efficacy of standalone or combination-therapy treatment that involve TREM-1-targeting therapies in various cancers by analyzing biological samples from cancer patients for the presence of myeloid cells and for the expression levels of TREM-1, CSF-1, CSF-1R, IL-6 and other markers. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe. In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with cancer using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer (NSCLC), pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system (CNS) cancer, stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, pigmented villonodular synovitis (PVNS) and other cancers in which myeloid cells are involved or recruited and cancer cachexia.
In some embodiments, cancer is selected from the list including but not limited to lung cancer including NSCLC, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system (CNS) cancer, stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor (TGCT; also referred to in the art as TSGCT), PVNS and other cancers in which myeloid cells are involved or recruited and cancer cachexia.
In some embodiments, the modulators of the TREM-1/DAP-12 signaling pathway are capable of suppressing tumor growth in the subject. In another aspect, the modulators are capable of delaying the development of cancer in the subject. In another aspect, the modulators are capable of reducing tumor size in the subject. In another aspect, the modulators are capable of treating cancer in the subject. In another aspect, the modulators are capable of treating cancer in the subject. In another aspect, the modulators are capable of increasing survival of the subject.
In some embodiments, the modulators are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation). In some embodiments, the modulators comprise peptide variants and compositions that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation) together with a pharmaceutically acceptable excipient, carrier, diluent, salt or a combination thereof In some embodiments, the modulators comprise antibodies or fragments thereof that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation) together with a pharmaceutically acceptable excipient, carrier, diluent, salt or a combination thereof.
The methods of combination therapy featured in the present invention may result in a synergistic effect, wherein the effect of a combination of compounds or other therapeutic agents is greater than the sum of the effects resulting from administration of any of the compounds or other therapeutic agents as single agents. A synergistic effect may also be an effect that cannot be achieved by administration of any of the compounds or other therapeutic agents as single agents. The synergistic effect may include, but is not limited to, an effect of treating cancer by reducing tumor size, inhibiting tumor growth, or increasing survival of the subject. The synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or delaying cancer cell growth.
In another aspect, the invention provides for a method of predicting the efficacy of TREM-1 targeted therapies in an individual with cancer by: (a) obtaining a biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample.
In another aspect, the invention provides for a method of detecting TREM-1 expression levels in an individual with cancer by: (a) administering to the individual the peptide variants and composition of the present invention having an affinity for TREM-1 and an imaging probe in a detectably effective amount; (b) imaging at least a portion of the patient;
(c) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition.
In certain embodiments, the invention provides for a diagnostic method of detecting TREM-1 expression levels in an individual with cancer by: (a) administering to the individual the modulators of TREM-1 transmembrane signaling having an affinity for TREM-1 and an imaging probe in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related cancer condition and correlates with the expression levels and the higher the levels are, the better the patient is predicted to respond to a TREM-1 inhibitory therapy using the modulators of the TREM-1/DAP-12 signaling pathway as standalone therapy or in combinations with other anticancer treatments.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth, or a combination thereof In one embodiment, said method further comprises administering the amount of the TREM-1 inhibitor together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof.
In one embodiment, said method further comprises administering to said patient an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy or a combination thereof.
In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof.
In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof.. In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c.. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I
inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT).
In some embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib).. In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, 'NO-.. 1001, ONO-2231 and the like. In some embodiments, said radiation therapy is administered to said patient.. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof. In some embodiments, said imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99111, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, p32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR1 2 and/or LP1 7 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-1 5 12 signaling pathway and suppressing tumor growth, or a combination thereof In some embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof In some embodiments, said a variant TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine. In some embodiments, said variant TREM-1 inhibitory peptide sequence is conjugated to at least one unmodified or modified amphipathic peptide sequence. In some embodiments, said an unmodified or modified amphipathic peptide sequence is derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, .. C-III, and E, and any combination thereof. In some embodiments, said a modified amphipathic peptide sequence derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof contains at least one amino acid residue which is chemically or enzymatically modified. In some embodiments, said a chemically or enzymatically modified amino acid residue is oxidized, halogenated or nitrated. In some embodiments, said an oxidized amino acid residue is the methionine residue. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is valine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met(0)-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said B is conjugated to an additional peptide sequence to enhance the targeting efficacy. In some embodiments, said an additional peptide sequence comprises amino acid sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp is asparagine. In some embodiments, said A is conjugated to at least one additional therapeutic agent to enhance the therapeutic efficacy. In some embodiments, said an additional therapeutic agent is selected from the group of anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, therapeutic peptides, and any combination thereof In some embodiments, said anticancer therapeutic agent is selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof In some embodiments, said A and/or B are conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, 11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic lipopeptide nanoparticle, said method comprising: a) co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a predetermined amount of triglycerides and/or cholesteryl ester; b) drying the mixture of step (a) under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a predetermined amount of sodium cholate; and ii. a predetermined amount of the compound of claim 1; for a time period sufficient to allow the components to self-assemble into synthetic lipopeptide particles; d) removing sodium cholate from the mixture of step (c); and e) isolating particles that have a size of between about 5 to about 200 nm diameter. In some embodiments, said lipid is conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42 In", Fe=59/
TC99111, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a triacylglycerol. In some embodiments, said phospholipid is selected from the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof. In some embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an affinity for TREM-1 and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) the compositions of claims 1, 3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 5, 6, and 21-25 having an affinity for TCR and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, said T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g.
autoimmune pericarditis, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g.
myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In some embodiments, the invention provides a method of treating a T cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) the compositions of claims 1, 5, 6, and 23 capable of inhibiting TCR; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In some embodiments, the invention provides a method of reducing pain in a subject with pigmented villonodular synovitis (PVNS) tumor, comprising administering to the subject an amount of a TREM-1 modulator that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and capable of reducing pain in PVNS subjects independently of tumor response. In some embodiments, said PVNS tumor has a tumor volume. In some embodiments, said inhibition reduces said PVNS tumor volume by at least 30% after administration of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses of the modulator that inhibits the TREM-1/DAP-12 signaling pathway. In some embodiments, said tumor volume is tumor volume in a single joint.In some embodiments, said single joint is selected from a hip joint and a knee joint.In some embodiments, said tumor volume is total tumor volume in all joints affected by PVNS. In some embodiments, said modulator is an antibody. In some embodiments, prior to administering the first dose of said antibody, the subject received a prior therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, joint replacement and CSF1/CSF1R
inhibitor. In some embodiments, said PVNS recurred or progressed after the prior therapy. In some embodiments, said antibody is administered prior to a therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, and joint replacement, or wherein the subject has a tumor that is unresectable. In some embodiments, said subject has not received prior treatment with a CSF1R inhibitor. In one embodiment, said method further comprises administering the amount of the TREM-1 modulator together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof.In one embodiment, said method further comprises administering the amount of the TREM-1 modulator together with an amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, or a combination thereof. In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating .. agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, CSF1/CSF1R inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT). In some embodiments, said topoisomerase inhibitor is a topoisomerase II
inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib).
In some .. embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP
inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231 and the like. In some embodiments, said CSF1/CSF1R inhibitor is selected from the group consisting of CSF1R kinase inhibitor, an antibody that binds CSF1R and the like. In some embodiments, said CSF1R kinase inhibitor is imatinib or nilotinib. In some embodiments, said CSF1R kinase inhibitor is PLX3397. In some embodiments, said anti-CSF1R antibody blocks binding of CSF1 and/or IL-34 to CSF1R. In some embodiments, said anti-CSF1R antibody inhibits ligand-induced CSF1R
phosphorylation in vitro. In some embodiments, said antibody is a humanized antibody. In some embodiments, a radiation therapy is administered to said patient. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof.
In some embodiments, the invention provides a method of predicting the efficacy of TREM-1 targeted therapies in an individual with the proliferative disorder by:
(a) obtaining a .. biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample; (d) measuring the level of soluble form of the human TREM-1 receptor in the biological sample.
In some embodiments, the invention provides a method of diagnosing disease of the proliferative disorder in a subject, wherein said disease is PVNS or TGCT, which method comprises the steps of (a) measuring a level of the soluble form of the human TREM-1 receptor in a biological sample obtained from said subject; (b) comparing the measured level of the soluble form of the human TREM-1 receptor in the sample with a mean level in a control population of individuals not PVNS or TGCT; (c) correlating elevated levels of the soluble form of the human TREM-1 receptor with the presence or extent of said proliferative disease. In some embodiments, said step of measuring the level of the soluble form of the human receptor comprises the steps of: (a) contacting said biological sample with a compound capable of binding the soluble form of the human TREM-1 receptor; (b) detecting the level of the soluble form of the human TREM-1 receptor present in the sample by observing the level of binding between said compound and the soluble form of the human TREM-1 receptor.In one embodiment, said method further comprises comprising the steps of measuring the level of the soluble form of the human TREM-1 receptor in a second or further sample from said subject, the first and second or further samples being obtained at different times; and comparing the levels in the samples to indicate the progression or remission of the proliferative disease. In some embodiments, said sample is selected from the group consisting of whole blood, blood serum, blood, plasma, urine, bronchoalveolar lavage fluid and synovial liquid. In some embodiments, said sample is from synovial fluid. In some embodiments, said sample is from blood serum or blood plasma. In some embodiments, said sample is a human sample. In some embodiments, said compound specifically binds the soluble form of the human TREM-1 receptor. In some embodiments, said compound capable of binding the soluble form of the human receptor is an antibody raised against all or part of the TREM-1 receptor. In some embodiments, said level of soluble form of the human TREM-1 receptor is measured by an immunochemical technique. In one embodiment, said method further comprisesan additional step of measuring the level of TREM-1-Ligand in one or more biological samples obtained from said subject. In some embodiments, said imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In111, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth and an amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, or a combination thereof. In one embodiment, said method further comprises administering the amount of the TREM-1 inhibitor together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS
347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT). In some embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor selected from the group consisting of Amsacrine, .. Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib). In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231 and the like. In some embodiments, a radiation therapy is administered to said patient. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof. In some embodiments, said an imaging probe is selected from the .. group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, ml ii, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth and an amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, or a combination thereof. In one embodiment, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said a variant TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine. In some embodiments, said variant TREM-1 inhibitory peptide sequence is conjugated to at least one unmodified or modified amphipathic peptide sequence. In some embodiments, said an unmodified or modified amphipathic peptide sequence is derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof. In some embodiments, said a modified amphipathic peptide sequence derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof contains at least one amino acid residue which is chemically or enzymatically modified. In some embodiments, said a chemically or enzymatically modified amino acid residue is oxidized, halogenated or nitrated. In some embodiments, said an oxidized amino acid residue is the methionine residue. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-Glu-Leu-Tyr-Arg-Gin-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is valine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met(0)-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said B is conjugated to an additional peptide sequence to enhance the targeting efficacy. In some embodiments, said an additional peptide sequence comprises amino acid sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp is asparagine. In some embodiments, said A is conjugated to at least one additional therapeutic agent to enhance the therapeutic efficacy. In some embodiments, said an additional therapeutic agent is selected from the group of anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, therapeutic peptides, and any combination thereof In some embodiments, said anticancer therapeutic agent is selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof In some embodiments, said A and/or B are conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In111, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic lipopeptide nanoparticle, said method comprising: a) co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a predetermined amount of triglycerides and/or cholesteryl ester; b) drying the mixture of step (a) under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a predetermined amount of sodium cholate; and ii. a predetermined amount of the compound of claim 1; for a time period sufficient to allow the components to self-assemble into synthetic lipopeptide particles; d) removing sodium cholate from the mixture of step (c); and e) isolating particles that have a size of between about 5 to about 200 nm diameter. In some embodiments, said lipid is conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, ml ii, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a triacylglycerol. In some .. embodiments, said phospholipid is selected from the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof In some embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an affinity for TREM-1 and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) the compositions of claims 1, 3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 5, 6, and 21-25 having an affinity for TCR and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, the invention provides a method of treating a T cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) the compositions of claims 1, 5, 6, and 23 capable of inhibiting TCR; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In another aspect, the invention provides for a method of predicting response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in standalone or combination-therapy regimen by: (a) obtaining a biological sample from the subject; (b) determining the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of CD68-positive TAMs or a combination thereof, wherein the higher is the expression of CSF-1, CSF-1R, IL-6, TREM-1 or the higher is number of CD68-positive TAMs or a combination thereof, the better the patient is predicted to respond to a therapy that involves the modulators.
In some embodiments, the invention provides for a method of diagnosing cancer in which myeloid cells are involved or recruited in the subject and/or predicting response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in standalone or combination-therapy regimen by: (a) administering to said patient an amount of at least one modulator capable of binding TREM-1 that is conjugated to at least one imaging probe, or a combination thereof, in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location and amount of the labeled probe corresponds to at least one symptom of the myeloid cell-related cancer condition and the TREM-1 expression levels and the higher the expression level is, the better the patient is predicted to respond to a therapy that involves the modulators.
The invention relates to personalized medical treatments for scleroderma (systemic sclerosis, SSc). More specifically, the invention provides for treatment of scleroderma or a related autoimmune or a fibrotic condition by using modulators of the TREM-pathway standalone or together with other antifibrotic therapies and the use of such combinations in the treatment of scleroderma. In certain embodiments, these modulators may possess the antifibrotic activity. In some embodiments, these modulators may not possess the antifibrotic activity. In certain embodiments, these modulators may possess the anti-inflammatory activity.
In one embodiment, these modulators include peptide variants and compositions that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation). In one embodiment these peptide variants and compositions modulate the TREM-1-mediated immunological responses beneficial for the treatment of scleroderma or a related autoimmune or a fibrotic condition. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on monocytes, macrophages and neutrophils. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on SSc-associated macrophages. In one embodiment, the invention provides a method for predicting the efficacy of standalone or combination-therapy treatment that involve TREM-1-targeting therapies in scleroderma by analyzing biological samples from cancer patients for the presence of myeloid cells and for the expression levels of TREM-1, CSF-1, CSF-1R, IL-6 and other markers. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe.
In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with scleroderma using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof In one embodiment, the peptides and compositions of the invention are used in combinations with other antifibrotic therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST).
The invention provides for a method of treating scleroderma (S Sc) or a related autoimmune or a fibrotic condition in an individual in need thereof by administering to the individual an effective amount of an inhibitor of the TREM-1/DAP-12 pathway.
In one aspect, the inhibitors are selected from peptide variants and compositions that suppress tumor growth by modulating the TREM-1/DAP-12 signaling pathway. In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to the peptide variants and compositions of the invention. In one embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MM). In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as a positron emission tomography (PET) imaging systems (and combined PET/computer tomography (CT) and PET/MRI systems). In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other antifibrotic therapeutic agents. In certain embodiments, the peptide variants and compositions of the present invention are incorporated into long half-life synthetic lipopeptide particles (SLP). In certain embodiments, the peptide variants and compositions of the invention may incorporate into lipopeptide particles (LP) in vivo upon administration to the individual. In certain embodiments, the peptides and compositions of the invention can cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention provides for a method for suppressing tumor growth in an individual in need thereof by administering to the individual an amount of a TREM-1 inhibitor that is effective for suppressing inflammation and fibrosis.
Some aspects of the invention provide methods for treating scleroderma or related autoimmune or a fibrotic condition in a subject by administering a therapeutically effective amount of a TREM-1 inhibitor to the subject in need of such a treatment. In some embodiments, scleroderma is a systemic sclerosis, which is a systemic autoimmune disease or systemic connective tissue disease. SSc is often characterized by deposition of collagen in the skin. In some cases, SSc involves deposition of collagen in organs, such as the kidneys, heart, lungs and/or stomach.
In other embodiments, scleroderma is a diffuse scleroderma. Diffuse scleroderma typically affects the skin and organs such as the heart, lungs, gastrointestinal tract, and kidneys.
Still in other embodiments, scleroderma is a limited scleroderma that affects primarily the skin including, but not limited to, that of the face, neck and distal elbows and knees. Still in other embodiments, scleroderma is a limited scleroderma. In some instances, the limited scleroderma includes clinical conditions that affect the hands, arms, and face. In other instances, clinical conditions associated with the limited scleroderma include, calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyl), telangiectasias and pulmonary arterial hypertension. Yet in other instances, scleroderma is a localized scleroderma.
In another aspect, the invention provides for a method of predicting the efficacy of TREM-1 targeted therapies in an individual with scleroderma by: (a) obtaining a biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample.
In another aspect, the invention provides for a method of detecting TREM-1 expression levels in an individual with scleroderma by: (a) administering to the individual the peptide variants and composition of the present invention having an affinity for TREM-1 and an imaging probe in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition.
The present invention provides the compounds and compositions for TREM-1-targeted treatment of SSc and the methods for predicting the efficacy of these compositions. The invention further provides a method of using these compounds and compositions.
These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth, or a combination thereof In one embodiment, said method further comprises administering the amount of the TREM-1 inhibitor together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof.In one embodiment, said method further comprises administering to said patient an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy or a combination thereof In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I
inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT).
In some embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib). In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, and the like.In one embodiment, said method further comprises a radiation therapy administered to said patient. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99m, Cr51, Ga67, Ga68, CU64, Rb82,M099, DY165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xel", 1125, 1131, 1123, p32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the signaling pathway and suppressing tumor growth, or a combination thereof. In some embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof In some embodiments, said variant TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val .. is valine. In some embodiments, said variant TREM-1 inhibitory peptide sequence is conjugated to at least one unmodified or modified amphipathic peptide sequence. In some embodiments, said unmodified or modified amphipathic peptide sequence is derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof. In some embodiments, said modified amphipathic peptide sequence derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof contains at least one amino acid residue which is chemically or enzymatically modified. In some embodiments, said chemically or enzymatically modified amino acid residue is oxidized, halogenated or nitrated. In some embodiments, said oxidized amino acid residue is the methionine residue. In some embodiments, said unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is .. methionine, Arg is arginine, and Val is valine. In some embodiments, said unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is valine. In some embodiments, said modified amphipathic peptide sequence is derived from an apolipoprotein A-I
amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met(0)-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said B is conjugated to an additional peptide sequence to enhance the targeting efficacy. In some embodiments, said an additional peptide sequence comprises amino acid sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp is asparagine. In some embodiments, said A is conjugated to at least one additional therapeutic agent to enhance the therapeutic efficacy. In some embodiments, said an additional therapeutic agent is selected from the group of anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, therapeutic peptides, and any combination thereof In some embodiments, said anticancer therapeutic agent is selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof In some embodiments, said A and/or B are conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In Fe.59, TC99M, cr51 , Ga67, Ga68, cu64 , Rb82,m099, DY165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, Cll, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic lipopeptide nanoparticle, said method comprising: a) co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a predetermined amount of triglycerides and/or cholesteryl ester; b) drying the mixture of step (a) under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a predetermined amount of sodium cholate; and ii. a predetermined amount of the compound of claim 1; for a time period sufficient to allow the components to self-assemble into synthetic lipopeptide particles; d) removing sodium cholate from the mixture of step (c); and e) isolating particles that have a size of between about 5 to about 200 nm diameter. In some embodiments, said lipid is conjugated to at least one imaging probe. In some embodiments, said imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In Fe.59, TC99M, Cr51, Ga67, Ga68, CU64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a triacylglycerol. In some embodiments, said phospholipid is selected from the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof. In some embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an affinity for TREM-1 and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. In some embodiments, said myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) the compositions of claims 1, 3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 5, 6, and 21-25 having an affinity for TCR and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, said T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g.
autoimmune pericarditis, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g.
myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In some embodiments, the invention provides a method of treating a T cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) the compositions of claims 1, 5, 6, and 23 capable of inhibiting TCR; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
The details of one or more embodiments of the invention are set forth in the accompanying Figures (Drawings) and Detailed Description of The Invention, as described herein and below. Other features, objects, and advantages of the invention will be apparent from the summary, description, figures and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The following figures form part of the present specification and are included to further illustrate embodiments of the present invention. The invention may be better understood by reference to the figures in combination with the detailed description of the specific embodiments presented herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 presents an exemplary schematic representation of one embodiment of a trifunctional peptide of the present invention comprising amino acid domains A and B where amino acid domain A represents a therapeutic peptide sequence with or without an attached drug compound and/or imaging probe that functions to treat, prevent and/or detect a disease or condition, whereas amino acid domain B represents an amphipathic alpha helical peptide sequence, with or without an additional targeting peptide sequence, and functions to 1) assist in the self-assembly of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with lipids or lipid mixtures in vitro, for use in transporting these trifunctional peptides as lipoprotein nanoparticles to sites of interest in vitro or in vivo and/or 2) form long half-life lipopeptide/lipoprotein particles upon interaction with endogenous lipoproteins for transporting these trifunctional peptides to the sites of interest. Endogenous lipoproteins may be lipoproteins added to or found in cell cultures, or lipoproteins in a mammalian body.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP). GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) comprises amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22 amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising amino acid domains A and B. Depending on lipid mixture compositions added to the peptides, sub 50 nm-sized SLP particles of discoidal (TREM-1/TRIOPEP-dSLP) or spherical (TREM-sSLP) morphology are self-assembled upon binding of the trifunctional peptide to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 4A illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (shown for atherosclerosis), whereas domain B represents a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of atherosclerosis. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP
of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by intraplaque macrophages and return to the circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (shown for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the trifunctional peptide of the present invention comprising two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence, whereas domain B is a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with TREM-1 in the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and a merged image (FIG. 6C).
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic lipopeptide particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that contain an equimolar mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 (TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The post 4 h incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified methionine residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4 (white bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001 to 0.001 as compared with 4 h incubation time.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production by lipopolysaccharide (LPS)-stimulated macrophages incubated for 24 hour (hr) at 37 C with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged macrophages.
FIG. 9A-C presents the exemplary data showing that scavenger receptors SR-A
and SR-B1 mediate the macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and the SCHOOL
mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1, right panel).
(FIG. 9A2, left panel) Activation of the TREM-1/DAP12 receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm; GF9 self-penetrates the cell membrane and blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing phosphorylation and the downstream signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774 macrophages were cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-HDL, cells were treated for 1 hour at 37 C with 40 i.tM cytochalasin D and either (FIG.
9B) 400 pg/mL
fucoidan or (FIG. 9C) 10 i.tM BLT-1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars) or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence intensities of lysates were measured and normalized to the protein content.
Results are expressed as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of GF9-HDL and GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo-oligomerization.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post lipopolysaccharide (LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline (PBS), dexamethasone (DEX), control peptide and with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
.. 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
Control peptide represents an equimolar mixture of two peptides, each of them comprising two amino acid domains A and B where domain A represents a non-functional 9 amino acids-long sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5 is substituted with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as compared with PBS-treated animals.
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *,p < 0.05; **,p < 0.01, ****,p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP-bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar = 200 p.m.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference .. between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *,p <0.05, **, p <0.01;
***,p <0.001 as compared with vehicle-treated or naive animals.
FIG. 18A-D presents the exemplary data showing reduction of pathological retinal neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-1 (FIG. 18C) and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-induced .. retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP) particles of spherical morphology (TREM-1/TRIOPEP-sSLP). ***,p < 0.001 as compared with vehicle-treated animals.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical self-assembled particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-sSLP) or -- an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B a--- Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
-- staining, and the lipid content was analyzed by ImageJ (FIG. 21B). *
indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
WooP < 0.01;*"/###P <0001; ****P < 0 .0001. *** , 0.001 > P> 0.0001; ##, 0.01 > P> 0.001.
FIG. 22 presents an exemplary schematic representation of one embodiment of a related trifunctional peptide (TREM-1/TRIOPEP) G-HV21 of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic peptide sequence GF9 and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B represents a 12 amino acids-long amino acid sequence GV12 that contains a sulfoxidized methionine residue and is derived from human apolipoprotein A-I amino acid sequence. While not being bound to any particular theory, it is believed that a resulting amphipathic alpha helical peptide G-HV21 upon interaction with native lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles and targets these particles to myeloid cells (e.g. macrophages). Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; TRIOPEP, trifunctional peptide.
FIG. 23 presents an exemplary schematic representation of one embodiment of a related trifunctional peptide (TREM-1/TRIOPEP) G-KV21 of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic peptide sequence GF9 and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B represents a 12 amino acids-long amino acid sequence WV12 that contains a sulfoxidized methionine residue and is derived from human apolipoprotein A-I amino acid sequence. While not being bound to any particular theory, it is believed that a resulting amphipathic alpha helical peptide G-KV21 upon interaction with native lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles and targets these particles to myeloid cells (e.g. macrophages). Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; TRIOPEP, trifunctional peptide.
FIG. 24 presents an exemplary schematic representation of one embodiment of a related control peptide G-TE21 of the present invention comprising amino acid domains A and B
where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic peptide sequence GF9, whereas domain B represents a 12 amino acids-long amino acid sequence TE12 that contains a sulfoxidized methionine residue and is derived from bovine serum albumin amino acid sequence. While not being bound to any particular theory, it is believed that a resulting non-amphipathic peptide G-TE21 does not interact with native lipoproteins and therefore does not form naturally long half-life lipopeptide/lipoprotein particles.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 25 presents an exemplary schematic representation of one embodiment of a TCR-related trifunctional peptide (TCR/TRIOPEP) M-VE32 of the present invention comprising amino acid domains A and B where domain A represents a 10 amino acids-long human TCR
inhibitory therapeutic peptide sequence MF10 and functions to treat and/or prevent a TCR-related disease or condition, whereas domain B represents a 22 amino acids-long amino acid sequence PE22 that is derived from human apolipoprotein A-I amino acid sequence. While not being bound to any particular theory, it is believed that a resulting amphipathic alpha helical peptide M-VE32 upon -- interaction with native lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles. Abbreviations: TCR, T cell receptor; TRIOPEP, trifunctional peptide.
FIG. 26 presents a schematic representation of one embodiment of a TCR-related control peptide M-TK32 of the present invention comprising amino acid domains A and B where domain A
represents a 10 amino acids-long human TCR inhibitory therapeutic peptide sequence MF10, whereas domain B represents a random 22 amino acids-long amino acid sequence LK22. While not being bound to any particular theory, it is believed that a resulting non-amphipathic peptide M-TK32 does not interact with native lipoproteins and therefore does not form naturally long half-life lipopeptide/lipoprotein particles. Abbreviations: TCR, T cell receptor.
FIG. 27 presents an exemplary schematic representation and the exemplary data showing that ultracentrifugation of whole mouse serum with added rho B-labeled TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) G-HV21 and G-KV21 results in floatation of these peptides with mouse lipoproteins. In contrast, when added to whole mouse serum, rho B-labeled TREM inhibitory peptide GF9 or rho B-labeled TREM-1-related control peptide G-sedimentate with serum proteins upon ultracentrifugation. When added to delipoproteinized mouse serum that does not contain lipoproteins, rho B-labeled TREM-1/TRIOPEP G-HV21 and G-KV21 sedimentate with serum proteins upon ultracentrifugation. While not being bound to any particular theory, it is believed that TREM-1/TRIOPEP G-HV21 and G-KV21 interact with native lipoproteins of a whole mouse serum and/or their lipid components and form lipopeptide/lipoprotein particles that mimic serum lipoproteins and float under the same ultracentrifugation conditions. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; rho B, rhodamine B.
FIG. 28 presents exemplary data showing the endocytosis of rho B-labeled GF9, G-TE21, G-HV-21 and G-KV21 by macrophages in the absence (white bars) or presence (black bars) of HDL. In contrast to GF9 and TREM-1-related control peptide G-TE21, the in vitro macrophage uptake of TREM-1/TRIOPEP G-HV21 and G-KV21 significantly increases in the presence of HDL. ***,p < 0.001 (presence vs. absence of HDL). Abbreviations: HDL, high density lipoproteins; rho B, rhodamine B; n.s., not significant.
FIG. 29A-C shows exemplary images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1/TRIOPEP G-KV21 (pre-incubated with HDL) with TREM-1 in the J774 cell membrane FIG. 29A. FIG. 29B TREM-1 immunostaining. FIG. 29C merged image.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL, high density -- lipoproteins.
FIG. 30A illustrates a hypothesized molecular mechanism of action of TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) of the present invention (shown for atherosclerosis).
While not being bound to any particular theory, it is believed that upon interaction with native lipoproteins including HDL, the modified methionine residue in the TREM-1/TRIOPEP domain B mediates the recognition of the formed lipopeptide/lipoprotein particles by macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TREM-1/TRIOPEP
released within these cells. In contrast, native HDL particles are not recognized by intraplaque macrophages and return to the circulation.
FIG. 30B Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL, high-density lipoproteins.
FIG. 31A illustrates a hypothesized molecular mechanism of action of TREM-1-related -- trifunctional peptides (TREM-1/TRIOPEP) of the present invention (shown for cancer). While not being bound to any particular theory, it is believed that upon intreaction with native lipoproteins including HDL, the modified methionine residue in the TREM-1/TRIOPEP domain B mediates the recognition of the formed lipopeptide/lipoprotein particles by macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by -- macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TREM-1/TRIOPEP
released within these cells. In contrast, native HDL particles are not recognized by intraplaque macrophages and return to the circulation.
FIG. 31B Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL, -- high-density lipoproteins.
FIG. 32 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention delivered to and released within TREM-1-expressing cells by the -- lipopeptide/lipoprotein particles formed upon interaction of TREM-1/TRIOPEP
with native lipoproteins. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; DAP-12, DNAX-activation protein 12; M--- CSF/CSF-1, macrophage colony stimulating factor-1; MCP-1/CCL2, monocyte chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor.
FIG. 33 presents exemplary data showing cytokine production by LPS-stimulated macrophages incubated for 24 h at 37 C with GF9, G-TE21, G-HV21 and G-KV21 in the presence of HDL. In -- contrast to GF9 and TREM-1-related control peptide G-TE21, TREM-1/TRIOPEP G-HV21 and G-KV21 significantly inhibit the cytokine release in the presence of HDL. In the absence of HDL, G-HV21 does not affect the cytokine production. ***,p <0.001 (vs. medium + HDL).
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; HDL, high density lipoproteins.
FIG. 34A-C presents exemplary LPS-challenged J774 macrophages: Cytokine release data showing that scavenger receptors SR-A and SR-B1 mediate the macrophage endocytosis of TREM-1/TRIOPEP G-HV21 and G-KV21 in the presence of HDL. (FIG. 34A) Schematic representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1 blockade.
Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated macrophage endocytosis of the lipopeptide/lipoprotein particles formed upon interaction of TREM-1/TRIOPEP
with native lipoproteins (shown for HDL) results in the release of TREM-1/TRIOPEP into the cytoplasm.
Then, the released TREM-1/TRIOPEP self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 34A, right panel).
Macrophage endocytosis of G-HV21 and G-KV21 in the presence of HDL in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (B, C). J774 macrophages were cultured at 37 C overnight with medium. Before adding G-HV21 and G-KV21, cells were treated for 1 h at 37 C with 40 [tM cytochalasin D, 400 [tg/mL fucoidan (FIG. 34B) or 10 [tM BLT-1 (FIG. 34C) as indicated. Cells were then incubated for either 4 h or 22 h with medium containing HDL and 2 [tM rho B-labeled G-KV21 (gray bars) or G-HV21 (black bars), respectively.
Cells were lysed and rho B fluorescence intensities of lysates were measured and normalized to the protein content. Results are expressed as the mean SEM (n = 3). *,p < 0.05; **,p <
0.01; ****, p <
0.0001 versus uptake of G-HV21 and G-KV21 in the absence of inhibitor.
Abbreviations:
TREM-1, triggering receptor expressed on myeloid cells-1; DAP-12, DNAX-activation protein 12; M-CSF/CSF-1, macrophage colony stimulating factor; MCP-1/CCL2, monocyte chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor; HDL, high density lipoproteins; BLT-1, blocker of lipid transport-1; rho B, rhodamine B; SR, scavenger receptor.
FIG. 35 presents exemplary data showing serum cytokine production at 90 min post LPS
challenge in mice treated at 1 h before LPS challenge with PBS, DEX, GF9, TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to GF9 and G-TE21, G-HV21 and G-KV21 significantly inhibit the LPS-induced cytokine release. ***,p < 0.001 as compared with PBS-treated animals.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; DEX, dexamethasone; PBS, phosphate-buffer saline.
FIG. 36A-B presents the exemplary data showing survival of LPS-challenged mice treated with PBS (vehicle), TREM-1-related control peptide G-TE21, TREM-1-related trifunctional peptides G-HV21 and G-KV21 (FIG. 36A) or with TREM-1 inhibitory peptide GF9 at different doses (FIG. 36B). In contrast to G-TE21, G-HV21 and G-KV21 significantly improve survival of septic mice (FIG. 36A). When administered at a dose of 5 mg/kg, GF9 does not affect survival of septic mice, while at 25 mg/g, GF9 improves survival. In contrast, high dose of GF9, 150 mg/kg, contributes to earlier death as compared with control animals treated with vehicle only (FIG.
36B). **,p < 0.01 as compared with vehicle-treated animals. Abbreviations:
TREM-1, triggering receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; PBS, phosphate-buffer saline.
FIG. 37A-B presents the exemplary data showing tumor growth in the human non-small cell lung cancer H292 mouse xenograft (FIG. 37A) and A549 mouse xenograft (FIG.
37B) xenograft mice treated with PBS (vehicle), PTX, TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-HV21 and G-KV21 significantly inhibit the tumor growth. ****,p < 0.0001 as compared with vehicle-treated animals. Abbreviations: PTX, paclitaxel; PBS, phosphate-buffer saline.
FIG. 38 presents exemplary A549 mouse xenograft data showing average tumor weights in the A549 xenograft mice treated with PBS (vehicle), PTX, TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-HV21 and G-KV21 significantly decrease the tumor weight. **,p < 0.01 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; PTX, paclitaxel; PBS, phosphate-buffer saline; n.s., not significant.
FIG. 39A-B presents exemplary data showing tumor growth (A) and,infiltration of macrophages into the tumor as evaluated by F4/80 staining (B) in the human pancreatic cancer BxPC-3 xenograft mice treated with PBS (vehicle), TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-HV21 and G-KV21 in a BxPC-3 mouse xenograft significantly inhibits the tumor growth (FIG. 389) and reduce macrophage infiltration into the tumor (FIG. 39B). **,p < 0.01, ****,p < 0.0001 (versus vehicle). Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1;
PBS, phosphate-buffer saline; n.s., not significant.
FIG. 40A-B presents exemplary data showing PANC-1 mouse xenograft tumor growth (FIG.
40A) and survival (FIG. 40B) in the human pancreatic cancer PANC-1 xenograft mice treated with PBS (vehicle) and TREM-1-related trifunctional peptide G-KV21 with or without chemotherapy treatment (GEM+ABX). G-KV21 sensitizes the tumor to chemotherapy (FIG.
40A) and significantly improves survival (FIG. 40B). The median survival times (FIG. 40B) are indicated in parentheses. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; PBS, phosphate-buffer saline; GEM, gemcitabine; ABX, Abraxane (nanoparticle albumin-bound paclitaxel).
FIG. 41 presents the exemplary data showing average weights of Healthy C57BL/6 mice treated with TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. No toxicity was observed for all three peptides.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 42A-B presents exemplary data showing average clinical arthritis score (Collagen-induced arthritis: Score FIG. 42A) and Collagen-induced arthritis: Body weight change mean BW
changes (FIG. 42B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the CIA mice treated with PBS (vehicle), DEX, TREM-1-related control peptide G-TE21, TCR-related control peptide M-TK32, TCR-related trifunctional peptide M-VE32 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to the relevant control peptides, G-HV21, G-KV21 and M-VE32 all ameliorate the disease (FIG. 42A) and are well-tolerated by arthritic mice (FIG. 42B). *,p < 0.05, **,p < 0.01;
***,p < 0.001 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; CIA, collagen-induced arthritis; PBS, phosphate-buffer saline; DEX, dexamethasone; TCR, T cell receptor; BW, body weight.
FIG. 43A-D Oxygen-induced retinopathy presents exemplary data showing pathological RNV
(FIG. 43A) and avascular (FIG. 43B) areas as well as expression of TREM-1 (FIG. 43C) and M-CSF (FIG. 43D) in the retina of the mice with OIR treated with PBS (vehicle), TREM-1-related control peptide G-TE21 or TREM-1-related trifunctional peptide G-KV21. In contrast to G-TE21, G-KV21 significantly suppresses pathological RNV and inhibits tissue expression of TREM-1 and M-CSF. *,p < 0.05, **,p < 0.01; ***,p < 0.001 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; OIR, oxygen-induced retinopathy; PBS, phosphate-buffer saline; M-CSF, macrophage colony stimulating factor; RNV, retinal neovascularization.
FIG. 44 presents exemplary data showing penetration of the BBB and BRB by systemically (mice ¨ intraperitoneally; rats and rabbits ¨ intravenously) administered rhodamine B-labeled TREM-1-related trifunctional peptide G-KV21. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; BBB, blood-brain barrier; BRB, blood-retinal barrier.
FIG. 45A-E TREM-1 pathway inhibition. TREM-1 pathway inhibition suppresses the expression of (FIG. 45A) TREM-1 and inflammatory cytokines (FIG. 45B) MCP-1, (FIG. 45C) TNF-a, (FIG. 45D) IL-113, and (FIG. 45E) MIP-la but not (FIG. 45F) RANTES at the mRNA
level as measured in whole-liver lysates by real-time quantitative PCR. *
indicates significance level compared to nontreated PF group; # indicates significance level compared to nontreated alcohol-fed group; o indicates significance level compared to vehicle-treated alcohol-fed group.
Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P < 0.01; ***
/000 P < 0.001;
****P < 0.0001. Abbreviation: CCL, chemokine (C-C motif) ligand.
FIG. 46AE-G TREM-1 blockade and inflammatory cytokine levels. TREM-1 blockade reduces inflammatory cytokine levels in (FIG. 46A) serum and (FIG. 46B-D) whole-liver lysates as measured with specific ELISA kits. (FIG. 46E-G) Total liver protein was analyzed for total SYK
and activated p-SYK Y525/526 expression by western blotting using 13-actin as a loading control.
Statistical analysis was performed by evaluating two blots (n = 4/group).*
indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##
P < 0.01; ***P <
0.001; **** / P < 0.0001.
FIG. 47A-H Effects of TREM-1 inhibition. (FIG. 47A, FIG. 47B) TREM-1 inhibition suppresses the mRNA expression of macrophage cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47C, FIG. 47D) Both TREM-1 inhibitors attenuated F4/80 as shown by IHC. (FIG. 47E, FIG. 47F) TREM-1 inhibition suppresses the mRNA
expression of neutrophil cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47G, H) Both TREM-1 inhibitors attenuated MPO-positive cell infiltration as shown by IHC. * indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P <
0.05; ** /## P < 0.01;
.. ### P < 0.001; **** / P < 0.0001.
FIG. 48A-F Measurement of mRNA expression. mRNA expression of genes involved in (FIG.
48A, FIG. 48B) lipid synthesis (SERBF1, ACC1), (FIG. 48C) the lipid accumulation marker (ADRP), and (FIG. 48D-F) lipid oxidation (PPARa, CPT1a, MCAD) were measured in whole liver.
* indicates significance level compared to the nontreated PF group; #
indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P
<0.01; ### P <0.001; ****P <0.0001.
FIG. 49A presents a schematic representation of one embodiment of the proposed role of inhibition of TREM-1 expressed on tumor-associated macrophages (TAMs) in pancreatic cancer.
Pancreatic ductal adenocarcinoma cells, cancer-associated fibroblasts (CAFs) and TAMs play a role in generating a tumor favorable microenvironment, in part by producing such cytokines and growth factors as interleukin (IL)-1a, IL-6 and macrophage colony-stimulating factor (M-CSF).
FIG. 49B presents a schematic representation of one embodiment of suppressing tumor favorable microenvironment by inhibition of TREM-1 expressed on tumor-associated macrophages (TAMs) and reduction of cytokines and growth factors including but not limited to interleukin (IL)-6, IL-1, monocyte chemoattractant protein-1 (MCP-1; also referred to in the art as CCL2) and macrophage colony-stimulating factor 1 (CSF-1; also referred to in the art as M-CSF). These prognostic factors are involved in tumorigenesis, cancer progression, metastasis, and even in the response to cancer treatment. The figure further presents a schematic representation of one embodiment of modulating the TREM-1/DAP-12 signaling pathway by type I TREM-1 inhibitors that bind either TREM-1 (type Ia inhibitors; e.g., anti-TREM-1 blocking antibodiesõ etc.) or its ligand (type Ib inhibitors; e.g., inhibitory peptides LP17 and LR12 that act as a decoy TREM-1 receptor), thereby blocking binding between TREM-1 and its yet uncertain ligand(s).
FIG. 50 presents a schematic representation of one embodiment of TREM-1 modulatory peptide variants and compositions of the present invention that are rationally designed using the Signaling Chain HOmoOLigomerization (SCHOOL approach) to inhibit TREM-1 in a ligand-independent manner by blocking intramembrane interactions between TREM-1 and its signaling partner DAP-12 (type II inhibitors). These SCHOOL peptides can be employed in either free form or incorporated into macrophage-targeted (macrophage-specific) synthetic lipopeptide particles (SLP), which allows them to reach their site of action from either outside (Route 1) or inside the cell (Route 2).
FIG. 51A-F shows images of one embodiment depicting colocalization of the TREM-modulatory peptide GF9 (GFLSKSLVF) with trifunctional TREM-1 in the cell membrane. Fig.
51A shows exemplary peptide GF9. Fig. 51B and 51E shows exemplary TREM-1. Fig.
51C and F shows exemplary merged Images. Fig. 51A shows exemplary inhibitory peptide ((GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue with TREM-1 in the cell membrane.
FIG. 51B shows images of one embodiment depicting colocalization of the TREM-1 modulatory peptide GF9 (GFLSKSLVF) and trifunctional TREM-1 FIG. 52 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into the a carrier, e.g.
synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology suppresses tumor growth in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares), GF9-loaded discoidal SLP
(GF9-dSLP, light gray circles) or GF9-loaded spherical SLP (GF9-sSLP, white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 6 mice per group). On the final day of treatment, tumor volumes were compared between the drug-treated and control groups. **, p < 0.01; ***, p <0.001; ****, p < 0.0001 (versus vehicle).
FIG. 53 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into the a carrier, e.g.
synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology (GF9-dSLP and GF9-sSLP, respectively) suppresses tumor growth in experimental pancreatic cancer without affecting body weight (well-tolerable in long term-treated mice). As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares), GF9-dSLP (light gray circles) or GF9-sSLP (white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All results are expressed as the mean SEM (n = 6 mice per group).
FIG. 54 presents the exemplary data of one embodiment showing that treatment with synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology loaded with an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-dSLP and GA/E31-sSLP, respectively) suppresses tumor growth in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 6 mice per group). On the final day of treatment, tumor volumes were compared between the drug-treated and control groups. **,p < 0.01; ***,p <0.001; ****, p < 0.0001 (versus vehicle).
FIG. 55 presents the exemplary data of one embodiment showing that treatment with synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology loaded with an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-dSLP and GA/E31-sSLP, respectively) suppresses tumor growth in experimental pancreatic cancer without affecting body weight (i.e. well tolerable by long term-treated mice). As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All results are expressed as the mean SEM
(n = 6 mice per group).
FIG. 56 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology (GF9-dSLP and GF9-sSLP, respectively) prolongs survival in experimental pancreatic cancer.
As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 MM , mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray circles), GF9-dSLP
(light gray circles) or GF9-sSLP (white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively.
Kaplan-Meier survival curves are shown for AsPC-1-, BxPC-3- or Capan-l-bearing mice (n = 6 mice per group). **, p < 0.01; ***,p < 0.001 by log-rank test (versus vehicle).
FIG. 57 presents the exemplary data of one embodiment showing that treatment with synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology loaded with an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-dSLP and GA/E31-sSLP, respectively) prolongs survival in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated doses.
Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Kaplan-Meier survival curves are shown for AsPC-1-, BxPC-3-or Capan-l-bearing mice (n = 6 mice per group). **, p < 0.01; ***, p < 0.001 by log-rank test (versus vehicle).
FIG. 58 presents the exemplary data of one embodiment showing that the antitumor efficacy of TREM-1 blockade correlates with the intratumoral macrophage content in experimental pancreatic cancer. Antitumor efficacy is expressed as percent treatment/control (% T/C) values calculated using the following formula: % T/C = 100 x AT/AC where T and C are the mean tumor volumes of the drug-treated and control groups, respectively, on the final day of the treatment; AT is the mean tumor volume of the drug-treated group on the final day of the treatment minus mean tumor volume of the drug-treated group on initial day of dosing; and AC is the mean tumor volume of the control group on the final day of the treatment minus mean tumor volume of the control group on initial day of dosing. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies. Data are shown for the groups of AsPC-1-, BxPC-3- and Capan-1- bearing mice treated with free and SLP-bound TREM-1 modulatory peptides GF9 (GFLSKSLVF), GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
where M(0) is a methionine sulfoxide residue) and (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide residue) (GA/E31-sSLP) (n = 4 mice per group).
FIG. 59 presents the exemplary data of one embodiment showing that TREM-1 blockade suppresses intratumoral macrophage infiltration in experimental pancreatic cancer. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies.
Data are shown for the groups of BxPC-3-bearing mice treated with either vehicle (black bars), free GF9 (GFLSKSLVF, dark grey bars), GF9 incorporated into a carrier, e.gs. synthetic lipopeptide particle of spherical morphology (GF9-sSLP, light grey bars) and sSLP that contain an equimolar mixture of TREM-1 modulatory peptides (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA where M(0) is a methionine sulfoxide residue) and GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide residue) (GA/E31-sSLP, white bars) (n = 4 mice per group).
FIG. 60 presents the exemplary data of one embodiment showing the representative F4/80 images demonstrating that TREM-1 blockade suppresses intratumoral macrophage infiltration in experimental pancreatic cancer. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies. Data are shown for the groups of BxPC-3-bearing mice treated with either vehicle, free GF9 (GFLSKSLVF), GF9 incorporated into synthetic lipopeptide particle of spherical morphology (GF9-sSLP) and sSLP that contain an equimolar mixture of TREM-1 modulatory peptides GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
where M(0) is a methionine sulfoxide residue) and (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide residue) (GA/E31-sSLP) (n = 4 mice per group).
FIG. 61 presents the exemplary data of one embodiment showing that TREM-1 blockade suppresses serum proinflammatory cytokines in xenograft mouse models of pancreatic cancer.
Serum interleukin- 1 a (IL-1a), IL-6 and macrophage colony-stimulating factor (M-CSF/CSF-1) levels were analyzed on study days 1 and 8 in AsPC-1-, BxPC-3- and Capan-l-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares) or GF9-loaded spherical synthetic lipopeptide particles (GF9-sSLP, white circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *,p < 0.05;
**,p < 0.01; ***,p <0.001; ****,p < 0.0001 (versus vehicle).
FIG. 62 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor 1 (CSF1) and interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the AsPC-1 xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were analyzed on study days 1 and 8 in AsPC-1-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <0.05; **, p <0.01;
***, p < 0.001;
****, p < 0.0001 (versus vehicle).
FIG. 63 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor 1 (CSF1) and interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the BxPC-3 xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were analyzed on study days 1 and 8 in BxPC-3-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <0.05; **, p <0.01;
***, p < 0.001;
****, p < 0.0001 (versus vehicle).
FIG. 64 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor 1 (CSF1) and interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were analyzed on study days 1 and 8 in CAPAN-1-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <
0.05; **, p <
0.01; ***, p <0.001; ****, p <0.0001 (versus vehicle).
FIG. 65 presents the exemplary data of one embodiment showing that combining of Gemcitabine and Abraxane chemotherapy with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP) has a synergistic effect in experimental pancreatic cancer. As described herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered with either vehicle (black diamonds; once daily 5 times per week, 5qw), GF9-sSLP (black squares; once daily 5 times per week, 5qw), Gemcitabine and Abraxane (black circles; days 1, 4, 8, 11, 15) or GF9-sSLP (once daily 5 times per week, 5qw) in combination with Gemcitabine and Abraxane (days 1, 4, 8, 11, 15) (Black triangles). Treatment with GF9-sSLP persisted for 28 days. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 9 mice per group). On the final day of treatment, tumor volumes were compared between the Gemcitabine+Abraxane-treated and GF9-sSLP+Gemcitabine+Abraxane-treated groups. **, p < 0.01 (versus chemotherapy alone treated group).
FIG. 66 presents the exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical synthetic lipopeptide particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 modulatory peptide GF9 (GF9-sSLP) or an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1 modulatory peptides, i.e. 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue GA 31 and GE 31 (GA/E31-sSLP).
Fig. 67 presents the exemplary data of one embodiment showing that TREM-1 blockade with GF9, GF9 incorporated into the carrier - spherical synthetic lipopeptide particles (GF9-sSLP) or sSLP that carried an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-sSLP) significantly reduces tissue expression of colony-stimulating factor 1 (C SF-1) and TREM-1 in the retina of mice with oxygen-induced retinopathy (OIR) at postnatal day 17 (P17). Representative Western blots of retinal lysates from OIR mice are shown. The membrane was probed for TREM-1, reprobed for CSF-1 and then for 13-actin.
Values in the bar graphs represent the mean SEM, n=6. *, p < 0.05, **, p < 0.01 vs. vehicle-treated mice.
Fig. 68 presents the exemplary data of one embodiment showing that combining gemcitabine (GEM) and abraxane (ABX) chemotherapy with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into a carrier, e.g. synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP) has a synergistic therapeutic effect in experimental pancreatic cancer. As described herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered at indicated doses with either vehicle (black diamonds; once daily 5 times per week, 5qw), GF9-LPC (black circles-black squares; once daily 5 times per week, 5qw), GEM and ABX (black squares-(black circles; days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX (days 1, 4, 8, 11, 15) (half black half white hexagons-Black triangles). Treatment with GF9-LPC persisted for 28 days. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 9 mice per group). On the day 88, tumor volumes were compared between the GEM+ABX-treated and GF9-sSLP+GEM+ABX-treated groups. *, p <
0.05 (versus GEM+ABX-treated group), second set of symbols are used in the longer term studies.
Fig. 69 presents the exemplary data of one embodiment showing that TREM-1 blockade treatment with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into a , e.g.
synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP) alone, lipopeptide complex (GF9-LPC) alone or in combination with gemcitabine (GEM) and abraxane (ABX) chemotherapy is well tolerable in mice with human PANC-1 pancreatic cancer xenografts. As described herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered at indicated doses with either vehicle (black diamonds; once daily 5 times per week, 5qw), GF9-LPC (black circles; once daily 5 times per week, 5qw), GEM and ABX (black squares; days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX
(days 1, 4, 8, 11, 15) (half black half white hexagons). Treatment with GF9-LPC (GF9-sSLP) persisted for 28 days. Body weighs are plotted. All results are expressed as the mean SEM (n =
6 mice per group).
Fig. 70 presents the exemplary data of one embodiment showing that treatment with TREM-1 modulatory peptide GF9 incorporated into a carrie, e.g. synthetic lipopeptide complex (GF9-LPC) and particle (SLP) of spherical (sSLP) morphology (GF9-sSLP), synergistically prolongs survival rate in experimental pancreatic cancer (e.g. PANC-1) when combined with gemcitabine (GEM) and abraxane (ABX) chemotherapy. As described herein, after tumors in bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered at indicated doses with either vehicle (once daily 5 times per week, 5qw), GF9-LPC (once daily 5 times per week, 5qw), GEM and ABX (days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX
(days 1, 4, 8, 11, 15). Treatment with GF9-LPC persisted for 28 days. Kaplan-Meier survival curves are shown for PANC-1-bearing mice (n = 6 mice per group). *, p < 0.05 by log-rank test (versus GEM+AB X).
Fig. 71 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) is well tolerable in mice up to at least 300 mg/kg. As described herein, healthy C57BL/6 mice were intraperitoneally (i.p.) administered daily for 7 consecutive days with GF9 at indicated doses Mouse body weight (BW) was measured daily.
Results are expressed as the mean SEM (n = 4 mice per group).
Fig.72 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) suppresses tumor growth in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered .. once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated doses.
Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated and plotted.
All results are expressed as the mean SEM (n = 6 mice per group).
Fig.73 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) is well tolerable in mice with human pancreatic cancer xenografts. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All results are expressed as the mean SEM (n = 6 mice per group).
Fig.74 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) suppresses tumor growth as effectively as 20 mg/kg paclitaxel and is well tolerable in mice with human non-small cell lung cancer xenografts. As described herein, after tumors in A549-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), paclitaxel (black squares), GF9 (white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated doses. Treatment persisted for 21 days. Mean tumor volumes are calculated and plotted. Body weighs are plotted.
All results are expressed as the mean SEM (n = 6 mice per group).
Fig.75 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) suppresses intratumoral macrophage infiltration in experimental pancreatic cancer. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies. Data are shown for the groups of BxPC-3-bearing mice treated with either vehicle, GF9, GF9-LPC or GA/E31-LPC at indicated doses. Treatment persisted for 21 days. All results are expressed as the mean SEM (n = 4 mice per group).
Scale bar = 200 Elm.
Fig.76 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) ameliorates arthritis in mice with collagen-induced arthritis (CIA). As described herein, starting on day 24 after immunization, mice with CIA were intraperitoneally (i.p.) administered daily for 14 consecutive days with vehicle (black diamonds), dexamethasone (black squares), GF9 (white circles), GF9-LPC (black circles) and GA/E31-LPC
(half black half white circles) at indicated doses. Daily clinical scores were given on a scale of 0-5 for each of the paws on days 24-38. On day 38, mice were killed and the histopathological examination of mouse joints was performed. Histopathological scores of inflammation (I), pannus (P), cartilage damage (CD), bone resorption (BR) and periosteal new bone formation (PBF) are shown. Summed histopathology scores were calculated as the sum of all five histopathological parameters. All results are expressed as the mean SEM (n =
The present disclosure provides novel peptides and compounds, which are capable of executing three functions: 1) assistance in the self-assembly of naturally long half-life lipopeptide particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In one embodiment, said peptides and compounds of the present invention form synthetic lipopeptide particles upon binding to lipid or lipid mixtures.
In some embodiments, the invention provides a synthetic trifunctional polypeptide comprising at least one peptide domain of 3 to 35 amino acids in length having a C-terminal amino acid and at least one amphipathic domain of 6 to 45 to amino acids in length comprising an amphipathic lipopeptide having an N-terminal amino acid, wherein said first domain's C-terminal amino acid is attached to said second domain's N-terminal amino acid.
In one embodiment, said synthetic trifunctional polypeptide further comprises an imaging agent. In one embodiment, said synthetic trifunctional polypeptide further comprises a therapeutic agent. In one embodiment, said synthetic trifunctional polypeptide further comprises a targeting agent. In one embodiment, said synthetic trifunctional polypeptide further comprises a lipopeptide nanoparticle.
In some embodiments, the invention provides a population of spherical lipopeptide nanoparticles or discoidal lipopeptide nanoparticles comprising a plurality of synthetic trifunctional polypeptides, wherein said synthetic trifunctional polypeptide comprising at least one peptide domain of 3 to 35 amino acids in length having a C-terminal amino acid and at least one amphipathic domain of 6 to 45 to amino acids in length comprising an amphipathic lipopeptide having an N-terminal amino acid, wherein said first domain's C-terminal amino acid is attached to said second domain's N-terminal amino acid.
In some embodiments, the invention provides a method of treating an immune-related disease or condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which immune cells are involved or recruited, and ii) a synthetic trifunctional polypeptide comprising at least one peptide domain of 3 to 35 amino acids in length having a C-terminal amino acid and at least one amphipathic domain of 6 to 45 to amino acids in length comprising an amphipathic lipopeptide having an N-terminal amino acid, wherein said first domain's C-terminal amino acid is attached to said second domain's N-terminal amino acid, wherein said trifunctional polypeptide is capable of modulating immune receptors; b) administering said synthetic trifunctional polypeptide to said patient under conditions such that said at least one symptom is reduced.
The invention relates to personalized medical treatments for cancer that involve targeting specific cancers by their tumor environment. More specifically, the invention provides for treatment of various cancers by using inhibitors of the TREM-1/DAP-12 pathway.
These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. In addition, the invention provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on macrophages. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe. In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with cancer using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention provides for a method of treating cancer in an individual in need thereof by administering to the individual an effective amount of an inhibitor of the pathway. In one aspect, the inhibitors are selected from peptide variants and compositions that suppress tumor growth by modulating the TREM-1/DAP-12 signaling pathway. In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to the peptide variants and compositions of the invention. In one embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MRI). In one embodiment, the imaging agent is .. a [64Cu]-containing imaging probe for imaging systems such as a positron emission tomography (PET) imaging systems (and combined PET/computer tomography (CT) and PET/MM
systems).
In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In certain embodiments, the peptide .. variants and compositions of the present invention are incorporated into long half-life synthetic lipopeptide particles (SLP). In certain embodiments, the peptide variants and compositions of the invention may incorporate into lipopeptide particles (LP) in vivo upon administration to the individual. In certain embodiments, the peptides and compositions of the invention can cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention provides for a method for suppressing tumor growth in an individual in need thereof by administering to the individual an amount of a TREM-1 inhibitor that is effective for suppressing tumor growth.
In some embodiments, methods of treating a proliferative disorder involving a synovial joint and/or tendon sheath in a subject are provided, comprising administering to the subject an effective amount of a compound or composition that modulates TREM-1/DAP-12 activity. In some embodiments, the proliferative disorder is selected from pigmented villonodular synovitis (PVNS), giant cell tumor of the tendon sheath (GCTTS), and tenosynovial giant cell tumor (TGCT) such as diffuse type tenosynovial gian cell tumor (dtTGCT). In some embodiments, the disorder is pigmented villonodular synovitis/diffuse type tenosynovial gian cell tumor (PVNS/dtTGCT).
In some embodiments, the PVNS tumor volume is reduced by at least 30% or at least 40% or at least 50% or at least 60% or at least 70% after administration of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses of the compound or composition that modulates TREM-1/DAP-12 activity. In some embodiments, the tumor volume is tumor volume in a single joint. In some embodiments, the single join is selected from a hip joint and a knee joint. In some embodiments, the tumor volume is total tumor volume in all joints affected by PVNS. In some embodiments, the subject experiences one or more than one of the following improvements in symptoms:
(a) a reduction in joint pain, (b) an increase range of motion in a joint, and (c) an increase in functional capacity of a joint, following at least one dose of the compound or composition.
In some embodiments, the compounds or compositions of the present invention are selected peptide variants and compositions (see, e.g., US 9,981,004; US
8,513,185; US
9,815,883; US 9,273,111; US 8,013,116) that modulate the TREM-1/DAP-12 signaling pathway.
In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein complexes and targets these complexes to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to the compounds and compositions of the invention.
In certain embodiments, the compounds and compositions of the present invention are incorporated into long half-life synthetic lipopeptide complexes (LPC). In certain embodiments, the compounds and compositions of the invention may incorporate into natural lipoprotein particles (LP) in vivo upon administration to the individual. See, e.g., US
20110256224 and (Sigalov 2014, Shen and Sigalov 2017, Shen et al. 2017, Rojas et al. 2018, Tornai et al. 2019).
In certain embodiments, the preferred TREM-1 modulatory compounds and compositions are TREM-1 inhibitory peptide sequences such e.g., as GF9 described in (described in (Sigalov 2014, Rojas et al. 2017, Shen and Sigalov 2017, Shen and Sigalov 2017) and disclosed in (US
8,513,185 and US 9,981,004) or LR12 and LP17 (described in Gibot, et al.
Infect Immun 2006, 74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J
Immunol 2007, 37:456-466; Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J
Clin Pharmacol 2018, in press; Zhou, et al. Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et .. al., US 8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US
9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136). In certain embodiments, the preferred TREM-1 modulatory compounds and compositions are antibodies that bind and block TREM-1 such e.g., as those disclosed in US 10,189,902. In some embodiments, combinations of different TREM-1 modulatory compounds and compositions of the invention is used.
In another aspect, the invention provides for a method of predicting the efficacy of TREM-1 targeted therapies in an individual with the proliferative disorder by:
(a) obtaining a biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample; (d) measuring the level of soluble form of the human TREM-1 receptor in the biological sample. See, e.g., US 8,021,836.
In some embodiments, prior to administering the first dose of the compound or composition that modulates the TREM-1/DAP-12 receptor complex signaling, the subject receives a first therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, and joint replacement. In some embodiments, the PVNS
recurred or progressed after the first therapy. In some embodiments, the compound or composition of the present invention is administered prior to a therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, and joint replacement. In some embodiments, the tumor is unresectable. In some embodiments, the subject has not received prior therapy with imatinib, nilotinib or a CSF1/CSF1R inhibitor, while in other embodiments the subject has received prior treatment with imatinib, nilotinib or a CSF1/CSF1R
inhibitor. In some embodiments, the subject has not received prior treatment with a CSF1/CSF1R
inhibitor, while in other embodiments the subject has received prior treatment with a CSF1/CSF1R inhibitor. In some embodiments, the compound or composition that modulates the TREM-1/DAP-12 receptor complex signaling is administered with imatinib, nilotinib, a CSF1/CSF1R
inhibitor, anti-programmed cell death protein 1 (anti-PD1) or anti-programmed cell death ligand 1 (PDL1) antibodies.
In one embodiment the compound or composition of the present invention is provided as a pharmaceutical composition for intravenous administration. In one embodiment, the compound or composition of the present invention is provided as a pharmaceutical composition for oral administration. In one embodiment, the compound is administered once a day. In one embodiment, the compound is administered twice a day. In one embodiment, the method includes administering to the patient one or more additional therapeutic compounds. In one embodiment, the one or more additional therapeutic compound is selected from one or more of a Btk tyrosine kinase inhibitor, an Erbb2 tyrosine kinase receptor inhibitor; an Erbb4 tyrosine kinase receptor inhibitor, an mTOR inhibitor, a thymidylate synthase inhibitor, an EGFR
tyrosine kinase receptor inhibitor, an epidermal growth factor antagonist, a Fyn tyrosine kinase inhibitor, a kit tyrosine kinase inhibitor, a Lyn tyrosine kinase inhibitor, a NK cell receptor modulator, a PDGF receptor antagonist, a PARP inhibitor, a poly ADP ribose polymerase inhibitor, a poly ADP ribose polymerase 1 inhibitor, a poly ADP ribose polymerase 2 inhibitor, a poly ADP ribose polymerase 3 inhibitor, a galactosyltransferase modulator, a dihydropyrimidine dehydrogenase inhibitor, an orotate phosphoribosyltransferase inhibitor, a telomerase modulator, a mucin 1 inhibitor, a mucin inhibitor, a secretin agonist, a TNF related apoptosis inducing ligand modulator, an IL-17 gene stimulator, an interleukin-17E ligand, a neurokinin receptor agonist, a cyclin G1 inhibitor, a checkpoint inhibitor, a PD-1 inhibitor, a PD-Li inhibitor, a CTLA4 inhibitor, a topoisomerase I inhibitor, an Alk-5 protein kinase inhibitor, a connective tissue growth factor ligand inhibitor, a notch-2 receptor antagonist, a notch-3 receptor antagonist, a hyaluronidase stimulator, a MEK-1 protein kinase inhibitor; MEK-2 protein kinase inhibitor, a GM-C SF receptor modulator; TNF alpha ligand modulator, a mesothelin modulator, an asparaginase stimulator, a caspase-3 stimulator; caspase-9 stimulator, a PKN3 gene inhibitor, a hedgehog protein inhibitor; smoothened receptor antagonist, an AKT1 gene inhibitor, a DHFR
inhibitor, a thymidine kinase stimulator, a CD29 modulator, a fibronectin modulator, an interleukin-2 ligand, a serine protease inhibitor, a D4OLG gene stimulator;
TNFSF9 gene stimulator, a 2-oxoglutarate dehydrogenase inhibitor, a TGF-beta type II
receptor antagonist, an Erbb3 tyrosine kinase receptor inhibitor, a cholecystokinin CCK2 receptor antagonist, a Wilms tumor protein modulator, a Ras GTPase modulator, an histone deacetylase inhibitor, a cyclin-dependent kinase 4 inhibitor A modulator, an estrogen receptor beta modulator, a 4-1BB
inhibitor, a 4-1BBL inhibitor, a PD-L2 inhibitor, a B7-H3 inhibitor, a B7-H4 inhibitor, a BTLA
inhibitor, a HVEM inhibitor, aTIM3 inhibitor, a GAL9 inhibitor, a LAG3 inhibitor, a VISTA
inhibitor, a KIR inhibitor, a 2B4 inhibitor, a CD160 inhibitor and a CD66e modulator. In one embodiment, the one or more additional therapeutic compounds is selected from one or more of bavituximab, IMM-101, CAP1-6D, Rexin-G, genistein, CVac, MM-D37K, PCI-27483, TG-01, .. mocetinostat, LOAd-703, CPI-613, upamostat, CRS-207, NovaCaps, trametinib, Atu-027, sonidegib, GRASPA, trabedersen, nastorazepide, Vaccell, oregovomab, istiratumab, refametinib, regorafenib, lapatinib, selumetinib, rucaparib, pelareorep, tarextumab, PEGylated hyaluronidase, varlitinib, aglatimagene besadenovec, GB S-01, GI-4000, WF-10, galunisertib, afatinib, RX-0201, FG-3019, pertuzumab, DCVax-Direct, selinexor, glufosfamide, virulizin, yttrium (90Y) clivatuzumab tetraxetan, brivudine, nimotuzumab, algenpantucel-L, tegafur+gimeracil+oteracil potassium+calcium folinate, olaparib, ibrutinib, pirarubicin, Rh-Apo2L, tertomotide, tegafur+gimeracil+oteracil potassium, tegafur +gimeracil +oteracil potassium, masitinib, Rexin-G, mitomycin, erlotinib, adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons, platinum derivatives, taxane, paclitaxel, vinca alkaloids, vinblastine, anthracyclines, doxorubicin, epipodophyllotoxins, etoposide, cisplatin, rapamycin, methotrexate, actinomycin D, dolastatin 10, colchicine, emetine, trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents, chlorambucil, 5-fluorouracil, campthothecin, metronidazole, Gleevec, Avastin, Vectibix, abarelix, aldesleukin, alemtuzumab, alitretinoin, allopurinol, altretamine, amifostine, anastrozole, arsenic trioxide, asparaginase, azacitidine, AZD9291, BCG Live, bevacuzimab, fluorouracil, bexarotene, bleomycin, bortezomib, busulfan, calusterone, capecitabine, camptothecin, carboplatin, carmustine, celecoxib, cetuximab, chlorambucil, cladribine, clofarabine, cyclophosphamide, cytarabine, dactinomycin, darbepoetin alfa, daunorubicin, denileukin, dexrazoxane, docetaxel, doxorubicin (neutral), doxorubicin hydrochloride, dromostanolone propionate, epirubicin, epoetin alfa, estramustine, etoposide phosphate, etoposide, exemestane, filgrastim, floxuridine fludarabine, fulvestrant, gefitinib, gemcitabine, gemtuzumab, goserelin acetate, histrelin acetate, hydroxyurea, ibritumomab, idarubicin, ifosfamide, imatinib mesylate, interferon alfa-2a, interferon alfa-2b, irinotecan, lenalidomide, letrozole, leucovorin, leuprolide acetate, levamisole, lomustine, megestrol acetate, melphalan, mercaptopurine, 6-MP, mesna, methotrexate, methoxsalen, mitomycin C, mitotane, mitoxantrone, nandrolone, nelarabine, nofetumomab, oprelvekin, oxaliplatin, paclitaxel, palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim, pemetrexed di sodium, pentostatin, pipobroman, plicamycin, porfimer sodium, procarbazine, quinacrine, rasburicase, rituximab, rociletinib, sargramostim, sorafenib, streptozocin, sunitinib maleate, talc, tamoxifen, temozolomide, teniposide, VM-26, testolactone, thioguanine, 6-TG, thiotepa, topotecan, toremifene, tositumomab, trastuzumab, tretinoin, ATRA, uracil mustard, valrubicin, vinblastine, vincristine, vinorelbine, zoledronate, zoledronic acid, pembrolizumab, nivolumab, D3I-308, mDX-400, BGB-108, MEDI-0680, SHR-1210, PF-06801591, PDR-001, GB-226, STI-1110, durvalumab, atezolizumab, avelumab, BMS-936559, ALN-PDL, TSR-042, KD-033, CA-170, STI-1014, FOLFIRINOX and KY-1003. In one embodiment, the one or more additional therapeutic compound is FOLFIRINOX. In one embodiment, the one or more additional therapeutic compounds are gemcitabine and paclitaxel.
In one embodiment, the one or more additional therapeutic compounds are gemcitabine and nab-paclitaxel.
In some embodiments, the invention provides diagnostic markers to prognose the response to TREM-1 therapy. In some embodiments, the invention provides prognostic markers to prognose the response to TREM-1 therapy. It is not meant to limit the markers to those described herein.
Accordingly, the invention provides for a method of treating cancer in an individual in need thereof by administering to the individual a therapeutically effective amount of at least one modulator which affects myeloid cells by action on the TREM-1/DAP-12 signaling pathway together with a therapeutically effective amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof. The subject of the present invention includes any human subject who has been diagnosed with, has symptoms of, or is at risk of developing a cancer or a pre- or post-cancerous condition.
The invention relates to personalized combination-therapy treatments for cancer that involve targeting specific cancers by their tumor environment. More specifically, the invention provides a method for treating various cancers by using modulators of the TREM-pathway together with other cancer therapies and the use of such combinations in the treatment of cancer. In certain embodiments, these modulators may possess the antitumor activity. In some embodiments, these modulators may not possess the antitumor activity. In one embodiment, these modulators include peptide variants and compositions that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation). In one embodiment these peptide variants and compositions modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on monocytes, macrophages and neutrophils. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on tumor-associated macrophages. In one embodiment, the invention provides a method for predicting the efficacy of standalone or combination-therapy treatment that involve TREM-1-targeting therapies in various cancers by analyzing biological samples from cancer patients for the presence of myeloid cells and for the expression levels of TREM-1, CSF-1, CSF-1R, IL-6 and other markers. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe. In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with cancer using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer (NSCLC), pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system (CNS) cancer, stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, pigmented villonodular synovitis (PVNS) and other cancers in which myeloid cells are involved or recruited and cancer cachexia.
In some embodiments, cancer is selected from the list including but not limited to lung cancer including NSCLC, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system (CNS) cancer, stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor (TGCT; also referred to in the art as TSGCT), PVNS and other cancers in which myeloid cells are involved or recruited and cancer cachexia.
In some embodiments, the modulators of the TREM-1/DAP-12 signaling pathway are capable of suppressing tumor growth in the subject. In another aspect, the modulators are capable of delaying the development of cancer in the subject. In another aspect, the modulators are capable of reducing tumor size in the subject. In another aspect, the modulators are capable of treating cancer in the subject. In another aspect, the modulators are capable of treating cancer in the subject. In another aspect, the modulators are capable of increasing survival of the subject.
In some embodiments, the modulators are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation). In some embodiments, the modulators comprise peptide variants and compositions that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation) together with a pharmaceutically acceptable excipient, carrier, diluent, salt or a combination thereof In some embodiments, the modulators comprise antibodies or fragments thereof that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation) together with a pharmaceutically acceptable excipient, carrier, diluent, salt or a combination thereof.
The methods of combination therapy featured in the present invention may result in a synergistic effect, wherein the effect of a combination of compounds or other therapeutic agents is greater than the sum of the effects resulting from administration of any of the compounds or other therapeutic agents as single agents. A synergistic effect may also be an effect that cannot be achieved by administration of any of the compounds or other therapeutic agents as single agents. The synergistic effect may include, but is not limited to, an effect of treating cancer by reducing tumor size, inhibiting tumor growth, or increasing survival of the subject. The synergistic effect may also include reducing cancer cell viability, inducing cancer cell death, and inhibiting or delaying cancer cell growth.
In another aspect, the invention provides for a method of predicting the efficacy of TREM-1 targeted therapies in an individual with cancer by: (a) obtaining a biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample.
In another aspect, the invention provides for a method of detecting TREM-1 expression levels in an individual with cancer by: (a) administering to the individual the peptide variants and composition of the present invention having an affinity for TREM-1 and an imaging probe in a detectably effective amount; (b) imaging at least a portion of the patient;
(c) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition.
In certain embodiments, the invention provides for a diagnostic method of detecting TREM-1 expression levels in an individual with cancer by: (a) administering to the individual the modulators of TREM-1 transmembrane signaling having an affinity for TREM-1 and an imaging probe in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related cancer condition and correlates with the expression levels and the higher the levels are, the better the patient is predicted to respond to a TREM-1 inhibitory therapy using the modulators of the TREM-1/DAP-12 signaling pathway as standalone therapy or in combinations with other anticancer treatments.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth, or a combination thereof In one embodiment, said method further comprises administering the amount of the TREM-1 inhibitor together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof.
In one embodiment, said method further comprises administering to said patient an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy or a combination thereof.
In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof.
In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof.. In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c.. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I
inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT).
In some embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib).. In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, 'NO-.. 1001, ONO-2231 and the like. In some embodiments, said radiation therapy is administered to said patient.. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof. In some embodiments, said imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99111, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, p32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR1 2 and/or LP1 7 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-1 5 12 signaling pathway and suppressing tumor growth, or a combination thereof In some embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof In some embodiments, said a variant TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine. In some embodiments, said variant TREM-1 inhibitory peptide sequence is conjugated to at least one unmodified or modified amphipathic peptide sequence. In some embodiments, said an unmodified or modified amphipathic peptide sequence is derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, .. C-III, and E, and any combination thereof. In some embodiments, said a modified amphipathic peptide sequence derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof contains at least one amino acid residue which is chemically or enzymatically modified. In some embodiments, said a chemically or enzymatically modified amino acid residue is oxidized, halogenated or nitrated. In some embodiments, said an oxidized amino acid residue is the methionine residue. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is valine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met(0)-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said B is conjugated to an additional peptide sequence to enhance the targeting efficacy. In some embodiments, said an additional peptide sequence comprises amino acid sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp is asparagine. In some embodiments, said A is conjugated to at least one additional therapeutic agent to enhance the therapeutic efficacy. In some embodiments, said an additional therapeutic agent is selected from the group of anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, therapeutic peptides, and any combination thereof In some embodiments, said anticancer therapeutic agent is selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof In some embodiments, said A and/or B are conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, 11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic lipopeptide nanoparticle, said method comprising: a) co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a predetermined amount of triglycerides and/or cholesteryl ester; b) drying the mixture of step (a) under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a predetermined amount of sodium cholate; and ii. a predetermined amount of the compound of claim 1; for a time period sufficient to allow the components to self-assemble into synthetic lipopeptide particles; d) removing sodium cholate from the mixture of step (c); and e) isolating particles that have a size of between about 5 to about 200 nm diameter. In some embodiments, said lipid is conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42 In", Fe=59/
TC99111, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a triacylglycerol. In some embodiments, said phospholipid is selected from the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof. In some embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an affinity for TREM-1 and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) the compositions of claims 1, 3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 5, 6, and 21-25 having an affinity for TCR and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, said T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g.
autoimmune pericarditis, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g.
myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In some embodiments, the invention provides a method of treating a T cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) the compositions of claims 1, 5, 6, and 23 capable of inhibiting TCR; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In some embodiments, the invention provides a method of reducing pain in a subject with pigmented villonodular synovitis (PVNS) tumor, comprising administering to the subject an amount of a TREM-1 modulator that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and capable of reducing pain in PVNS subjects independently of tumor response. In some embodiments, said PVNS tumor has a tumor volume. In some embodiments, said inhibition reduces said PVNS tumor volume by at least 30% after administration of at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten doses of the modulator that inhibits the TREM-1/DAP-12 signaling pathway. In some embodiments, said tumor volume is tumor volume in a single joint.In some embodiments, said single joint is selected from a hip joint and a knee joint.In some embodiments, said tumor volume is total tumor volume in all joints affected by PVNS. In some embodiments, said modulator is an antibody. In some embodiments, prior to administering the first dose of said antibody, the subject received a prior therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, joint replacement and CSF1/CSF1R
inhibitor. In some embodiments, said PVNS recurred or progressed after the prior therapy. In some embodiments, said antibody is administered prior to a therapy selected from surgical synovectomy, radiation beam therapy, radio isotope synovectomy, and joint replacement, or wherein the subject has a tumor that is unresectable. In some embodiments, said subject has not received prior treatment with a CSF1R inhibitor. In one embodiment, said method further comprises administering the amount of the TREM-1 modulator together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof.In one embodiment, said method further comprises administering the amount of the TREM-1 modulator together with an amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, or a combination thereof. In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating .. agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, CSF1/CSF1R inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT). In some embodiments, said topoisomerase inhibitor is a topoisomerase II
inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib).
In some .. embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP
inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231 and the like. In some embodiments, said CSF1/CSF1R inhibitor is selected from the group consisting of CSF1R kinase inhibitor, an antibody that binds CSF1R and the like. In some embodiments, said CSF1R kinase inhibitor is imatinib or nilotinib. In some embodiments, said CSF1R kinase inhibitor is PLX3397. In some embodiments, said anti-CSF1R antibody blocks binding of CSF1 and/or IL-34 to CSF1R. In some embodiments, said anti-CSF1R antibody inhibits ligand-induced CSF1R
phosphorylation in vitro. In some embodiments, said antibody is a humanized antibody. In some embodiments, a radiation therapy is administered to said patient. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof.
In some embodiments, the invention provides a method of predicting the efficacy of TREM-1 targeted therapies in an individual with the proliferative disorder by:
(a) obtaining a .. biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample; (d) measuring the level of soluble form of the human TREM-1 receptor in the biological sample.
In some embodiments, the invention provides a method of diagnosing disease of the proliferative disorder in a subject, wherein said disease is PVNS or TGCT, which method comprises the steps of (a) measuring a level of the soluble form of the human TREM-1 receptor in a biological sample obtained from said subject; (b) comparing the measured level of the soluble form of the human TREM-1 receptor in the sample with a mean level in a control population of individuals not PVNS or TGCT; (c) correlating elevated levels of the soluble form of the human TREM-1 receptor with the presence or extent of said proliferative disease. In some embodiments, said step of measuring the level of the soluble form of the human receptor comprises the steps of: (a) contacting said biological sample with a compound capable of binding the soluble form of the human TREM-1 receptor; (b) detecting the level of the soluble form of the human TREM-1 receptor present in the sample by observing the level of binding between said compound and the soluble form of the human TREM-1 receptor.In one embodiment, said method further comprises comprising the steps of measuring the level of the soluble form of the human TREM-1 receptor in a second or further sample from said subject, the first and second or further samples being obtained at different times; and comparing the levels in the samples to indicate the progression or remission of the proliferative disease. In some embodiments, said sample is selected from the group consisting of whole blood, blood serum, blood, plasma, urine, bronchoalveolar lavage fluid and synovial liquid. In some embodiments, said sample is from synovial fluid. In some embodiments, said sample is from blood serum or blood plasma. In some embodiments, said sample is a human sample. In some embodiments, said compound specifically binds the soluble form of the human TREM-1 receptor. In some embodiments, said compound capable of binding the soluble form of the human receptor is an antibody raised against all or part of the TREM-1 receptor. In some embodiments, said level of soluble form of the human TREM-1 receptor is measured by an immunochemical technique. In one embodiment, said method further comprisesan additional step of measuring the level of TREM-1-Ligand in one or more biological samples obtained from said subject. In some embodiments, said imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In111, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth and an amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, or a combination thereof. In one embodiment, said method further comprises administering the amount of the TREM-1 inhibitor together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS
347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT). In some embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor selected from the group consisting of Amsacrine, .. Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib). In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, ONO-2231 and the like. In some embodiments, a radiation therapy is administered to said patient. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof. In some embodiments, said an imaging probe is selected from the .. group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, ml ii, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth and an amount of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, or a combination thereof. In one embodiment, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said a variant TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val is valine. In some embodiments, said variant TREM-1 inhibitory peptide sequence is conjugated to at least one unmodified or modified amphipathic peptide sequence. In some embodiments, said an unmodified or modified amphipathic peptide sequence is derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof. In some embodiments, said a modified amphipathic peptide sequence derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof contains at least one amino acid residue which is chemically or enzymatically modified. In some embodiments, said a chemically or enzymatically modified amino acid residue is oxidized, halogenated or nitrated. In some embodiments, said an oxidized amino acid residue is the methionine residue. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is methionine, Arg is arginine, and Val is valine. In some embodiments, said an unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-Glu-Leu-Tyr-Arg-Gin-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gin is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is valine. In some embodiments, said a modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met(0)-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said B is conjugated to an additional peptide sequence to enhance the targeting efficacy. In some embodiments, said an additional peptide sequence comprises amino acid sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp is asparagine. In some embodiments, said A is conjugated to at least one additional therapeutic agent to enhance the therapeutic efficacy. In some embodiments, said an additional therapeutic agent is selected from the group of anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, therapeutic peptides, and any combination thereof In some embodiments, said anticancer therapeutic agent is selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof In some embodiments, said A and/or B are conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In111, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic lipopeptide nanoparticle, said method comprising: a) co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a predetermined amount of triglycerides and/or cholesteryl ester; b) drying the mixture of step (a) under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a predetermined amount of sodium cholate; and ii. a predetermined amount of the compound of claim 1; for a time period sufficient to allow the components to self-assemble into synthetic lipopeptide particles; d) removing sodium cholate from the mixture of step (c); and e) isolating particles that have a size of between about 5 to about 200 nm diameter. In some embodiments, said lipid is conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, ml ii, Fe.59, Tc99m, Cr51, Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a triacylglycerol. In some .. embodiments, said phospholipid is selected from the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof In some embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an affinity for TREM-1 and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) the compositions of claims 1, 3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 5, 6, and 21-25 having an affinity for TCR and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, the invention provides a method of treating a T cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) the compositions of claims 1, 5, 6, and 23 capable of inhibiting TCR; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said a T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In another aspect, the invention provides for a method of predicting response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in standalone or combination-therapy regimen by: (a) obtaining a biological sample from the subject; (b) determining the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of CD68-positive TAMs or a combination thereof, wherein the higher is the expression of CSF-1, CSF-1R, IL-6, TREM-1 or the higher is number of CD68-positive TAMs or a combination thereof, the better the patient is predicted to respond to a therapy that involves the modulators.
In some embodiments, the invention provides for a method of diagnosing cancer in which myeloid cells are involved or recruited in the subject and/or predicting response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in standalone or combination-therapy regimen by: (a) administering to said patient an amount of at least one modulator capable of binding TREM-1 that is conjugated to at least one imaging probe, or a combination thereof, in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location and amount of the labeled probe corresponds to at least one symptom of the myeloid cell-related cancer condition and the TREM-1 expression levels and the higher the expression level is, the better the patient is predicted to respond to a therapy that involves the modulators.
The invention relates to personalized medical treatments for scleroderma (systemic sclerosis, SSc). More specifically, the invention provides for treatment of scleroderma or a related autoimmune or a fibrotic condition by using modulators of the TREM-pathway standalone or together with other antifibrotic therapies and the use of such combinations in the treatment of scleroderma. In certain embodiments, these modulators may possess the antifibrotic activity. In some embodiments, these modulators may not possess the antifibrotic activity. In certain embodiments, these modulators may possess the anti-inflammatory activity.
In one embodiment, these modulators include peptide variants and compositions that are capable of binding TREM-1 and reducing or blocking TREM-1 activity (signaling and/or activation). In one embodiment these peptide variants and compositions modulate the TREM-1-mediated immunological responses beneficial for the treatment of scleroderma or a related autoimmune or a fibrotic condition. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on monocytes, macrophages and neutrophils. In one embodiment, the peptides and compositions of the present invention modulate TREM-1/DAP-12 receptor complex expressed on SSc-associated macrophages. In one embodiment, the invention provides a method for predicting the efficacy of standalone or combination-therapy treatment that involve TREM-1-targeting therapies in scleroderma by analyzing biological samples from cancer patients for the presence of myeloid cells and for the expression levels of TREM-1, CSF-1, CSF-1R, IL-6 and other markers. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe.
In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with scleroderma using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof In one embodiment, the peptides and compositions of the invention are used in combinations with other antifibrotic therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST).
The invention provides for a method of treating scleroderma (S Sc) or a related autoimmune or a fibrotic condition in an individual in need thereof by administering to the individual an effective amount of an inhibitor of the TREM-1/DAP-12 pathway.
In one aspect, the inhibitors are selected from peptide variants and compositions that suppress tumor growth by modulating the TREM-1/DAP-12 signaling pathway. In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to the peptide variants and compositions of the invention. In one embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MM). In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as a positron emission tomography (PET) imaging systems (and combined PET/computer tomography (CT) and PET/MRI systems). In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other antifibrotic therapeutic agents. In certain embodiments, the peptide variants and compositions of the present invention are incorporated into long half-life synthetic lipopeptide particles (SLP). In certain embodiments, the peptide variants and compositions of the invention may incorporate into lipopeptide particles (LP) in vivo upon administration to the individual. In certain embodiments, the peptides and compositions of the invention can cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention provides for a method for suppressing tumor growth in an individual in need thereof by administering to the individual an amount of a TREM-1 inhibitor that is effective for suppressing inflammation and fibrosis.
Some aspects of the invention provide methods for treating scleroderma or related autoimmune or a fibrotic condition in a subject by administering a therapeutically effective amount of a TREM-1 inhibitor to the subject in need of such a treatment. In some embodiments, scleroderma is a systemic sclerosis, which is a systemic autoimmune disease or systemic connective tissue disease. SSc is often characterized by deposition of collagen in the skin. In some cases, SSc involves deposition of collagen in organs, such as the kidneys, heart, lungs and/or stomach.
In other embodiments, scleroderma is a diffuse scleroderma. Diffuse scleroderma typically affects the skin and organs such as the heart, lungs, gastrointestinal tract, and kidneys.
Still in other embodiments, scleroderma is a limited scleroderma that affects primarily the skin including, but not limited to, that of the face, neck and distal elbows and knees. Still in other embodiments, scleroderma is a limited scleroderma. In some instances, the limited scleroderma includes clinical conditions that affect the hands, arms, and face. In other instances, clinical conditions associated with the limited scleroderma include, calcinosis, Raynaud's phenomenon, esophageal dysfunction, sclerodactyl), telangiectasias and pulmonary arterial hypertension. Yet in other instances, scleroderma is a localized scleroderma.
In another aspect, the invention provides for a method of predicting the efficacy of TREM-1 targeted therapies in an individual with scleroderma by: (a) obtaining a biological sample from the individual; (b) determining the number of myeloid cells in the biological sample; (c) determining the expression levels of TREM-1 in the cells contained within the biological sample.
In another aspect, the invention provides for a method of detecting TREM-1 expression levels in an individual with scleroderma by: (a) administering to the individual the peptide variants and composition of the present invention having an affinity for TREM-1 and an imaging probe in a detectably effective amount; (b) imaging at least a portion of the patient; (c) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition.
The present invention provides the compounds and compositions for TREM-1-targeted treatment of SSc and the methods for predicting the efficacy of these compositions. The invention further provides a method of using these compounds and compositions.
These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the TREM-1/DAP-12 signaling pathway and suppressing tumor growth, or a combination thereof In one embodiment, said method further comprises administering the amount of the TREM-1 inhibitor together with a pharmaceutically acceptable excipient, carrier, diluents, or a combination thereof.In one embodiment, said method further comprises administering to said patient an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy or a combination thereof In some embodiments, said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge. In some embodiments, said anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumab, Nivolumab, Pembrolizumab, Rituximab, Interferon, Interleukin, and a combination thereof In some embodiments, said anti-cancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and a combination thereof In some embodiments, said additional anti-cancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK1 inhibitor, a CHK2 inhibitor, PARP inhibitor, doxorubicin, epirubicin, vinblastine, etoposide, topotecan, bleomycin, and mytomycin c. In some embodiments, said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and Oxaliplatin. In some embodiments, said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52, and IDN-5109. In some embodiments, said topoisomerase inhibitor is a topoisomerase I
inhibitor selected from the group consisting of Irinotecan, Topotecan, and Camptothecins (CPT).
In some embodiments, said topoisomerase inhibitor is a topoisomerase II inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, and ellipticine. In some embodiments, said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib). In some embodiments, said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-64 1397. In some embodiments, said PARP inhibitor is selected from the group consisting of Olaparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, and the like.In one embodiment, said method further comprises a radiation therapy administered to said patient. In some embodiments, said at least one said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for detecting TREM-1/DAP-expression levels in a patient with cancer in need thereof, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is conjugated to at least one imaging probe, or a combination thereof. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, Tc99m, Cr51, Ga67, Ga68, CU64, Rb82,M099, DY165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xel", 1125, 1131, 1123, p32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof. In some embodiments, said at least one said TREM-1 inhibitor comprises LR12 and/or LP17 peptide variants and the like.
In some embodiments, the invention provides a method for treating cancer in a patient in need thereof by modulating immune system activity, said method comprising administering to said patient an amount of a TREM-1 inhibitor that is effective for inhibiting the signaling pathway and suppressing tumor growth, or a combination thereof. In some embodiments, said TREM-1 inhibitor comprises a variant TREM-1 inhibitory peptide sequence derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, thereof In some embodiments, said variant TREM-1 inhibitory peptide sequence comprises amino acid sequence Gly-Phe-Leu-Ser-Lys-Ser-Leu-Val-Phe, wherein Gly is glycine, Phe is phenylalanine, Leu is leucine, Ser is serine, Lys is lysine, and Val .. is valine. In some embodiments, said variant TREM-1 inhibitory peptide sequence is conjugated to at least one unmodified or modified amphipathic peptide sequence. In some embodiments, said unmodified or modified amphipathic peptide sequence is derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof. In some embodiments, said modified amphipathic peptide sequence derived from amino acid sequences of apolipoproteins selected from the group consisting of A-I, A-II, A-IV, B, C-I, C-II, C-III, and E, and any combination thereof contains at least one amino acid residue which is chemically or enzymatically modified. In some embodiments, said chemically or enzymatically modified amino acid residue is oxidized, halogenated or nitrated. In some embodiments, said oxidized amino acid residue is the methionine residue. In some embodiments, said unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met is .. methionine, Arg is arginine, and Val is valine. In some embodiments, said unmodified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said modified amphipathic peptide sequence is derived from an apolipoprotein A-I amino acid sequence and comprises amino acid sequence Pro-Tyr-Leu-Asp-Asp-Phe-Gln-Lys-Lys-Trp-Gln-Glu-Glu-Met(0)-Glu-Leu-Tyr-Arg-Gln-Lys-Val-Glu, wherein Pro is proline, Tyr is tyrosine, Leu is leucine, Asp, asparagine, Phe is phenylalanine, Gln is glutamine, Lys is lysine, Trp is tryptophan, Glu is glutamic acid, Met(0) is methionine sulfoxide, Arg is arginine, and Val is valine. In some embodiments, said modified amphipathic peptide sequence is derived from an apolipoprotein A-I
amino acid sequence and comprises amino acid sequence Pro-Leu-Gly-Glu-Glu-Met(0)-Arg-Asp-Arg-Ala-Arg-Ala-His-Val-Asp-Ala-Leu-Arg-Thr-His-Leu-Ala, wherein Pro is proline, Leu is leucine, Gly is glycine, Glu is glutamic acid, Met is methionine sulfoxide, Arg is arginine, Asp, asparagine, Ala is alanine, His is histidine, Val is valine, and Thr is threonine. In some embodiments, said B is conjugated to an additional peptide sequence to enhance the targeting efficacy. In some embodiments, said an additional peptide sequence comprises amino acid sequence Arg-Gly-Asp (RGD), wherein Arg is arginine; Gly is glycine; and Asp is asparagine. In some embodiments, said A is conjugated to at least one additional therapeutic agent to enhance the therapeutic efficacy. In some embodiments, said an additional therapeutic agent is selected from the group of anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, therapeutic peptides, and any combination thereof In some embodiments, said anticancer therapeutic agent is selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof In some embodiments, said A and/or B are conjugated to at least one imaging probe. In some embodiments, said an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In Fe.59, TC99M, cr51 , Ga67, Ga68, cu64 , Rb82,m099, DY165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, Cll, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol.
In some embodiments, the invention provides a method of making a synthetic lipopeptide nanoparticle, said method comprising: a) co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with: i. a predetermined amount of cholesterol;
and ii. a predetermined amount of triglycerides and/or cholesteryl ester; b) drying the mixture of step (a) under nitrogen; c) co-dissolving the dried mixture of step (b) with: i. a predetermined amount of sodium cholate; and ii. a predetermined amount of the compound of claim 1; for a time period sufficient to allow the components to self-assemble into synthetic lipopeptide particles; d) removing sodium cholate from the mixture of step (c); and e) isolating particles that have a size of between about 5 to about 200 nm diameter. In some embodiments, said lipid is conjugated to at least one imaging probe. In some embodiments, said imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In Fe.59, TC99M, Cr51, Ga67, Ga68, CU64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 1131, 1123, P32, C11, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol. In some embodiments, said lipid is selected from the group comprising cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, and a triacylglycerol. In some embodiments, said phospholipid is selected from the group comprising phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), phosphatidic acid (PA), and any combination thereof. In some embodiments, said lipid is polyethylene glycol(PEG)ylated.
In some embodiments, the invention provides a method of imaging a myeloid cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 3, 4, and 21-25 having an affinity for TREM-1 and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. In some embodiments, said myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of treating a myeloid cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which myeloid cells are involved or recruited, and ii) the compositions of claims 1, 3, 4, and 23 capable of inhibiting TREM-1; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said myeloid cell-related condition is selected from the group comprising cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases (e.g., rheumatoid arthritis, Sjogrens, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as interstitial pneumonitis and asthma, inflammatory bowel disease such as Crohn's disease, inflammatory arthritis retinopathy such as retinopathy of prematurity and diabetic retinopathy, Alzheimer's, Parkinson's and Huntington's diseases), transplant (e.g., heart/lung transplants) rejection reactions, and other diseases and conditions where myeloid cells are involved or recruited.
In some embodiments, the invention provides a method of imaging a T cell-related condition, comprising a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) a labeled probe, wherein the labeled probe includes the compositions of claims 1, 5, 6, and 21-25 having an affinity for TCR and an imaging probe; b) administering said composition to said patient in a detectably effective amount c) imaging at least a portion of the patient; and d) detecting the labeled probe, wherein the location of the labeled probe corresponds to at least one symptom of the T
cell-related condition.
In some embodiments, said T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g.
autoimmune pericarditis, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g.
myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
In some embodiments, the invention provides a method of treating a T cell-related condition, comprising: a) providing; i) a patient having at least one symptom of a disease or condition in which T cells are involved or recruited, and ii) the compositions of claims 1, 5, 6, and 23 capable of inhibiting TCR; b) administering said composition to said patient under conditions such that said at least one symptom is reduced. In some embodiments, said T cell-related condition is selected from the group including but not limited to include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, scleroderma, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac disease, Guillain-Barre syndrome, Hashimoto's disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection, and other diseases and conditions where T cells are involved or recruited.
The details of one or more embodiments of the invention are set forth in the accompanying Figures (Drawings) and Detailed Description of The Invention, as described herein and below. Other features, objects, and advantages of the invention will be apparent from the summary, description, figures and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
The following figures form part of the present specification and are included to further illustrate embodiments of the present invention. The invention may be better understood by reference to the figures in combination with the detailed description of the specific embodiments presented herein.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
FIG. 1 presents an exemplary schematic representation of one embodiment of a trifunctional peptide of the present invention comprising amino acid domains A and B where amino acid domain A represents a therapeutic peptide sequence with or without an attached drug compound and/or imaging probe that functions to treat, prevent and/or detect a disease or condition, whereas amino acid domain B represents an amphipathic alpha helical peptide sequence, with or without an additional targeting peptide sequence, and functions to 1) assist in the self-assembly of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with lipids or lipid mixtures in vitro, for use in transporting these trifunctional peptides as lipoprotein nanoparticles to sites of interest in vitro or in vivo and/or 2) form long half-life lipopeptide/lipoprotein particles upon interaction with endogenous lipoproteins for transporting these trifunctional peptides to the sites of interest. Endogenous lipoproteins may be lipoproteins added to or found in cell cultures, or lipoproteins in a mammalian body.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP). GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) comprises amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22 amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising amino acid domains A and B. Depending on lipid mixture compositions added to the peptides, sub 50 nm-sized SLP particles of discoidal (TREM-1/TRIOPEP-dSLP) or spherical (TREM-sSLP) morphology are self-assembled upon binding of the trifunctional peptide to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 4A illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (shown for atherosclerosis), whereas domain B represents a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of atherosclerosis. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP
of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by intraplaque macrophages and return to the circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (shown for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the trifunctional peptide of the present invention comprising two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence, whereas domain B is a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with TREM-1 in the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and a merged image (FIG. 6C).
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic lipopeptide particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that contain an equimolar mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 (TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The post 4 h incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified methionine residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4 (white bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001 to 0.001 as compared with 4 h incubation time.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production by lipopolysaccharide (LPS)-stimulated macrophages incubated for 24 hour (hr) at 37 C with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged macrophages.
FIG. 9A-C presents the exemplary data showing that scavenger receptors SR-A
and SR-B1 mediate the macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and the SCHOOL
mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1, right panel).
(FIG. 9A2, left panel) Activation of the TREM-1/DAP12 receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm; GF9 self-penetrates the cell membrane and blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing phosphorylation and the downstream signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774 macrophages were cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-HDL, cells were treated for 1 hour at 37 C with 40 i.tM cytochalasin D and either (FIG.
9B) 400 pg/mL
fucoidan or (FIG. 9C) 10 i.tM BLT-1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars) or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence intensities of lysates were measured and normalized to the protein content.
Results are expressed as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of GF9-HDL and GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo-oligomerization.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post lipopolysaccharide (LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline (PBS), dexamethasone (DEX), control peptide and with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
.. 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
Control peptide represents an equimolar mixture of two peptides, each of them comprising two amino acid domains A and B where domain A represents a non-functional 9 amino acids-long sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5 is substituted with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as compared with PBS-treated animals.
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *,p < 0.05; **,p < 0.01, ****,p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP-bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar = 200 p.m.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference .. between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *,p <0.05, **, p <0.01;
***,p <0.001 as compared with vehicle-treated or naive animals.
FIG. 18A-D presents the exemplary data showing reduction of pathological retinal neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-1 (FIG. 18C) and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-induced .. retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP) particles of spherical morphology (TREM-1/TRIOPEP-sSLP). ***,p < 0.001 as compared with vehicle-treated animals.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical self-assembled particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-sSLP) or -- an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B a--- Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
-- staining, and the lipid content was analyzed by ImageJ (FIG. 21B). *
indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
WooP < 0.01;*"/###P <0001; ****P < 0 .0001. *** , 0.001 > P> 0.0001; ##, 0.01 > P> 0.001.
FIG. 22 presents an exemplary schematic representation of one embodiment of a related trifunctional peptide (TREM-1/TRIOPEP) G-HV21 of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic peptide sequence GF9 and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B represents a 12 amino acids-long amino acid sequence GV12 that contains a sulfoxidized methionine residue and is derived from human apolipoprotein A-I amino acid sequence. While not being bound to any particular theory, it is believed that a resulting amphipathic alpha helical peptide G-HV21 upon interaction with native lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles and targets these particles to myeloid cells (e.g. macrophages). Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; TRIOPEP, trifunctional peptide.
FIG. 23 presents an exemplary schematic representation of one embodiment of a related trifunctional peptide (TREM-1/TRIOPEP) G-KV21 of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic peptide sequence GF9 and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B represents a 12 amino acids-long amino acid sequence WV12 that contains a sulfoxidized methionine residue and is derived from human apolipoprotein A-I amino acid sequence. While not being bound to any particular theory, it is believed that a resulting amphipathic alpha helical peptide G-KV21 upon interaction with native lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles and targets these particles to myeloid cells (e.g. macrophages). Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; TRIOPEP, trifunctional peptide.
FIG. 24 presents an exemplary schematic representation of one embodiment of a related control peptide G-TE21 of the present invention comprising amino acid domains A and B
where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic peptide sequence GF9, whereas domain B represents a 12 amino acids-long amino acid sequence TE12 that contains a sulfoxidized methionine residue and is derived from bovine serum albumin amino acid sequence. While not being bound to any particular theory, it is believed that a resulting non-amphipathic peptide G-TE21 does not interact with native lipoproteins and therefore does not form naturally long half-life lipopeptide/lipoprotein particles.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 25 presents an exemplary schematic representation of one embodiment of a TCR-related trifunctional peptide (TCR/TRIOPEP) M-VE32 of the present invention comprising amino acid domains A and B where domain A represents a 10 amino acids-long human TCR
inhibitory therapeutic peptide sequence MF10 and functions to treat and/or prevent a TCR-related disease or condition, whereas domain B represents a 22 amino acids-long amino acid sequence PE22 that is derived from human apolipoprotein A-I amino acid sequence. While not being bound to any particular theory, it is believed that a resulting amphipathic alpha helical peptide M-VE32 upon -- interaction with native lipoproteins, forms naturally long half-life lipopeptide/lipoprotein particles. Abbreviations: TCR, T cell receptor; TRIOPEP, trifunctional peptide.
FIG. 26 presents a schematic representation of one embodiment of a TCR-related control peptide M-TK32 of the present invention comprising amino acid domains A and B where domain A
represents a 10 amino acids-long human TCR inhibitory therapeutic peptide sequence MF10, whereas domain B represents a random 22 amino acids-long amino acid sequence LK22. While not being bound to any particular theory, it is believed that a resulting non-amphipathic peptide M-TK32 does not interact with native lipoproteins and therefore does not form naturally long half-life lipopeptide/lipoprotein particles. Abbreviations: TCR, T cell receptor.
FIG. 27 presents an exemplary schematic representation and the exemplary data showing that ultracentrifugation of whole mouse serum with added rho B-labeled TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) G-HV21 and G-KV21 results in floatation of these peptides with mouse lipoproteins. In contrast, when added to whole mouse serum, rho B-labeled TREM inhibitory peptide GF9 or rho B-labeled TREM-1-related control peptide G-sedimentate with serum proteins upon ultracentrifugation. When added to delipoproteinized mouse serum that does not contain lipoproteins, rho B-labeled TREM-1/TRIOPEP G-HV21 and G-KV21 sedimentate with serum proteins upon ultracentrifugation. While not being bound to any particular theory, it is believed that TREM-1/TRIOPEP G-HV21 and G-KV21 interact with native lipoproteins of a whole mouse serum and/or their lipid components and form lipopeptide/lipoprotein particles that mimic serum lipoproteins and float under the same ultracentrifugation conditions. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; rho B, rhodamine B.
FIG. 28 presents exemplary data showing the endocytosis of rho B-labeled GF9, G-TE21, G-HV-21 and G-KV21 by macrophages in the absence (white bars) or presence (black bars) of HDL. In contrast to GF9 and TREM-1-related control peptide G-TE21, the in vitro macrophage uptake of TREM-1/TRIOPEP G-HV21 and G-KV21 significantly increases in the presence of HDL. ***,p < 0.001 (presence vs. absence of HDL). Abbreviations: HDL, high density lipoproteins; rho B, rhodamine B; n.s., not significant.
FIG. 29A-C shows exemplary images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1/TRIOPEP G-KV21 (pre-incubated with HDL) with TREM-1 in the J774 cell membrane FIG. 29A. FIG. 29B TREM-1 immunostaining. FIG. 29C merged image.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL, high density -- lipoproteins.
FIG. 30A illustrates a hypothesized molecular mechanism of action of TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) of the present invention (shown for atherosclerosis).
While not being bound to any particular theory, it is believed that upon interaction with native lipoproteins including HDL, the modified methionine residue in the TREM-1/TRIOPEP domain B mediates the recognition of the formed lipopeptide/lipoprotein particles by macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TREM-1/TRIOPEP
released within these cells. In contrast, native HDL particles are not recognized by intraplaque macrophages and return to the circulation.
FIG. 30B Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL, high-density lipoproteins.
FIG. 31A illustrates a hypothesized molecular mechanism of action of TREM-1-related -- trifunctional peptides (TREM-1/TRIOPEP) of the present invention (shown for cancer). While not being bound to any particular theory, it is believed that upon intreaction with native lipoproteins including HDL, the modified methionine residue in the TREM-1/TRIOPEP domain B mediates the recognition of the formed lipopeptide/lipoprotein particles by macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by -- macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TREM-1/TRIOPEP
released within these cells. In contrast, native HDL particles are not recognized by intraplaque macrophages and return to the circulation.
FIG. 31B Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; HDL, -- high-density lipoproteins.
FIG. 32 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention delivered to and released within TREM-1-expressing cells by the -- lipopeptide/lipoprotein particles formed upon interaction of TREM-1/TRIOPEP
with native lipoproteins. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; DAP-12, DNAX-activation protein 12; M--- CSF/CSF-1, macrophage colony stimulating factor-1; MCP-1/CCL2, monocyte chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor.
FIG. 33 presents exemplary data showing cytokine production by LPS-stimulated macrophages incubated for 24 h at 37 C with GF9, G-TE21, G-HV21 and G-KV21 in the presence of HDL. In -- contrast to GF9 and TREM-1-related control peptide G-TE21, TREM-1/TRIOPEP G-HV21 and G-KV21 significantly inhibit the cytokine release in the presence of HDL. In the absence of HDL, G-HV21 does not affect the cytokine production. ***,p <0.001 (vs. medium + HDL).
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; HDL, high density lipoproteins.
FIG. 34A-C presents exemplary LPS-challenged J774 macrophages: Cytokine release data showing that scavenger receptors SR-A and SR-B1 mediate the macrophage endocytosis of TREM-1/TRIOPEP G-HV21 and G-KV21 in the presence of HDL. (FIG. 34A) Schematic representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1 blockade.
Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated macrophage endocytosis of the lipopeptide/lipoprotein particles formed upon interaction of TREM-1/TRIOPEP
with native lipoproteins (shown for HDL) results in the release of TREM-1/TRIOPEP into the cytoplasm.
Then, the released TREM-1/TRIOPEP self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 34A, right panel).
Macrophage endocytosis of G-HV21 and G-KV21 in the presence of HDL in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (B, C). J774 macrophages were cultured at 37 C overnight with medium. Before adding G-HV21 and G-KV21, cells were treated for 1 h at 37 C with 40 [tM cytochalasin D, 400 [tg/mL fucoidan (FIG. 34B) or 10 [tM BLT-1 (FIG. 34C) as indicated. Cells were then incubated for either 4 h or 22 h with medium containing HDL and 2 [tM rho B-labeled G-KV21 (gray bars) or G-HV21 (black bars), respectively.
Cells were lysed and rho B fluorescence intensities of lysates were measured and normalized to the protein content. Results are expressed as the mean SEM (n = 3). *,p < 0.05; **,p <
0.01; ****, p <
0.0001 versus uptake of G-HV21 and G-KV21 in the absence of inhibitor.
Abbreviations:
TREM-1, triggering receptor expressed on myeloid cells-1; DAP-12, DNAX-activation protein 12; M-CSF/CSF-1, macrophage colony stimulating factor; MCP-1/CCL2, monocyte chemoattractant protein-1; IL, interleukin; TNF, tumor necrosis factor; HDL, high density lipoproteins; BLT-1, blocker of lipid transport-1; rho B, rhodamine B; SR, scavenger receptor.
FIG. 35 presents exemplary data showing serum cytokine production at 90 min post LPS
challenge in mice treated at 1 h before LPS challenge with PBS, DEX, GF9, TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to GF9 and G-TE21, G-HV21 and G-KV21 significantly inhibit the LPS-induced cytokine release. ***,p < 0.001 as compared with PBS-treated animals.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; IL, interleukin; TNF, tumor necrosis factor; DEX, dexamethasone; PBS, phosphate-buffer saline.
FIG. 36A-B presents the exemplary data showing survival of LPS-challenged mice treated with PBS (vehicle), TREM-1-related control peptide G-TE21, TREM-1-related trifunctional peptides G-HV21 and G-KV21 (FIG. 36A) or with TREM-1 inhibitory peptide GF9 at different doses (FIG. 36B). In contrast to G-TE21, G-HV21 and G-KV21 significantly improve survival of septic mice (FIG. 36A). When administered at a dose of 5 mg/kg, GF9 does not affect survival of septic mice, while at 25 mg/g, GF9 improves survival. In contrast, high dose of GF9, 150 mg/kg, contributes to earlier death as compared with control animals treated with vehicle only (FIG.
36B). **,p < 0.01 as compared with vehicle-treated animals. Abbreviations:
TREM-1, triggering receptor expressed on myeloid cells-1; LPS, lipopolysaccharide; PBS, phosphate-buffer saline.
FIG. 37A-B presents the exemplary data showing tumor growth in the human non-small cell lung cancer H292 mouse xenograft (FIG. 37A) and A549 mouse xenograft (FIG.
37B) xenograft mice treated with PBS (vehicle), PTX, TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-HV21 and G-KV21 significantly inhibit the tumor growth. ****,p < 0.0001 as compared with vehicle-treated animals. Abbreviations: PTX, paclitaxel; PBS, phosphate-buffer saline.
FIG. 38 presents exemplary A549 mouse xenograft data showing average tumor weights in the A549 xenograft mice treated with PBS (vehicle), PTX, TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-HV21 and G-KV21 significantly decrease the tumor weight. **,p < 0.01 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; PTX, paclitaxel; PBS, phosphate-buffer saline; n.s., not significant.
FIG. 39A-B presents exemplary data showing tumor growth (A) and,infiltration of macrophages into the tumor as evaluated by F4/80 staining (B) in the human pancreatic cancer BxPC-3 xenograft mice treated with PBS (vehicle), TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to G-TE21, G-HV21 and G-KV21 in a BxPC-3 mouse xenograft significantly inhibits the tumor growth (FIG. 389) and reduce macrophage infiltration into the tumor (FIG. 39B). **,p < 0.01, ****,p < 0.0001 (versus vehicle). Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1;
PBS, phosphate-buffer saline; n.s., not significant.
FIG. 40A-B presents exemplary data showing PANC-1 mouse xenograft tumor growth (FIG.
40A) and survival (FIG. 40B) in the human pancreatic cancer PANC-1 xenograft mice treated with PBS (vehicle) and TREM-1-related trifunctional peptide G-KV21 with or without chemotherapy treatment (GEM+ABX). G-KV21 sensitizes the tumor to chemotherapy (FIG.
40A) and significantly improves survival (FIG. 40B). The median survival times (FIG. 40B) are indicated in parentheses. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; PBS, phosphate-buffer saline; GEM, gemcitabine; ABX, Abraxane (nanoparticle albumin-bound paclitaxel).
FIG. 41 presents the exemplary data showing average weights of Healthy C57BL/6 mice treated with TREM-1-related control peptide G-TE21 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. No toxicity was observed for all three peptides.
Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1.
FIG. 42A-B presents exemplary data showing average clinical arthritis score (Collagen-induced arthritis: Score FIG. 42A) and Collagen-induced arthritis: Body weight change mean BW
changes (FIG. 42B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the CIA mice treated with PBS (vehicle), DEX, TREM-1-related control peptide G-TE21, TCR-related control peptide M-TK32, TCR-related trifunctional peptide M-VE32 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to the relevant control peptides, G-HV21, G-KV21 and M-VE32 all ameliorate the disease (FIG. 42A) and are well-tolerated by arthritic mice (FIG. 42B). *,p < 0.05, **,p < 0.01;
***,p < 0.001 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; CIA, collagen-induced arthritis; PBS, phosphate-buffer saline; DEX, dexamethasone; TCR, T cell receptor; BW, body weight.
FIG. 43A-D Oxygen-induced retinopathy presents exemplary data showing pathological RNV
(FIG. 43A) and avascular (FIG. 43B) areas as well as expression of TREM-1 (FIG. 43C) and M-CSF (FIG. 43D) in the retina of the mice with OIR treated with PBS (vehicle), TREM-1-related control peptide G-TE21 or TREM-1-related trifunctional peptide G-KV21. In contrast to G-TE21, G-KV21 significantly suppresses pathological RNV and inhibits tissue expression of TREM-1 and M-CSF. *,p < 0.05, **,p < 0.01; ***,p < 0.001 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; OIR, oxygen-induced retinopathy; PBS, phosphate-buffer saline; M-CSF, macrophage colony stimulating factor; RNV, retinal neovascularization.
FIG. 44 presents exemplary data showing penetration of the BBB and BRB by systemically (mice ¨ intraperitoneally; rats and rabbits ¨ intravenously) administered rhodamine B-labeled TREM-1-related trifunctional peptide G-KV21. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; BBB, blood-brain barrier; BRB, blood-retinal barrier.
FIG. 45A-E TREM-1 pathway inhibition. TREM-1 pathway inhibition suppresses the expression of (FIG. 45A) TREM-1 and inflammatory cytokines (FIG. 45B) MCP-1, (FIG. 45C) TNF-a, (FIG. 45D) IL-113, and (FIG. 45E) MIP-la but not (FIG. 45F) RANTES at the mRNA
level as measured in whole-liver lysates by real-time quantitative PCR. *
indicates significance level compared to nontreated PF group; # indicates significance level compared to nontreated alcohol-fed group; o indicates significance level compared to vehicle-treated alcohol-fed group.
Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P < 0.01; ***
/000 P < 0.001;
****P < 0.0001. Abbreviation: CCL, chemokine (C-C motif) ligand.
FIG. 46AE-G TREM-1 blockade and inflammatory cytokine levels. TREM-1 blockade reduces inflammatory cytokine levels in (FIG. 46A) serum and (FIG. 46B-D) whole-liver lysates as measured with specific ELISA kits. (FIG. 46E-G) Total liver protein was analyzed for total SYK
and activated p-SYK Y525/526 expression by western blotting using 13-actin as a loading control.
Statistical analysis was performed by evaluating two blots (n = 4/group).*
indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##
P < 0.01; ***P <
0.001; **** /
FIG. 47A-H Effects of TREM-1 inhibition. (FIG. 47A, FIG. 47B) TREM-1 inhibition suppresses the mRNA expression of macrophage cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47C, FIG. 47D) Both TREM-1 inhibitors attenuated F4/80 as shown by IHC. (FIG. 47E, FIG. 47F) TREM-1 inhibition suppresses the mRNA
expression of neutrophil cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47G, H) Both TREM-1 inhibitors attenuated MPO-positive cell infiltration as shown by IHC. * indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P <
0.05; ** /## P < 0.01;
.. ### P < 0.001; **** /
FIG. 48A-F Measurement of mRNA expression. mRNA expression of genes involved in (FIG.
48A, FIG. 48B) lipid synthesis (SERBF1, ACC1), (FIG. 48C) the lipid accumulation marker (ADRP), and (FIG. 48D-F) lipid oxidation (PPARa, CPT1a, MCAD) were measured in whole liver.
* indicates significance level compared to the nontreated PF group; #
indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P
<0.01; ### P <0.001; ****P <0.0001.
FIG. 49A presents a schematic representation of one embodiment of the proposed role of inhibition of TREM-1 expressed on tumor-associated macrophages (TAMs) in pancreatic cancer.
Pancreatic ductal adenocarcinoma cells, cancer-associated fibroblasts (CAFs) and TAMs play a role in generating a tumor favorable microenvironment, in part by producing such cytokines and growth factors as interleukin (IL)-1a, IL-6 and macrophage colony-stimulating factor (M-CSF).
FIG. 49B presents a schematic representation of one embodiment of suppressing tumor favorable microenvironment by inhibition of TREM-1 expressed on tumor-associated macrophages (TAMs) and reduction of cytokines and growth factors including but not limited to interleukin (IL)-6, IL-1, monocyte chemoattractant protein-1 (MCP-1; also referred to in the art as CCL2) and macrophage colony-stimulating factor 1 (CSF-1; also referred to in the art as M-CSF). These prognostic factors are involved in tumorigenesis, cancer progression, metastasis, and even in the response to cancer treatment. The figure further presents a schematic representation of one embodiment of modulating the TREM-1/DAP-12 signaling pathway by type I TREM-1 inhibitors that bind either TREM-1 (type Ia inhibitors; e.g., anti-TREM-1 blocking antibodiesõ etc.) or its ligand (type Ib inhibitors; e.g., inhibitory peptides LP17 and LR12 that act as a decoy TREM-1 receptor), thereby blocking binding between TREM-1 and its yet uncertain ligand(s).
FIG. 50 presents a schematic representation of one embodiment of TREM-1 modulatory peptide variants and compositions of the present invention that are rationally designed using the Signaling Chain HOmoOLigomerization (SCHOOL approach) to inhibit TREM-1 in a ligand-independent manner by blocking intramembrane interactions between TREM-1 and its signaling partner DAP-12 (type II inhibitors). These SCHOOL peptides can be employed in either free form or incorporated into macrophage-targeted (macrophage-specific) synthetic lipopeptide particles (SLP), which allows them to reach their site of action from either outside (Route 1) or inside the cell (Route 2).
FIG. 51A-F shows images of one embodiment depicting colocalization of the TREM-modulatory peptide GF9 (GFLSKSLVF) with trifunctional TREM-1 in the cell membrane. Fig.
51A shows exemplary peptide GF9. Fig. 51B and 51E shows exemplary TREM-1. Fig.
51C and F shows exemplary merged Images. Fig. 51A shows exemplary inhibitory peptide ((GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue with TREM-1 in the cell membrane.
FIG. 51B shows images of one embodiment depicting colocalization of the TREM-1 modulatory peptide GF9 (GFLSKSLVF) and trifunctional TREM-1 FIG. 52 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into the a carrier, e.g.
synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology suppresses tumor growth in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares), GF9-loaded discoidal SLP
(GF9-dSLP, light gray circles) or GF9-loaded spherical SLP (GF9-sSLP, white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 6 mice per group). On the final day of treatment, tumor volumes were compared between the drug-treated and control groups. **, p < 0.01; ***, p <0.001; ****, p < 0.0001 (versus vehicle).
FIG. 53 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into the a carrier, e.g.
synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology (GF9-dSLP and GF9-sSLP, respectively) suppresses tumor growth in experimental pancreatic cancer without affecting body weight (well-tolerable in long term-treated mice). As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares), GF9-dSLP (light gray circles) or GF9-sSLP (white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All results are expressed as the mean SEM (n = 6 mice per group).
FIG. 54 presents the exemplary data of one embodiment showing that treatment with synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology loaded with an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-dSLP and GA/E31-sSLP, respectively) suppresses tumor growth in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 6 mice per group). On the final day of treatment, tumor volumes were compared between the drug-treated and control groups. **,p < 0.01; ***,p <0.001; ****, p < 0.0001 (versus vehicle).
FIG. 55 presents the exemplary data of one embodiment showing that treatment with synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology loaded with an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-dSLP and GA/E31-sSLP, respectively) suppresses tumor growth in experimental pancreatic cancer without affecting body weight (i.e. well tolerable by long term-treated mice). As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All results are expressed as the mean SEM
(n = 6 mice per group).
FIG. 56 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology (GF9-dSLP and GF9-sSLP, respectively) prolongs survival in experimental pancreatic cancer.
As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 MM , mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray circles), GF9-dSLP
(light gray circles) or GF9-sSLP (white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively.
Kaplan-Meier survival curves are shown for AsPC-1-, BxPC-3- or Capan-l-bearing mice (n = 6 mice per group). **, p < 0.01; ***,p < 0.001 by log-rank test (versus vehicle).
FIG. 57 presents the exemplary data of one embodiment showing that treatment with synthetic lipopeptide particle (SLP) of discoidal (dSLP) or spherical (sSLP) morphology loaded with an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-dSLP and GA/E31-sSLP, respectively) prolongs survival in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GA/E31-dSLP (light gray triangles) or GA/E31-sSLP (white triangles) at indicated doses.
Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Kaplan-Meier survival curves are shown for AsPC-1-, BxPC-3-or Capan-l-bearing mice (n = 6 mice per group). **, p < 0.01; ***, p < 0.001 by log-rank test (versus vehicle).
FIG. 58 presents the exemplary data of one embodiment showing that the antitumor efficacy of TREM-1 blockade correlates with the intratumoral macrophage content in experimental pancreatic cancer. Antitumor efficacy is expressed as percent treatment/control (% T/C) values calculated using the following formula: % T/C = 100 x AT/AC where T and C are the mean tumor volumes of the drug-treated and control groups, respectively, on the final day of the treatment; AT is the mean tumor volume of the drug-treated group on the final day of the treatment minus mean tumor volume of the drug-treated group on initial day of dosing; and AC is the mean tumor volume of the control group on the final day of the treatment minus mean tumor volume of the control group on initial day of dosing. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies. Data are shown for the groups of AsPC-1-, BxPC-3- and Capan-1- bearing mice treated with free and SLP-bound TREM-1 modulatory peptides GF9 (GFLSKSLVF), GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
where M(0) is a methionine sulfoxide residue) and (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide residue) (GA/E31-sSLP) (n = 4 mice per group).
FIG. 59 presents the exemplary data of one embodiment showing that TREM-1 blockade suppresses intratumoral macrophage infiltration in experimental pancreatic cancer. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies.
Data are shown for the groups of BxPC-3-bearing mice treated with either vehicle (black bars), free GF9 (GFLSKSLVF, dark grey bars), GF9 incorporated into a carrier, e.gs. synthetic lipopeptide particle of spherical morphology (GF9-sSLP, light grey bars) and sSLP that contain an equimolar mixture of TREM-1 modulatory peptides (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA where M(0) is a methionine sulfoxide residue) and GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide residue) (GA/E31-sSLP, white bars) (n = 4 mice per group).
FIG. 60 presents the exemplary data of one embodiment showing the representative F4/80 images demonstrating that TREM-1 blockade suppresses intratumoral macrophage infiltration in experimental pancreatic cancer. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies. Data are shown for the groups of BxPC-3-bearing mice treated with either vehicle, free GF9 (GFLSKSLVF), GF9 incorporated into synthetic lipopeptide particle of spherical morphology (GF9-sSLP) and sSLP that contain an equimolar mixture of TREM-1 modulatory peptides GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
where M(0) is a methionine sulfoxide residue) and (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE where M(0) is a methionine sulfoxide residue) (GA/E31-sSLP) (n = 4 mice per group).
FIG. 61 presents the exemplary data of one embodiment showing that TREM-1 blockade suppresses serum proinflammatory cytokines in xenograft mouse models of pancreatic cancer.
Serum interleukin- 1 a (IL-1a), IL-6 and macrophage colony-stimulating factor (M-CSF/CSF-1) levels were analyzed on study days 1 and 8 in AsPC-1-, BxPC-3- and Capan-l-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (dark gray squares) or GF9-loaded spherical synthetic lipopeptide particles (GF9-sSLP, white circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *,p < 0.05;
**,p < 0.01; ***,p <0.001; ****,p < 0.0001 (versus vehicle).
FIG. 62 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor 1 (CSF1) and interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the AsPC-1 xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were analyzed on study days 1 and 8 in AsPC-1-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <0.05; **, p <0.01;
***, p < 0.001;
****, p < 0.0001 (versus vehicle).
FIG. 63 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor 1 (CSF1) and interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the BxPC-3 xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were analyzed on study days 1 and 8 in BxPC-3-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <0.05; **, p <0.01;
***, p < 0.001;
****, p < 0.0001 (versus vehicle).
FIG. 64 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) suppresses serum proinflammatory cytokines colony-stimulating factor 1 (CSF1) and interleukin 6 (IL-6) but not vascular endothelial growth factor (VEGF) in the xenograft mouse model of pancreatic cancer. Serum CSF1, VEGF and IL-6 levels were analyzed on study days 1 and 8 in CAPAN-1-bearing mice treated daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles) or GF9-LPC (black circles) at indicated doses. Results are expressed as the mean SEM (n = 5 mice per group). *, p <
0.05; **, p <
0.01; ***, p <0.001; ****, p <0.0001 (versus vehicle).
FIG. 65 presents the exemplary data of one embodiment showing that combining of Gemcitabine and Abraxane chemotherapy with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP) has a synergistic effect in experimental pancreatic cancer. As described herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered with either vehicle (black diamonds; once daily 5 times per week, 5qw), GF9-sSLP (black squares; once daily 5 times per week, 5qw), Gemcitabine and Abraxane (black circles; days 1, 4, 8, 11, 15) or GF9-sSLP (once daily 5 times per week, 5qw) in combination with Gemcitabine and Abraxane (days 1, 4, 8, 11, 15) (Black triangles). Treatment with GF9-sSLP persisted for 28 days. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 9 mice per group). On the final day of treatment, tumor volumes were compared between the Gemcitabine+Abraxane-treated and GF9-sSLP+Gemcitabine+Abraxane-treated groups. **, p < 0.01 (versus chemotherapy alone treated group).
FIG. 66 presents the exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical synthetic lipopeptide particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 modulatory peptide GF9 (GF9-sSLP) or an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1 modulatory peptides, i.e. 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue GA 31 and GE 31 (GA/E31-sSLP).
Fig. 67 presents the exemplary data of one embodiment showing that TREM-1 blockade with GF9, GF9 incorporated into the carrier - spherical synthetic lipopeptide particles (GF9-sSLP) or sSLP that carried an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-sSLP) significantly reduces tissue expression of colony-stimulating factor 1 (C SF-1) and TREM-1 in the retina of mice with oxygen-induced retinopathy (OIR) at postnatal day 17 (P17). Representative Western blots of retinal lysates from OIR mice are shown. The membrane was probed for TREM-1, reprobed for CSF-1 and then for 13-actin.
Values in the bar graphs represent the mean SEM, n=6. *, p < 0.05, **, p < 0.01 vs. vehicle-treated mice.
Fig. 68 presents the exemplary data of one embodiment showing that combining gemcitabine (GEM) and abraxane (ABX) chemotherapy with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into a carrier, e.g. synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP) has a synergistic therapeutic effect in experimental pancreatic cancer. As described herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered at indicated doses with either vehicle (black diamonds; once daily 5 times per week, 5qw), GF9-LPC (black circles-black squares; once daily 5 times per week, 5qw), GEM and ABX (black squares-(black circles; days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX (days 1, 4, 8, 11, 15) (half black half white hexagons-Black triangles). Treatment with GF9-LPC persisted for 28 days. Mean tumor volumes are calculated and plotted. All results are expressed as the mean SEM (n = 9 mice per group). On the day 88, tumor volumes were compared between the GEM+ABX-treated and GF9-sSLP+GEM+ABX-treated groups. *, p <
0.05 (versus GEM+ABX-treated group), second set of symbols are used in the longer term studies.
Fig. 69 presents the exemplary data of one embodiment showing that TREM-1 blockade treatment with TREM-1 modulatory peptide GF9 (GFLSKSLVF) incorporated into a , e.g.
synthetic lipopeptide particle (SLP) of spherical (sSLP) morphology (GF9-sSLP) alone, lipopeptide complex (GF9-LPC) alone or in combination with gemcitabine (GEM) and abraxane (ABX) chemotherapy is well tolerable in mice with human PANC-1 pancreatic cancer xenografts. As described herein, after tumors in PANC-1-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered at indicated doses with either vehicle (black diamonds; once daily 5 times per week, 5qw), GF9-LPC (black circles; once daily 5 times per week, 5qw), GEM and ABX (black squares; days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX
(days 1, 4, 8, 11, 15) (half black half white hexagons). Treatment with GF9-LPC (GF9-sSLP) persisted for 28 days. Body weighs are plotted. All results are expressed as the mean SEM (n =
6 mice per group).
Fig. 70 presents the exemplary data of one embodiment showing that treatment with TREM-1 modulatory peptide GF9 incorporated into a carrie, e.g. synthetic lipopeptide complex (GF9-LPC) and particle (SLP) of spherical (sSLP) morphology (GF9-sSLP), synergistically prolongs survival rate in experimental pancreatic cancer (e.g. PANC-1) when combined with gemcitabine (GEM) and abraxane (ABX) chemotherapy. As described herein, after tumors in bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered at indicated doses with either vehicle (once daily 5 times per week, 5qw), GF9-LPC (once daily 5 times per week, 5qw), GEM and ABX (days 1, 4, 8, 11, 15) or GF9-LPC (once daily 5 times per week, 5qw) in combination with GEM and ABX
(days 1, 4, 8, 11, 15). Treatment with GF9-LPC persisted for 28 days. Kaplan-Meier survival curves are shown for PANC-1-bearing mice (n = 6 mice per group). *, p < 0.05 by log-rank test (versus GEM+AB X).
Fig. 71 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) is well tolerable in mice up to at least 300 mg/kg. As described herein, healthy C57BL/6 mice were intraperitoneally (i.p.) administered daily for 7 consecutive days with GF9 at indicated doses Mouse body weight (BW) was measured daily.
Results are expressed as the mean SEM (n = 4 mice per group).
Fig.72 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) suppresses tumor growth in experimental pancreatic cancer. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered .. once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated doses.
Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Mean tumor volumes are calculated and plotted.
All results are expressed as the mean SEM (n = 6 mice per group).
Fig.73 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) is well tolerable in mice with human pancreatic cancer xenografts. As described herein, after tumors in AsPC-1-, BxPC-3- or Capan-l-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated doses. Treatment persisted for 31, 29 and 29 days for mice containing AsPC-1, BxPC-3 and Capan-1 tumor xenografts, respectively. Body weighs are plotted. All results are expressed as the mean SEM (n = 6 mice per group).
Fig.74 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) suppresses tumor growth as effectively as 20 mg/kg paclitaxel and is well tolerable in mice with human non-small cell lung cancer xenografts. As described herein, after tumors in A549-bearing mice reached a volume of 150-200 mm3, mice were randomized into groups and intraperitoneally (i.p.) administered once daily 5 times per week (5qw) with either vehicle (black diamonds), paclitaxel (black squares), GF9 (white circles), GF9-LPC (black circles) or GA/E31-LPC (half black-half white circles) at indicated doses. Treatment persisted for 21 days. Mean tumor volumes are calculated and plotted. Body weighs are plotted.
All results are expressed as the mean SEM (n = 6 mice per group).
Fig.75 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) suppresses intratumoral macrophage infiltration in experimental pancreatic cancer. Intratumoral macrophage content was quantified by F4/80 staining using F4/80 antibodies. Data are shown for the groups of BxPC-3-bearing mice treated with either vehicle, GF9, GF9-LPC or GA/E31-LPC at indicated doses. Treatment persisted for 21 days. All results are expressed as the mean SEM (n = 4 mice per group).
Scale bar = 200 Elm.
Fig.76 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) ameliorates arthritis in mice with collagen-induced arthritis (CIA). As described herein, starting on day 24 after immunization, mice with CIA were intraperitoneally (i.p.) administered daily for 14 consecutive days with vehicle (black diamonds), dexamethasone (black squares), GF9 (white circles), GF9-LPC (black circles) and GA/E31-LPC
(half black half white circles) at indicated doses. Daily clinical scores were given on a scale of 0-5 for each of the paws on days 24-38. On day 38, mice were killed and the histopathological examination of mouse joints was performed. Histopathological scores of inflammation (I), pannus (P), cartilage damage (CD), bone resorption (BR) and periosteal new bone formation (PBF) are shown. Summed histopathology scores were calculated as the sum of all five histopathological parameters. All results are expressed as the mean SEM (n =
10 mice per group).
Fig.77 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue is well tolerable in mice with collagen-induced arthritis (CIA). Mouse body weight (BW) was measured every other day from day 24 to day 38. Mean BW
changes were calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the CIA mice intraperitoneally (i.p.) treated daily for 14 consecutive days with vehicle, dexamethasone, GF9, GF9-LPC and GA/E31-LPC at indicated doses. All results are expressed as the mean SEM (n = 10 mice per group).
Fig.78 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) prevents pathological appearances from collagen-induced arthritis (CIA) in mice. As described herein, toluidine blue staining of the joints from mice with CIA treated with TREM-1 inhibitory GF9 sequences or control peptide GF9-G
(GFLSGSLVF) was performed. Photomicrographs of fore paws, hind paws, knees and ankles from representative mice are shown for each treatment group. For paws (original magnification 16x) and ankles (original magnification 40x), arrows identify affected joints. For knees (original magnification 100x), large arrow identifies cartilage damage, small arrow identifies pannus and arrowhead identifies bone resorption. W, wrist; S, synovium.
Fig.79 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) reduces plasma cytokines in mice with collagen-induced arthritis (CIA). Plasma was collected on days 24, 30 and 38 from arthritic mice treated with vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) and GA/E31-LPC (half black half white circles). Plasma samples were analyzed for concentrations of interleukin-lb (IL-lb), IL-6, and colony-stimulating factor 1 (CSF1). Results are expressed as the mean SEM (n =
mice per group).
Fig. 80 presents the exemplary data of one embodiment showing that treatment with free TREM-5 1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) significantly reduces tissue expression of colony-stimulating factor 1 (CSF1) and TREM-1 in the retina of mice with oxygen-induced retinopathy (OIR) at postnatal day 17 (P17). Representative Western blots of retinal lysates from OIR mice are shown. The membrane was probed for TREM-1, reprobed for CSF1 and then for 13-actin. Values in the bar graphs represent the mean SEM, n=6. *, p < 0.05, **, p < 0.01 vs.
vehicle-treated mice.
Fig. 81 shows exemplary illustrations of peptide GF9 blocking TREM-1 signaling by disruption of intramembrane interactions with its signaling partner, DAP-12. One example of a comparision of current approaches (upper) with a SCHOOL approach (lower), e.g. Route 1.
Fig. 82 shows exemplary illustrations of LPC delivering of peptide GF9 to macrophages, as two exemplary embodiments, e.g. each as Route 2.
Fig. 83 shows exemplary results using Pancreas Cancer: PANC-1 Xenografts demonstrating GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM
+ nab-PTX, ABX) and Adding GF9 treatment sensitizes the tumor to chemotherapy and at least triples survival rate.
Fig. 84 shows exemplary results using Pancreas Cancer: AsPC-1 Xenografts demonstrating GF9 treatment alone does not inhibit tumor growth.Adding of the GF9 treatment sensitizes the tumor to chemotherapy. NOTE: Most tumors ¨ abscessed.
Fig. 85 shows exemplary results using Pancreas Cancer: MiaPaca-2 Xenografts demonstrating GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM + nab-PTX, ABX) and Adding of the GF9 treatment to chemo does not affect.
Fig. 86 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts demonstrating GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM
+ nab-PTX, ABX) and Adding of the GF9 treatment to chemo does not significantly affect survival rate.
Fig. 87 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts demonstrating GF9 treatment reduces macrophage content in the tumor, Vehicle, 2.5 mg/kg GF9-LPC
(5 qw, 4 wk).
Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 88 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts demonstrating GF9 treatment reduces serum cytokine levels, Vehicle, 2.5 mg/kg GF9-LPC. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 89 shows exemplary results using Pancreas Cancer: Xenografts demonstrating GF9 Treatment is Non-Toxic. Free GF9 tolerability (upper). GF9-LPC* tolerability (lower). * Shown for PANC-1 xenograft Fig. 90 shows exemplary results demonstrating that GF9 peptide is well-tolerable by healthy mice up to at least, 300 mg/kg.
Fig. 91 shows exemplary results demonstrating that in mice with collagen-induced arthritis (CIA), GF9 suppresses arthritis as effectively as dexamethasone (DEX). Study Day (Treatment:
Days 24-38). I, inflammation; P, pannus; CD, cartilage damage; BR, bone resorption; PBF, periosteal new bone formation. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 92 shows exemplary results demonstrating that in mice with collagen-induced arthritis (CIA), GF9 treatment reduces serum IL-lb\ TNFal, IL-6 and CSF-1. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 93 shows exemplary results demonstrating that in mice with collagen-induced arthritis (CIA), GF9 treatment is well-tolerable: no body weight changes or other clinical symptoms are observed. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 94 shows exemplary results demonstrating that in NSCLC: A549 Xenografts, GF9 inhibits tumor growth as effectively aschemo (20 mg/kg Paclitaxel, PTX). Sigalov, 2014, 21:208.
Fig. 95 shows exemplary results demonstrating that in Capan-1 xenografts, GF9 inhibits tumor growth and reduces serum cytokines, including CSF-1 (but not VEGF). Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 96 shows exemplary results demonstrating that GF9 is well-tolerated by long term treated cancer mice inCapan-1 Xenografts and A549 Xenografts, GF9 inhibits tumor growth as effectively aschemo (20 mg/kg Paclitaxel, PTX). Sigalov, 2014, 21:208.
Fig. 97A-C shows exemplary current approaches for blocking TREM-1 binding to its uncertain ligand (Bouchon et al. 2001, Schenk et al. 2007, Gibot et al. 2008, Gibot et al. 2009, Murakami et al. 2009, Luo et al. 2010, Derive et al. 2013, Derive et al. 2014) Fig. 97A
In contrast, GF9 self-penetrates into the membrane and disrupts TREM-1 / DAP12 interactions Fig. 97B when colocalizes with TREM-1 Fig. 97C. Fig. 97A. CURRENT. Fig. 97B. SCHOOL Fig.
97C.
CONFOCAL.
Fig. 98A-B shows exemplary results demonstrating that GF9 is non-toxic in healthy mice Fig.
98A and reduces TREM-1 and M-CSF overexpression in the retina of mice with oxygen-induced retinopathy Fig. 98B. Fig. 98A Graph. Fig. 98B Blot.
Fig. 99A-C shows exemplary results demonstrating that Oxidized apo A-I
peptides in LPC
increase J774 intracellular uptake of GF9-LPC in vitro Fig. 99A, 99B and enable in vivo delivery to macrophages Fig. 99C (as shown using magnetic resonance imaging (Mill) and confocal microscopy (Sigalov 2014, Sigalov 2014, Shen and Sigalov 2017)). Fig. 99A. IN
VITRO. Fig.
.. 99B. CONFOCAL red: Rho B-PE; green: 488-GF9; blue: 405-apo A-I PE22. Fig.
99C. MOUSE
AORTA.
Fig. 100A-D shows exemplary results demonstrating that GF9-dLPC (disks) and GF9-sLPC
(spheres) reduce LPS-induced cytokine release in vitro Fig. 100A and in vivo Fig. 100B and prolong survival Fig. 100C (Sigalov 2014). In cancer mice, GF9 and GF9-LPC
treatments inhibit production of CSF-1/M-CSF but not VEGF Fig. 100D (Shen and Sigalov 2017). Fig.
100A.
CYTOKINES IN VITRO. Fig. 100B. CYTOKINES IN VIVO. Fig. 100C. SURVIVAL IN LPS-INDUCED SEPTIC MICE. Fig. 100D. M-CSF / VEGF RELEASE IN CANCER MICE.
Fig. 101 shows exemplary results demonstrating that Different rate and efficiency of GF9-dLPC
and GF9-sLPC in vitro uptake by J774 macrophages (Sigalov 2014).
Fig. 102 shows exemplary results demonstrating that Stability of GF9-LPC. GF9-LPC AT 4 C.
Fig. 103A-D shows exemplary results demonstrating that GF9-LPC daily i.p.
administered at 2.5 mg/kg suppress the expression of TREM-1, MCP-1/CCL2 and early fibrosis marker molecules in mice with ALD. Indicates significance level compared to nontreated pair-fed group; # indicates significance level compared to nontreated alcohol-fed group. Significance levels are as follows:
<0.05; **/#4, p < 0.01; ***,p <0.001; ****/ p <0.0001. Fig. 102A. TREM-1.
Fig.
102B. MCP-1/CCL2. Fig. 102C. Pro-Colllalpha. Fig. 102D. alpha-SMA.
Fig. 104A-D shows exemplary results demonstrating that GF9 and GF9-LPC daily i.p.
administered are well-tolerated Fig. 104A, suppress macrophage infiltration into the tumor Fig.
104B, 104C and inhibit release of CSF-1/M-CSF but not VEGF Fig. 104D. Scale bar = 200 pm.
*,p< 0.05; **,p < 0.01; ***,p < 0.001; ****,p < 0.0001 (vs vehicle). Fig.
104A. BODY
WEIGHT. Fig. 104B. INTRATITMORAL MACROPHAGE INFILTRATION ¨ INHIBITION
BY GF9 AND GF9-LPC. Fig. 104C. MACROPHAGE INFILTRATION. Fig. 104D. M-CSF /
VEGF RELEASE IN CANCER MICE.
Fig. 105A- C shows exemplary results demonstrating that in mice with autoimmune arthritis, GF9, discoidal GF9-LPC (GF9-dHDL) and spherical GF9-LPC (GF9-sHDL) i.p.
administered daily are well-tolerated Fig. A, ameliorate the disease Fig. 105B and inhibit production of cytokines and M-CSF Fig. C (Shen and Sigalov 2017). Fig. 105A. BODY WEIGHT
CHANGES.
Fig. 105B. ARTHRITIS AMELIORATION. Fig. 105C. CYTOKINE RELEASE IN
ARTHRITIC MICE.
DEFINITIONS
The term, "composition", as used herein, refers to any mixture of substances comprising a peptide and/or compound contemplated by the present invention. Such a composition may include the substances individually or in any combination.
As used herein the term "lipoprotein" such as VLDL (very low density lipoproteins), LDL (low density lipoproteins) and HDL (high density lipoproteins), refers to lipoproteins found in the serum, plasma and lymph, in vivo, related to lipid transport. The chemical composition of each lipoprotein differs, for examples, HDL has a higher proportion of protein versus lipid, whereas the VLDL has a lower proportion of protein versus lipid. When referring to lipoproteins, the term "native" refers to naturally-occurring (e.g., a "wild-type") lipoproteins.
The terms "AP0A1 HUMAN", "Apolipoprotein A-I", "Apolipoprotein A-1", "AP0A1", "ApoA-I", "Apo-AI", "ApoA-1", "apo-Al", "apoA-1" and "Apo-Al" refer to the naturally occurring human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name "AP0A1 HUMAN". The protein amino acid sequence can be found under the entry UniProt KB/Swiss-Prot P02647 (www.uniprot.org/uniprot/P02647). The terms "AP0A2 HUMAN", "Apolipoprotein A-II", Apolipoprotein A-2", "AP0A2", "ApoA-II", "Apo-All", "ApoA-2", "apo-A2", "apoA-2" and "Apo-A2" refer to the naturally occurring human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name "AP0A2 HUMAN". The protein amino acid sequence can be found under the entry .. UniProt KB/Swiss-Prot P02652 (http://www.uniprot.org/uniprot/P02652).
The term "TREM receptor", as used herein, refers to a member of TREM receptor family including: TREM-1, TREM-2, TREM-3 and TREM-4. The terms "TREM1 HUMAN", "TREM-1 receptor", "TREM-1 receptor subunit", "TREM-1 subunit", and "TREM-1 recognition subunit" refer to the naturally occurring human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name "TREM1 HUMAN". The protein amino acid sequence can be found under the entry UniProt KB/Swiss-Prot Q9NP99.
The term "TREM receptor", as used herein, refers to a member of TREM receptor family: TREM-1, TREM-2, TREM-3 and TREM-4.
As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y/6) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain, in some embodiments, the TCR may be modified on any ceil comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T ceil, regulatory T
cell, natural killer T
cell, and gamma delta T cell.
As employed herein and understood by the ordinary skill in the art, "amino acid domain"
is a contiguous polymer of at least 2 amino acids joined by peptide bond(s).
The domain may be joined to another amino acid or amino acid domain by one or more peptide bonds. An amino acid domain can constitute at least two amino acids at the N-terminus or C-terminus of a peptide or can constitute at least two amino acids in the middle of a peptide.
The term "antibody" herein refers to a protein, derived from a germline immunoglobulin sequence, which is capable of specifically binding to an antigen (TREM-1) or a portion thereof.
The term includes full length antibodies of any class or isotype (that is, IgA, IgE, IgG, IgM
and/or IgY) and any single chain or fragment thereof. An antibody that specifically binds to an antigen, or portion thereof, may bind exclusively to that antigen, or portion thereof, or it may bind to a limited number of homologous antigens, or portions thereof.
As used herein, a "peptide" and "polypeptide" comprises a string of at least two amino acids linked together by peptide bonds. A peptide generally represents a string of between approximately 2 and 200 amino acids, more typically between approximately 6 and 64 amino acids. Peptide may refer to an individual peptide or a collection of peptides.
Inventive peptides typically contain natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain and/
or amino acid analogs as are known in the art may alternatively be employed. In particular, D-amino acids may be used.
As employed herein and understood by the ordinary skill in the art, "peptide sequence", or "amino acid sequence", is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group. "Peptide sequence" is often called "protein sequence" if it represents the primary structure of a protein (http://en.wikipedia.org/wiki/Peptide sequence).
Peptides and compositions of the present invention made synthetically may include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or .. unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH2(CH2)õCOOH
wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;
citrulline and .. methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
Naturally occurring residues are divided into groups based on common side chain properties: as described herein. Analogues may be generated by substitutional mutagenesis and retain the biological activity of the original trifunctional peptides.
Examples of substitutions identified as "conservative substitutions" are shown in TABLE 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in TABLE 1, or as further described herein in reference to amino acid classes, are introduced and the products screened for their capability of executing three functions.
The term "amphipathic" is used herein to describe a molecule that has both polar and .. non-polar parts and as such, has two different affinities, as a polar end that is attracted to water and a nonpolar end that is repelled by it. An amphipathic helix is defined as an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix.
As well known in the art, amino acid sequences can be screened for amphipathic helixes and an amphipathicity score can be calculated using a variety of computer programs available online (see, for example, http ://www.tcdb . org/progs/?tool=pepwheel, http ://lbqp.unb . br/NetWhe el s/, https ://np s a-prabi.ibcp.fr/cgi-bin/npsa automat.pl?page=/NPSA/npsa amphipaseek.html, http ://rzlab .ucr. e du/scripts/wheel/whe el .cgi, http ://heli quest. ipm c.
cnrs.fr/cgi-bin/ComputParams.py) or other techniques including but not limiting to those described in Jones, et al. J Lipid Res 1992, 33:287-296.
As used herein, the term "aptamer" or "specifically binding oligonucleotide"
refers to an oligonucleotide that is capable of forming a complex with an intended target substance.
In the present disclosure, the term "modified peptide" is used to describe chemically or enzymatically, or chemically and enzymatically modified oligopeptides, oligopseudopeptides, polypeptides, and pseudopolypeptides (synthetic or otherwise derived), regardless of the nature of the chemical and/or enzymatic modification. The term "pseudopeptide" refers to a peptide where one or more peptide bonds are replaced by non-amido bonds such as ester or one or more amino acids are replaced by amino acid analogs. The term "peptides" refers not only to those comprised of all natural amino acids, but also to those which contain unnatural amino acids or other non-coded structural units. The terms "peptides", when used alone, include pseudopeptides.
It is worth mentioning that "modified peptides" have utility in many biomedical applications because of their increased stability against in vivo degradation, superior pharmacokinetics, and altered immunogenicity compared to their native counterparts.
The term "modified peptides," as employed herein, also includes oxidized peptides.
The term "oxidized peptide" refers to a peptide in which at least one amino acid residue is oxidized.
The term "analog", as used herein, includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
The term "conservative substitution", as used herein, also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite inhibitory function on myeloid cells as specified herein. The term derivative includes any chemical derivative of the peptide of the invention having one or more residues chemically derivatized by reaction of side chains or functional groups.
The term "homolog" or "homologous" when used in reference to a polypeptide refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structures or to a high degree of similarity between the active site and the mechanism of action. In a preferred embodiment, a homolog has a greater than 60%
sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as, for example, by the programs KALIGN, DOTLET, LALIGN and DIALIGN (https://www.expasy.org/tools) using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
The term "modified peptides," as employed herein, also includes oxidized peptides. The term "oxidized peptide" refers to a peptide in which at least one amino acid residue is oxidized.
The term "oxidation status" refers to a metric of the extent to which specific amino acid residues are replaced by corresponding oxidized amino acid residues in a peptide. The term "extent of oxidation" refers to the degree to which potentially oxidizable amino acids in a peptide have undergone oxidation. For example, if the peptide contains a single tyrosine residue which is potentially oxidized to 3-chlorotyrosine, then an increase in mass of about 34 Dalton (i.e., the approximate difference in mass between chlorine and hydrogen) indicates oxidation of tyrosine to 3-chlorotyrosine. Similarly, if the peptide contains a single methionine residue which is potentially oxidized to methionine sulfoxide, then an increase in mass of 16 Dalton (i.e., the difference in mass between methionine and methionine containing one extra oxygen) indicates oxidation of methionine to methionine sulfoxides.
The term "oxidation status" refers to a metric of the extent to which specific amino acid residues are replaced by corresponding oxidized amino acid residues in a peptide. The term "extent of oxidation" refers to the degree to which potentially oxidizable amino acids in a peptide have undergone oxidation. For example, if the peptide contains a single tyrosine residue which is potentially oxidized to 3-chlorotyrosine, then an increase in mass of about 34 Dalton (i.e., the approximate difference in mass between chlorine and hydrogen) indicates oxidation of tyrosine to 3-chlorotyrosine. Similarly, if the peptide contains a single methionine residue which is potentially oxidized to methionine sulfoxide, then an increase in mass of 16 Dalton (i.e., the difference in mass between methionine and methionine containing one extra oxygen) indicates oxidation of methionine to methionine sulfoxides.
The oxidation status can be measured by metrics known to the arts of protein and peptide chemistry (as disclosed in Caulfield, US 8,114,613 and Hazen, et al., US
8,338,110, herein incorportaed by reference) including, without limitation, assay of the number of oxidized residues, mass spectral peak intensity, mass spectral integrated area, and the like. In some embodiments, oxidation status is reported as a percentage, wherein 0% refers to no oxidation and 100% refers to complete oxidation of potentially oxidizable amino acid residues within apo A-I
or apo A-II peptide fragments.
The term "potentially subject to oxidation," "potentially oxidizable amino acid residues", and the like refer to an amino acid which can undergo oxidation, for example by nitration or chlorination.
A "biologically active peptide motif' is a peptide that induces a phenotypic response or change in an appropriate cell type when the cell is contacted with the peptide. The peptide may be present either in isolated form or as part of a larger polypeptide or other molecule. The ability of the peptide to elicit the response may be determined, for example, by comparing the relevant parameter in the absence of the peptide (e.g., by mutating or removing the peptide when normally present within a larger polypeptide). Phenotypic responses or changes include, but are not limited to, enhancement of cell spreading, attachment, adhesion, proliferation, secretion of an extracellular matrix (ECM) molecule, or expression of a phenotype characteristic of a particular differentiated cell type.
As used herein, a "minimal biologically active sequence" refers to the minimum length of a sequence of a peptide that has a specific biological function. In a first example, -IVILLAGGFLSKSLVFSVLFA- (e.g., Domain A, SEQ ID NO. 47) is a biologically active TREM-1 inhibitory sequence corresponding to the human TREM-1 transmembrane domain, wherein -GFLSKSLVF- (e.g. Domain A, SEQ ID NO. 1) has the sole function of inhibition. Thus, in this case, -GFLSKSLVF- (Domain A, SEQ ID NO. 1) is a "minimal biologically active sequence." In a second example, the sequence ¨
PLGEEMRDRARAHVDALRTHLARGD, and an internal sequence -GEEMRDRARAHVRGD-(Domain B, SEQ ID NO. 5) contains the sequence -RGD-; -RGD- has a cell attachment function.
However, -PLGEEMRDRARAHVDALRTHLARGD and -GEEMRDRARAHVRGD- (Domain B, SEQ ID NO. 5) also functions to assist in the formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with native lipoproteins and to promote binding of these particles with scavenger receptor type I (SR-B1). Thus, in this case, both -PLGEEMRDRARAHVDALRTHLARGD- andGEEMRDRARAHVRGD- (Domain B, SEQ ID
NO. 5) in addition to -RGD- are considered a "minimal biologically active sequence." In another example, the sequence -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO.
...) contains the sequence -RGD-; -RGD-has a cell attachment function.
However, -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO. ...) also has the functions of inhibition of TREM-1, assistance in the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures particle and of interaction with scavenger receptor type I (SRBI). Thus, in this case, both -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO. ...) and -RGD- are considered a "minimal biologically active sequence." As is understood from the present invention, the first and second amino acid domains of a resulting peptide contain at least one minimal biologically active sequence. This minimal biologically active sequence is any length of sequence from an original peptide sequence. Moreover, with the exception of the amino acids of the minimal biologically active sequence, the amino acids of any or both amino acid domain can be exchanged, added or removed according to the design of the molecule to adjust its overall hydrophilicity and/or net charge. In certain embodiments, the minimal biologically active sequence refers to any one of the sequences provided in TABLE 2.
The term "imaging agent" or "imaging probe" as used herein refers to contrast agents used in imaging techniques such as computed tomography (CT), gamma-scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MM), and combined imaging techniques in order to improve diagnostic performance of medical imaging.
The term "labeling substance or label or labeled probe" refers to a substance that can image whether there is a binding between the modulator and the cellular component (e.g., TREM-1/DAP-12 receptor complex), and can visualize the binding by a pattern.
It may include radioactive materials, fluorescent or emitting materials.
The term "carrier" as used herein, refers to a biocompatible nanoparticle that facilitates administration of a pharmaceutical agent to an individual.
The term "encapsulation" as used herein refers to the enclosure of a molecule, such as trifunctional peptides and compounds of the present invention, inside the nanoparticle. The term "incorporation" as used herein refers to imbibing or adsorbing the trifunctional peptides and compounds onto the nanoparticle. The terms "reconstituted" and "recombinant"
as used herein both refer to synthetic lipopeptide particles that represent both discoidal and spherical nanoparticles and mimic native HDL particles.
As used herein, "naturally occurring" means found in nature. A naturally occurring biomolecule is, in general, synthesized by an organism that is found in nature and is unmodified by the hand of man, or is a degradation product of such a molecule. A molecule that is synthesized by a process that involves the hand of man (e.g., through chemical synthesis not involving a living organism or through a process that involves a living organism that has been manipulated by the hand of man or is descended from such an organism) but that is identical to a molecule that is synthesized by an organism that is found in nature and is unmodified by the hand of man is also considered a naturally occurring molecule.
A "site of interest" on a target as used herein is a site to which modified peptides and compounds of the present invention bind.
The term "target site", as used herein, refers to sites/tissue areas of interest.
As used in this invention, the terms "target cells" or "target tissues" refer to those cells or tissues, respectively that are intended to be targeted using the compositions of the present invention delivered in accord with the invention. Target cells or target tissues take up or link with the modified peptides and compounds of the invention. As used in this invention, the terms "target cells" or "target tissues" refer to those cells or tissues, respectively that are intended to be treated and/or visualized in imaging techniques such as CT, gamma-scintigraphy, PET, SPECT, Mill, and combined imaging techniques, using the compositions of the present invention delivered in accord with the invention. Target cells are cells in target tissue, and the target tissue includes, but is not limited to, atherosclerotic plaques, vascular endothelial tissue, abnormal vascular walls of tumors, solid tumors, tumor-associated macrophages, and other tissues or cells related to cancer, cardiovascular, inflammatory, autoimmune diseases, and the like. Further, target cells include virus-containing cells, and parasite-containing cells.
Also included among target cells are cells undergoing substantially more rapid division as compared to non-target cells.
The term "target cells" also includes, but is not limited to, microorganisms such as bacteria, viruses, fungi, parasites, and infectious agents. Thus, the term "target cell" is not limited to living cells but also includes infectious organic particles such as viruses. "Target compositions" or "target biological components" include, but are not be limited to: toxins, peptides, polymers, and other compounds that may be selectively and specifically identified as an organic target that is intended to be visualized in imaging techniques using the compositions of the present invention.
The term "therapeutic agent" or "drug" as used herein refers to any compound or composition having preventive, therapeutic or diagnostic activity, primarily but not exclusively in the treatment of patients with macrophage (myeloid cell)-related diseases.
The term "myeloid cells" include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets.
The terms "macrophage-associated", "macrophage-mediated", and "macrophage-related diseases" include diseases associated with macrophages as disclosed in Low and Turk, US
8,916,167, herein incorportaed by reference in its entirety.
The term "plaque" includes, for example, an atherosclerotic plaque.
The term "myeloid cell-mediated pathology" (or "myeloid cell-related pathologies", or "myeloid cell-mediated disorder, or "myeloid cell-related disease"), as used herein, refers to any condition in which an inappropriate myeloid cell response is a component of the pathology. The term is intended to include both diseases directly mediated by myeloid cells, and also diseases in which an inappropriate myeloid cell response contributes to the production of abnormal antibodies, antibodies, as well as graft rejection.
The term "ligand-induced myeloid cell activation", as used herein, refers to myeloid cell activation in response to the stimulation by the specific ligand.
The term "stimulation", as used herein, refers to a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation entails the ligation of a receptor and a subsequent signal transduction event. With respect to stimulation of a myeloid cell, such stimulation refers to the ligation of a myeloid cell surface moiety that in one embodiment subsequently induces a signal transduction event, such as binding the TREM-1/DAP- 12 complex. Further, the stimulation event may activate a cell and up-regulate or down-regulate expression or secretion of a molecule.
The term "ligand", or "antigen", as used herein, refers to a stimulating molecule that binds to a defined population of cells. The ligand may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population. The ligand may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), or the like.
Within the specification and in the context of myeloid cell stimulation, the ligand (or antigen) binds the TREM receptor and this binding activates the myeloid cell.
The term "activation", as used herein, refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of myeloid cells, such activation, refers to the state of a myeloid cell that has been sufficiently stimulated to induce production of interleukin (IL) 1, 6 and/or 8 (IL-1, IL-6 and/or IL-8, respectively) and tumor necrosis factor alpha (TNF-alpha), differentiation of primary monocytes into immature dendritic cells, and enhancement of inflammatory responses to microbial products. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process.
The term "inhibiting myeloid cell activation" (or "TREM-mediated cell activation"), as used herein, refers to the slowing of myeloid cell activation, as well as completely eliminating and/or preventing myeloid cell activation.
The term, "treating a disease or condition", as used herein, refers to modulating myeloid cell activation including, but not limited to, decreasing cytokine production and differentiation of primary monocytes into immature dendritic cells and/or slowing myeloid cell activation, as well as completely eliminating and/or preventing myeloid cell activation. Myeloid cell-related diseases and/ or conditions treatable by modulating myeloid cell activation include, but are not limited to, cancer including but not limited to lung cancer, pancreatic cancer, multiple myeloma, melanoma, leukemia, prostate cancer, breast cancer, liver cancer, bladder cancer, stomach cancer, prostate cancer, colon cancer, colorectal cancer, CNS cancer, melanoma, ovarian cancer, gastrointestinal cancer, renal cancer, or osteosarcoma and other cancers, brain and skin cancers, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor (TGCT;
also referred to in the art as TSGCT), PVNS and other cancers in which myeloid cells are involved or recruited, cancer cachexia, in addition to ALD, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other rheumatic diseases, sepsis, diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases, and other myeloid cell-related inflammatory conditions eg myositis, tissue/organ rejection, brain and spinal cord injuries. Other exemplary cancers include, but are not limited to, adrenocortical carcinoma, acquired immune deficiency syndrome (AIDS)-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, uringary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodeimal tumors, visual pathway and hypothalamic glioma, bronchial adenomas/carcinoids, carcinoid tumor, nervous system cancer, nervous system lymphoma, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, islet cell pancreatic cancer, paranasal sinus and nasal .. cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterine cancer, .. uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, .. uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm's Tumor.
The term "detectable" refers to the ability to detect a signal over the background signal.
In accordance with the present disclosure, "a detectably effective amount" of the labeled probe of the present disclosure is defined as an amount sufficient to yield an acceptable image using equipment that is available for clinical use. A detectably effective amount of the labeled probe of the present disclosure may be administered in more than one injection. The detectably effective amount of the labeled probe of the present disclosure can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, and the like.
Detectably effective amounts of the probe of the present disclosure can also vary according to instrument and film-related factors. Optimization of such factors is well within the level of skill in the art.
The term "in vivo imaging" as used herein refers to methods or processes in which the structural, functional, molecular, or physiological state of a living being is examinable without the need for a life-ending sacrifice.
The term "inhibiting T cell activation", as used herein, refers to the slowing of T cell activation, as well as completely eliminating and/or preventing T cell activation.
The term "T cell-mediated pathology" (or "T cell-related pathologies", or "T
cell-mediated disorder, or "T cell-related disease"), as used herein, refers to any condition in which an inappropriate T cell response is a component of the pathology. The term is intended to include both diseases directly mediated by T cells, and also diseases in which an inappropriate T cell response contributes to the production of abnormal antibodies, as well as graft rejection.
The term "treating a T cell-mediated disease or condition", as used herein, refers to modulating T cell activation including, but not limited to, decreasing cellular proliferation, cytokine production and performance of regulatory or cytolytic effector functions and/or slowing T cell activation, as well as completely eliminating and/or preventing T cell activation. T cell-related diseases and/or conditions treatable by modulating T cell activation include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac, Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection.
The term, "subject" or "patient", as used herein, refers to any individual organism. For example, the organism may be a mammal, such as a primate (i.e., for example, a human) or a laboratory animal. Further, the organism may be a domesticated animal (i.e., for example, cats, dogs, etc.), livestock (i.e., for example, cattle, horses, pigs, sheep, goats, etc.), or a laboratory animal (i.e., for example, mouse, rabbit, rat, guinea pig, etc.).
The term, "therapeutically effective amount", "therapeutically effective dose"
or "effective amount", as used herein, refers to an amount needed to achieve a desired clinical result .. or results (e.g. inhibiting receptor-mediated cell activation) based upon trained medical observation and/or quantitative test results. The potency of any administered peptide or compound determines the "effective amount" which can vary for the various compounds that inhibit myeloid cell activation (i.e., for example, compounds inhibiting TREM
ligand-induced myeloid cell activation and/or TCR-mediated T cell activation). Additionally, the "effective amount" of a compound may vary depending on the desired result, for example, the level of myeloid cell activation inhibition desired. The "therapeutically effective amount" necessary for inhibiting differentiation of primary monocytes into immature dendritic cells may differ from the "therapeutically effective amount" necessary for preventing or inhibiting cytokine production.
The term, "agent", as used herein, refers to any natural or synthetic compound (i.e., for example, a peptide, a peptide variant, or a small molecule).
The term, "intrinsic helicity", as used herein, refers to the helicity which is adopted by a peptide in an aqueous solution. The term, "induced helicity", as used herein, refers to the helicity which is adopted by a peptide when in the presence of a helicity inducer, including, but not limited to, trifluoroethanol (TFE), detergents (e.g., sodium dodecyl sulfate, SDS) or lipids.
The term "therapeutic drug", as used herein, refers to any pharmacologically active substance capable of being administered which achieves a desired effect. Drugs or compounds can be synthetic or naturally occurring, non-peptide, proteins or peptides, oligonucleotides or nucleotides, polysaccharides or sugars. Drugs or compounds may have any of a variety of activities, which may be stimulatory or inhibitory, such as antibiotic activity, antiviral activity, antifungal activity, steroidal activity, cytotoxic, cytostatic, anti-proliferative, anti-inflammatory, analgesic or anesthetic activity, or can be useful as contrast or other diagnostic agents.
The term "effective dose" as used herein refers to the concentration of any compound or drug contemplated herein that results in a favorable clinical response. In solution, an effective dose may range between approximately 1 ng/ml and 100 mg/ml, preferably between 100 ng/ml and 10 mg/ml, but more preferably between 500 ng/ml and 1 mg/ml.
The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a subject. In certain embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of the compound or composition of the invention that modulate TREM-1/DAP-12 receptor complex signaling may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound or composition to elicit a desired response in the individual. A
therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the compound or composition are outweighed by the therapeutically beneficial effects.
As one example, in some embodiments, the expression "effective amount" refers to an amount of the compound or composition that is effective for treating cancer.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
A "induction therapy" refers to the first treatment given for a disease. It is often part of a standard set of treatments, such as surgery followed by chemotherapy and radiation. When used by itself, induction therapy is the one accepted as the best treatment. If it doesn't cure the disease or it causes severe side effects, other treatment may be added or used instead. Also called first-line therapy, primary therapy, and primary treatment.
A "maintenance therapy" refers to a medical therapy that is designed to help a primary treatment succeed. For example, maintenance chemotherapy may be given to people who have a cancer in remission in an attempt to prevent a relapse. In other words, treatment that is given to help keep cancer from coming back after it has disappeared following the initial therapy. It may include treatment with drugs, vaccines, or antibodies that kill cancer cells or keep tumor unfavorable microenvironment, and it may be given for a long time. This form of treatment is also a common approach for the management of many incurable, chronic diseases such as periodontal disease, Crohn's disease or ulcerative colitis.
Administration "in combination with" one or more further therapeutic agents includes .. simultaneous (concurrent) and consecutive (sequential) administration in any order.
A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.
The term "administered" or "administering" a drug or compound, as used herein, refers to any method of providing a drug or compound to a patient such that the drug or compound has its intended effect on the patient. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, syringe etc. A second exemplary method of administering is by a direct mechanism such as, local tissue administration (i.e., for example, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository etc.) The term, "agent", as used herein, refers to any natural or synthetic compound (i.e., for example, a peptide, a peptide variant, or a small molecule).
The term, "composition", as used herein, refers to any mixture of substances comprising a peptide and/or compound contemplated by the present invention. Such a composition may include the substances individually or in any combination.
The term "modulator" used in this invention refers to a substance and/or compositions contemplated by the present invention or a combination thereof with capacity to inhibit (e.g., "antagonist" activity) a functional property of biological activity or process (e.g., reducing or blocking TREM-1/DAP-12 activity ¨ signaling and/or activation); such inhibition can be contingent on the occurrence of a specific event, such as reduction or blockade of a signal transduction pathway, and/or can be manifest only in particular cell types.
For instance, small molecules such as drugs, proteins such as antibodies, hormones or growth factors, protein domains, protein motifs, and peptides or a combination thereof can act as a modulator.
The term "tissue sample" refers to a collection of similar cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, synovial fluid, or interstitial fluid; cells from any time in gestation or development of the subject. In some embodiments, a tissue sample is a synovial biopsy tissue sample and/or a synovial fluid sample. In some .. embodiments, a tissue sample is a synovial fluid sample. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue sample is obtained from a disease tissue/organ. The tissue sample may contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. A "control sample" or "control tissue", as used herein, refers to a sample, cell, or tissue obtained from a source known, or believed, not to be afflicted with the disease for which the subject is being treated.
For the purposes herein a "section" of a tissue sample means a part or piece of a tissue sample, such as a thin slice of tissue or cells cut from a solid tissue sample.
The term "anti-inflammatory drug" means any compound, composition, or drug useful for preventing or treating inflammatory disease.
The term "medical device", as used herein, refers broadly to any apparatus used in relation to a medical procedure. Specifically, any apparatus that contacts a patient during a medical procedure or therapy is contemplated herein as a medical device.
Similarly, any apparatus that administers a drug or compound to a patient during a medical procedure or therapy is contemplated herein as a medical device. "Direct medical implants"
include, but are not limited to, urinary and intravascular catheters, dialysis catheters, wound drain tubes, skin sutures, vascular grafts and implantable meshes, intraocular devices, implantable drug delivery systems and heart valves, and the like. "Wound care devices" include, but are not limited to, general wound dressings, non-adherent dressings, burn dressings, biological graft materials, tape closures and dressings, surgical drapes, sponges and absorbable hemostats.
"Surgical devices"
include, but are not limited to, surgical instruments, endoscope systems (i.e., catheters, vascular catheters, surgical tools such as scalpels, retractors, and the like) and temporary drug delivery devices such as drug ports, injection needles etc. to administer the medium. A
medical device is "coated" when a medium comprising an anti-inflammatory drug (i.e., for example, the peptides, compositions, and compounds of the present invention) becomes attached to the surface of the medical device. This attachment may be permanent or temporary. When temporary, the attachment may result in a controlled release of an inflammatory drug.
DETAILED DESCRIPTION OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-signaling pathway. The compositions, including peptides and peptide variants, modulate TREM-1-mediated immunological response as standalone and combination-therapy treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
The present disclosure describes novel amphipathic trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating diseases related to activated macrophages. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages.
As described herein, surprisingly it was found that potentially therapeutic trifunctional peptides of the present invention are capable of executing at least, three functions (trifunctional peptides): 1) assistance in the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures in vitro, i.e. incorporation of the trifunctional peptides as part of the lipid portion of synthetic/recombinant HDLs, then after administration; 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. Thus in some embodiments, trifunctional peptides, after mixing with lipids in vitro, may assist in the self-assembly of synthetic lipopeptide particles (SLP) upon binding to a lipid or to lipids in mixtures. In the methods of the present invention, the SLP of interest are synthetic nanoparticles that mimic human lipoproteins as recombinant (r)HDLs. While not being bound to any particular theory, it is believed that this interaction and ability to form lipopeptide/lipoprotein particles is mediated by the amphipathic alpha helical sequences of the trifunctional peptides described herein.
Another surprising discovery was that administration of potentially therapeutic trifunctional peptides of the present invention, that were not in rHDL
formulations, showed: 1) mediation of formation of naturally long half-life lipopeptide/lipoprotein particles (LP) upon interaction with native lipoproteins in vivo, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. Thus in some embodiments, free trifunctional peptides (i.e. not in rHDL formulations) as part of compounds of the present invention, after administration to populations of cells or administration to a mammal, may interact with native lipoproteins and form trifunctional peptide .. containing lipopeptide/lipoprotein particles in vivo.
Thus, potentially therapeutic trifunctional peptides of the present invention were synthesized and used for targeted treatment and imaging in vivo, as either formulations with HDLs or without, i.e. trifunctional peptides in a pharmaceutical formulation without HDLs.
Advantageous of using the trifunctional peptides described herein in order to solve numerous problems administering therapeutic or diagnostic compounds include avoiding high dosages of other TAs (therapeutic agents) and imaging probes required; and the lack of control and reproducibility of formulations, especially in large-scale production. In other words, using trifunctional peptides described herein, including trifunctional peptide formulations including therapeutic drug compounds, would potentially lower the amount of drug needed to reduce symptoms of a disease.
Another advantage is economic. Therapeutic peptides have relatively high synthetic and production costs, For example, the production cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500 Da molecular mass small molecule, which in turn exceeds the production cost of a 500 Da molecular mass small molecule by more than 10-fold up to less than 100-fold for each increase in magnitude of size. By combining three functions in one peptide significantly simplifies the manufacture of these trifunctional peptides as targeted drugs, and as delivery agents for drug compounds and imaging probes.
I. Trifunctional Peptides.
The present invention encompasses the discovery that it is possible to combine multiple functions in one polypeptide amino acid sequence, i.e. a trifunctional peptide, in order to confer a variety of properties on the resulting amphipathic multipeptide.
The present disclosure describes novel amphipathic trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating diseases related to activated immune cells. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In some embodiments, each trifunctional peptide is capable of at least three functions:
1) mediating the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages. The invention further relates to personalized medical treatments for cancer that involve targeting specific cancers by their tumor environment.
In preferred embodiments, trifunctional peptides of the present invention comprise two amino acid domains (See FIG. 1): domain A that confers therapeutic and/or diagnostic benefits in the context of the treatment, prevention, and/or detection of a disease or condition; and domain B that confers multiple benefits in the context of: 1A) formation of long half-life lipopeptide particles upon binding to lipid or lipid mixtures in vitro 1B) formation of long half-life LP upon interaction with lipoproteins in vivo, and 2) the targeted delivery of the particles formed to cells of interest and/or sites of disease or condition.
In one embodiment, the present invention includes a resulting trifunctional peptide comprising: (a) one amino acid domain that confers therapeutic and/or diagnostic benefits in the context of the treatment, prevention, and/or detection of a disease or condition; and (b) another amino acid domain that confers multiple benefits in the context of the self-assembly of naturally long half-life SLP and LP upon binding to lipid or lipid mixtures and targeted delivery of the particles formed to cells of interest and/or sites of disease or condition. In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence.
In one embodiment, the first amino acid domain comprises a cyclic peptide sequence.
In one embodiment, the first amino acid domain comprises a disulfide-linked dimer. In one embodiment, any or both of the amino acid domains include amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to a drug compound (therapeutic agent:
TA). In one .. embodiment, a therapeutic agent is selected from the group including, but not limited to, anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, and therapeutic peptides. In one embodiment, the therapeutic agent is a hydrophobic therapeutic agent. The therapeutic agent may also be selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to an imaging probe. In one embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MM). In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as a positron emission tomography (PET) imaging systems (and combined PET/computer tomography (CT) and PET/MM systems). In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to any or both of the domains. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof Although many examples describe or show results of using trifunctional peptides in formulations with rHDLs, it is not meant to limit the use of these trifunctional peptide sequences in HDL formulations. Conversely, examples describing or showing results of using trifunctional peptides alone, or in formulations without rHDLs is not meant to limit the use of such trifunctional peptides without rHDLs. Thus, in certain embodiments, the trifunctional peptides of the present invention may be administered within rHDLs, or administered in pharmaceutical formulations as part of rHDLs. In other embodiments, the trifunctional peptides of the present invention may be administered without rHDLs, or administered in pharmaceutical formulations without rHDLs.
In one embodiment, the peptides of the present invention form lipopeptide particles in vitro. In one embodiment, the peptides of the present invention form lipopeptide particles in vivo.
In certain embodiments, the present invention relates to peptides consisting of two amino acid domains, wherein upon binding to lipid or lipid mixtures, one amino acid domain assists in the self-assembly of naturally long half-life lipopeptide particles and targets these particles to macrophages, whereas another amino acid domain inhibits TREM-1/DAP-12 receptor complex expressed on macrophages.
In certain embodiments, the present invention relates to peptides comprising at least two amino acid domains, wherein upon binding to lipid or lipid mixtures, the first amino acid domain assists in the self-assembly of naturally long half-life lipopeptide particles and targets these particles to macrophages, whereas the second amino acid domain inhibits TREM-receptor complex expressed on macrophages.
In certain embodiments, the peptides of the present invention self-assemble upon binding to lipid or lipid mixtures in vitro to form synthetic lipopeptide particles (SLP) that mimic human lipoproteins and have a long half-life in a bloodstream. In one embodiment, the peptides and compounds of the present invention interact with endogenous lipoproteins in vivo and form long half-life LP. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof.
The peptides and compounds of the present invention and combinations thereof alone as well as the SLP formed upon their binding to lipid or lipid mixtures have a wide variety of uses, particularly in the areas of oncology, transplantology, dermatology, hepatology, ophthalmology, cardiovascular diseases, sepsis, autoimmune diseases, neurodegenerative diseases and other diseases and conditions. They also are useful in the production of medical devices (for example, medical implants and implantable devices).
The invention disclosed herein provides for methods of treating cancer using inhibitors of the TREM-1 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. The invention also provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using inhibitors of the TREM-1 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. The invention also provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using modulators of the TREM-1/DAP-12 signaling pathway. These compounds and compositions modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer in standalone and combination-therapy treatment regimen. The invention also provides for predicting the efficacy of TREM-1 modulatory therapies in patients with various cancers. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system cancer, stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, .. neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, and other cancers in which myeloid cells are involved or recruited and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using inhibitors of the TREM-1 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. The invention also provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating scleroderma using modulators of the TREM-1/DAP-12 signaling pathway. These compounds and compositions modulate the TREM-1-m edi ated immunological responses beneficial for the treatment of scleroderma or a related autoimmune or a fibrotic condition in standalone and combination-therapy treatment regimen. The invention also provides for predicting the efficacy of TREM-1 modulatory therapies in patients with scleroderma. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or tel angi ectasi a syndrome (CREST).
In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis and other rheumatic diseases, sepsis and other inflammatory diseases or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, including but not limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases.
The disclosure also provides for a method of treating, preventing and/or detecting an immune-related condition. The method comprises providing a composition comprising peptides and compounds of the present disclosure and/or a synthetic nanoparticle formed upon their binding to lipid or lipid mixtures, a patient having at least one symptom of a disease or condition in which the immune system is involved, and administering the composition to the patient under conditions such that said one symptom is reduced. The immune-related condition of the method may include a heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, multiple sclerosis, the cancers (e.g., sarcoma, lymphoma, leukemia, carcinoma and melanoma), bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, autoimmune diseases (e.g., atopic dermatitis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as chronic obstructive .. pulmonary disease ( COPD ), interstitial pneumonitis and asthma, retinopathy such as diabetic retinopathy and retinopathy of prematurity, inflammatory bowel disease such as Crohn's disease, and inflammatory arthritis), liver diseases (e.g., alcoholic liver disease and nonalcoholic fatty liver disease), neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases, and transplant (e.g., heart/lung transplants) rejection reactions.
The invention relates to personalized medical treatments for scleroderma. More specifically, the invention provides for treatment of scleroderma or a related autoimmune or a fibrotic condition by using inhibitors of the TREM-1/DAP-12 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of scleroderma. In addition, the invention provides for predicting the efficacy of TREM-1-targeted therapies in scleroderma by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the peptides and compositions of the present invention modulate receptor complex expressed on macrophages. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe. In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with scleroderma using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof In one embodiment, the peptides and compositions of the invention are used in combinations with other antifibrotic therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST).
Trifunctional Peptides In rHDL (SLP) Formulations.
In one embodiment, the SLP self-assembled upon binding of the peptides and compounds .. of the present invention and combinations thereof to lipid or lipid mixtures are discoidal or spherical in shape. While the size of the particles is preferably between 5 nm and 50 nm, the diameter may be up to 200 nm. In one embodiment, the lipid of the particles may include cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, or a triacylglycerol. And further, the phospholipid may include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), or phosphatidic acid (PA). And even further, the cationic lipid can be 1,2-dioleoy1-3-trimethylammonium-propane (DOTAP). The lipid of the synthetic nanoparticle may be polyethylene glycol(PEG)ylated. In certain embodiments, the peptides and compounds of the present invention and/or the SLP and LP formed by these peptides and compounds may pass the blood-brain barrier (BBB). In one embodiment, the peptides and compounds of the present invention and/or the SLP and LP formed by these peptides and compounds may pass the blood-retinal barrier (BRB). In one embodiment, the peptides and compounds of the present invention and/or the SLP and LP formed by these peptides and compounds may pass the blood-tumor barrier (BTB).
In certain embodiments, the peptides and compounds of the present invention include an amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E. In one embodiment, the peptides and compounds of the present invention include an amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E and Arginine-glycine-aspartic acid (RGD)-peptide sequence. In certain embodiments, the peptides and compounds of the present invention include an amino acid sequence derived from transmembrane domain sequences of human or animal cell-surface receptors and of signaling subunits thereof In certain embodiments, the peptides and compounds of the present invention include an amino acid sequence derived from virus membrane fusion and structural proteins. In one embodiment, the peptides and compounds of the present invention include an amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E conjugated to a targeting moiety to enhance the targeting efficacy of the therapeutic agent. The targeting moiety may include a polypeptide, an antibody, a receptor, a ligand, a peptidomimetic agent, an aptamer or a product of phage display.
In one embodiment, the amino acid domains of the peptides and compounds of the present invention comprise unmodified or modified peptide sequences. The modified peptide sequence may contain at least one amino acid residue which is chemically or enzymatically modified. The modified amino acid residue may be an oxidized amino acid residue. The oxidized amino acid residue may be a methionine residue. The modified peptide sequence may contain at least one amino acid residue, which is oxidized, halogenated, or nitrated. The modified peptide sequence may include an amphipathic amino acid sequence.
In certain embodiments, the present invention relates to the targeted treatment or prevention of inflammatory or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, such as cancer including but not limited to, lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, sepsis, diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis and other rheumatic diseases.
In one embodiment, the present invention provides a pharmaceutical composition comprising the peptides and compounds and combinations thereof alone or the SLP
nanoparticles self-assembled upon binding of these peptides and compounds to lipid or lipid mixtures.
A. TREM-1-related Trifunctional peptides.
TREM-1 is expressed on the majority of innate immune cells and to a lesser extent on parenchymal cells. Upon activation, TREM-1 can directly amplify an inflammatory response.
Although it was initially demonstrated that TREM-1 was predominantly associated with infectious diseases, recent evidences demonstrate that TREM-1 receptor and its signaling pathways contribute to the pathology of non-infectious acute and chronic inflammatory diseases, including but not limiting to, rheumatoid arthritis, atherosclerosis, ischemia reperfusion-induced tissue injury, colitis, fibrosis, neurodegenerative diseases, liver diseases, retinopathies, and cancer (see e.g., Tammaro, et al. Pharmacol Ther 2017, 177:81-95; Saadipour.
Neurotox Res 2017, 32:14-16; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov.
US 8,513,185;
Sigalov. US 9,981,004; Rojas, et al. Biochim Biophys Acta 2018, 1864: 2761-2768, Tornai, et al.
Hepatology Communications 2018, in press, and Kuai, et al. US 2008/0247955).
In certain embodiments, a resulting trifunctional peptide of the present invention comprises two amino acid domains, wherein one domain comprises a variant TREM-1 inhibitory amino acid sequence and functions to inhibit TREM-1/DAP-12 receptor complex expressed on myeloid cells (e.g. macrophages), whereas another amino acid domain comprises the chemically and/or enzymatically modified amino acid sequence derived from apolipoprotein amino acid sequences and functions to assist in the self-assembly of SLP upon binding to lipid or lipid mixtures in vitro and/or to form LP in vivo, respectively, and to target these particles to myeloid cells (e.g. macrophages). In one embodiment, the TREM-1 inhibitory amino acid domain is the .. N-terminal domain of a resulting peptide. In one embodiment, the TREM-1 inhibitory amino acid domain is the C-terminal domain of a resulting peptide. In one embodiment, the TREM-1 inhibitory amino acid domain comprises a cyclic peptide sequence. In one embodiment, the TREM-1 inhibitory amino acid domain comprises a disulfide-linked dimer. In one embodiment, the TREM-1 inhibitory amino acid domain includes the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging agent is conjugated to the TREM-1 inhibitory amino acid domain or to the apolipoprotein amino acid sequence-derived domain or to both.
In some preferred embodiments, TREM-1-related peptides and associated compositions of the present invention have a domain A conjugated to a domain B. See, Fig.
1. Domain A
comprises a TREM-1 modulatory peptide sequence designed using a known model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TREM-1 receptor expressed on myeloid cells (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 8,513,185; and Sigalov. US 9,981,004), all of which are herein incorporated by reference in their entirety.
In some preferred embodiments, peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic alpha helical peptide fragment, such as a apo A-I and/or A-II peptide fragment, to form upon interaction with lipid and/or lipid mixtures. In certain embodiments, exemplary trifunctional peptides comprise the domain B comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
FIG. 1 presents an exemplary schematic representation of one embodiment of a trifunctional peptide of the present invention comprising amino acid domains A and B where amino acid domain A represents a therapeutic peptide sequence with or without an attached drug compound and/or imaging probe that functions to treat, prevent and/or detect a disease or condition, whereas amino acid domain B represents an amphipathic alpha helical peptide sequence, with or without an additional targeting peptide sequence, and functions to 1) assist in the self-assembly of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with lipids or lipid mixtures in vitro, for use in transporting these trifunctional peptides as lipoprotien nanoparticles to sites of interest in vitro or in vivo and/or 2) form long half-life lipopeptide/lipoprotein particles upon interaction with endogenous lipoproteins for transporting these trifunctional peptides to the sites of interest. Endogenous lipoproteins may be lipoproteins added to or found in cell cultures, or lipoprotiens in a mammalian body.
In certain embodiments, FIG. 2 shows the structures of representative TREM-1-related trifunctional peptides, TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ
ID NO. 2) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. GFLSKSLVF, is found within isoform 1 of human (UniProtKB - Q9NP99 (TREM1 HUMAN), and in human TREM-1 isoform CRA a (UniProtKB - Q38L15 (Q38L15 HUMAN), both downloaded 10-24-2018)). Peptide GFLSKSLVF is also described without an attached apo I peptide domain, in, for examples, WO
2011/047097 "Inhibition of trem receptor signaling with peptide variants."
Publication Date:
21.04.2011, U59981004B2 "Inhibition of TREM receptor signaling with peptide variants."
Published June 5, 2014, each of which is herein incorporated by reference in its entirety.
Sequence information was downloaded 10-25-, 10-26- or 10-27-2019.
Q9NP991TREM1 HUMAN Isoform 1 Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
Q38L15938L15 HUMAN Triggering receptor expressed on myeloid cells 1, Homo sapiens isoform CRA a:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF S GTP GSNENS T QNVYKIPP TT TKAL CPLYT SPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVF SVLF AVTLR SF VP
GFLSKSLVF, is not found within human TREM-1 isoforms 2 or 3.
Q9NP99-21TREM1 HUMAN Isoform 2 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFRCSTL SF SWLVDS
Q9NP99-31TREM1 HUMAN Isoform 3 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLW GLLWMLF V SELRAATKLTEEKYELKEGQ TLDVKCDYTLEKF A S S QKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF S GTP GSNENS T QNVYKIPP TT TKAL CPLYT SPRTVTQAPP
KST.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP). GE31 (GFL SKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) comprises amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22 amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Imaging of TREM-1 expression.
Another way to evaluate the TREM-1 expression level is to use imaging (visualization) techniques and procedures. In one embodiment, FIG. 50 shows that the fluorescently labeled TREM-1/TRIOPEP peptide GE31 delivered to macrophages by the SLP particles colocalizes with TREM-1 expressed on these cells. See also (Rojas et al. 2018). As described herein and in (Rojas et al. 2018), TREM-1 inhibitory therapy using the modulators of the signaling pathway results in reduction of tissue TREM-1 expression as measured by Western Blot (See Fig. 13).
In certain embodiments, the capability of the modulators of the TREM-1/DAP-12 signaling pathway described herein, including but not limited to, anti-TREM-1 blocking antibodies and fragments thereof, TREM-1 inhibitory SCHOOL peptides (e.g., GF9) and trifunctional TREM-1 inhibitory peptides including but not limited to, GA31 and GE31, to colocalize with TREM-1 can be used to visualize (image) this receptor and evaluate its expression/level in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu-.j, see TABLE 3) can be conjugated to the peptide sequences, GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 27) and/or GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide) (SEQ ID NO. 26). In one embodiment, methionine residues of the peptides GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 25) and GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 24) are unmodified. In one embodiment, imaging (visualization) of TREM-1 levels using the labeled modulators described herein and the PET and/or other imaging techniques can be used to diagnose GBM
and/or to select and monitor novel GBM therapies as disclosed in WO
2017083682A1 and described in (Johnson et al. 2017, Liu et al. 2019). In certain embodiments, imaging (visualization) of TREM-1 levels can be used to diagnose other TREM-1-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
GF9 immunotherapy targets pathways restricted to pathological conditions and is highly competitive. In some embodiments, safe and effective GF9 therapies are contemplated for use on pancreatic cancer (PC) to be used in combination with standard first-line treatments:
FOLFIRINOX (5-FU, leucovorin, irinotecan and oxaliplatin) or Gemzar +
ABRAXANE .
In some embodiments, advantages for using free GF9 peptide for treating PVNS
include but are not limited to: Low toxicity; Proven efficacy in vivo, including joints; easy formulation development; easy scale-up process; Easy and fast GMP production; Low cost of production;
and Stable and easy to store.
Therapy*
* Shown for Cancer Acute toxicity Risk of side effects Administration Cost Indications Systemic /
GF9 immunotherapy Intranasal /
LOW LOW LOW
(as described herein) Pulmonary /
Oral Cytotoxic drugs (Gemzar, Abraxane, HIGH HIGH Systemic / Oral HIGH
Temozol omi de) Biologies LOW HIGH
Systemic HIGH
(Bevacizumab, Canakinumab) In certain embodiments, other preferred TREM-1-related trifunctional peptides and compositions of this class comprise the domain A comprising the TREM-1 inhibitory peptide sequences LR12 and LP17 (described in Gibot, et al. Infect Immun 2006, 74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J Immunol 2007, 37:456-466;
Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J Clin Pharmacol 2018, in press; Zhou, et al.
Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et al., US
8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US 9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136, each of which is herein incorporated by reference in its entirety) while the domain B comprises at least one modified or unmodified amphipathic apo A-I
and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal. In some embodiments, resulting trifunctional peptide sequences may be radiolabeled and/or contain unmodified or modified methionine residues (TABLE 2) including but not limiting to, the following sequences:
LQEEDAGEYGCNIPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide (SEQ ID NO 7), LQEEDAGEYGCNIPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO
8), LQVTDSGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA (M(0), methinone sulfoxide (SEQ ID NO 9), LQVTDSGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO 10).
SLP (rHDL) structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US
20130039948;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). The inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 2) and TREM-1/TRIOPEP
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. In one embodiment, interaction of TREM-1/TRIOPEP GA31 with lipids results in self-assembly of nanosized SLP of discoidal or spherical morphology (dSLP and sSLP, respectively) (see FIG. 3).
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising amino acid domains A and B. Depending on lipid mixture compositions added to the peptides, sub 50 nm-sized SLP particles of discoidal (TREM-1/TRIOPEP-dSLP) or spherical (TREM-sSLP) morphology are self-assembled upon binding of the trifunctional peptide to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to intraplaque macrophages in vivo (FIG. 4A). In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to tumor-associated macrophages (TAMs) in vivo (FIG. 4B).
FIG. 4A illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for atherosclerosis), whereas domain B represents a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of atherosclerosis. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP
of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by intraplaque macrophages and return to the circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 5), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety.
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the trifunctional peptide of the present invention comprising two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence, whereas domain B is a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
In one embodiment, FIG. 6 shows that the fluorescently labeled TREM-1/TRIOPEP
peptide GE31 delivered to macrophages by the SLP particles of the present invention colocalizes with TREM-1 expressed on these cells (see also Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). In certain embodiments, the capability of the TREM-1-related trifunctional peptides and compounds of the present invention including but not limiting to, TREM-1/TRIOPEP GA31 and TREM-1/TRIOPEP GE31, to colocalize with TREM-1 can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64Cu], see TABLE 2) can be conjugated to the TREM-1/TRIOPEP
sequences, GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, imaging (visualization) of TREM-1 levels using PET and/or other imaging techniques can be used to diagnose glioblastoma multiforme (GBM) and/or to select and monitor novel GBM therapies (see e.g., Johnson, et al.
Neuro Oncol 2017, 19:vi249 and James and Andreasson, WO 2017083682A1). In certain embodiments, imaging (visualization) of TREM-1 levels can be used to diagnose other TREM-1-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with TREM-1 in the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and a merged image (FIG. 6C).
As described herein (see FIG. 7), sulfoxidation of methionine residues in the TREM-1/TRIOPEP peptides GE31 and GA31 results in increased macrophage endocytosis of the SLP
containing an equimolar mixture of these peptides (designated as TREM-1/TRIOPEP), TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP. Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety.
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic lipopeptide particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that contain an equimolar mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 (TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The post 4 h incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified methionine residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4 (white bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001 to 0.001 as compared with 4 h incubation time.
In certain embodiments, FIGS. 8 and 10 demonstrate that TREM-1/TRIOPEP in free and SLP-bound forms inhibits TREM-1 function as shown by reduction of TREM-1-mediated release of pro-inflammatory cytokines, both in vitro (FIG. 8) and in vivo (in serum) (FIG. 10).
.. While not being bound to any particular theory, it is believed that this indicates that similarly to TREM-1-inhibitory peptide GF9 (see e.g., Sigalov. Int Immunopharmacol 2014, 21:208-219;
Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety), TREM-1-related trifunctional peptides can reach their site of action from both outside (free TREM-1/TRIOPEP) and inside (SLP-bound TREM-1/TRIOPEP) the cell. It is also believed that upon administration, free TREM-1/TRIOPEP may form LP in vivo and/or interact with native lipoproteins, resulting in formation of HDL-mimicking LP. In one embodiment, these LP may further target the cells of interest delivering their content to the areas of interest in a body.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production by lipopolysaccharide (LPS)-stimulated macrophages incubated for 24 h at 37 C with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged macrophages.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post lipopolysaccharide (LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline (PBS), dexamethasone (DEX), control peptide and with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
Control peptide represents an equimolar mixture of two peptides, each of them comprising two amino acid domains A and B where domain A represents a non-functional 9 amino acids-long sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5 is substituted with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as compared with PBS-treated animals.
While not being bound to any particular theory, it is believed that increased uptake described herein, is mediated by macrophage scavenger receptors (SR) including, but not limiting to, SR-A and SR-B1 (see FIG. 9A1,A2-C). While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 9A), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety.
FIG. 9A1-A2-C shows schematic representations of activation of the TREM-1/DAP12 receptor complex expressed on macrophages and presents the exemplary data showing that scavenger receptors SR-A and SR-B1 mediate the macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1, right panel).
FIG. 9A2, left panel shows schematic representations of activation of the TREM-receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm;
GF9 self-penetrates the cell membrane and blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and the downstream signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774 macrophages were cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-HDL, cells were treated for 1 hour at 37 C with 40 [tM cytochalasin D and either (FIG.
9B) 400 g/mL
fucoidan or (FIG. 9C) 10 M BLT-1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars) or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence intensities of lysates were measured and normalized to the protein content.
Results are expressed as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of GF9-HDL and GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo-oligomerization.
In certain embodiments, FIGS. 11A-B -14 demonstrate that TREM-1/TRIOPEP in free .. and SLP-bound forms inhibits tumor growth, reduces infiltration of macrophages into the tumor in mouse models of NSCLC and PC and is well-tolerated by cancer mice during the treatment period (see also Shen and Sigalov. Mol Pharm 2017, 14:4572-4582, each of which is herein incorporated by reference in its entirety).
IG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****, p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *, p < 0.05; **, p < 0.01, ****, p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP-bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar = 200 m.
In embodiment, FIG. 15 demonstrates that TREM-1/TRIOPEP in free and SLP-bound forms significantly prolongs survival in mice with lipopolysaccharide (LPS)-induced septic shock.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
In one embodiment, FIG. 16 shows that TREM-1/TRIOPEP is non-toxic in healthy mice at least up to 400 mg/kg.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
In one embodiment, FIG. 17 demonstrates that TREM-1/TRIOPEP in free and SLP-bound form ameliorates arthritis in mice with collagen-induced arthritis (CIA) and is well-tolerated by arthritic mice during the treatment period of 2 weeks (see Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety).
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *, p <0.05, **, p <0.01;
***, p <0.001 as compared with vehicle-treated or naive animals.
In certain embodiments, FIG. 18 demonstrates that TREM-1/TRIOPEP-sSLP prevents pathological RNV in mice with oxygen-induced retinopathy and is well-tolerated by these mice during the treatment period (see Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety).
FIG. 18A-D presents the exemplary data showing reduction of pathological retinal neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-1 (FIG. 18C) and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-induced retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP) particles of spherical morphology (TREM-1/TRIOPEP-sSLP). * * *, p < 0.001 as compared with vehicle-treated animals.
As described in Stukas, et al. J Am Heart Assoc 2014, 3:e001156, herein incorporated by reference in its entirety, systemically administered human apo A-I accumulates in murine brain.
It is also known that transcytosis of HDL in brain microvascular endothelial cells is mediated by SRBI (see Fung, et al. Front Physiol 2017, 8:841, herein incorporated by reference in its entirety). However, until tested as described herein, it was not known that a self-assembled SLP
of the present invention comprising a trifunctional peptide was capable of crossing the BBB.
In certain embodiments, FIG. 19 shows that the self-assembled SLP of the present invention may cross the BBB, BRB and BTB, thus delivering their constituents including but not limiting to, TREM-1/TRIOPEP, GF9, GA31 and GE31, to the areas of interest in the brain, retina and tumor. In certain embodiments, FIG. 63 demonstrates that the fluorescently labeled sSLP described herein may cross the BBB, BRB and BTB, thus delivering their constituents including but not limiting to, GBCA imaging probe to the areas of interest in the brain, retina and tumor.
While not being bound to any particular theory, it is believed that the brain-, retina-, and tumor-penetrating capabilities of these SLP can be mediated by interaction of SRBI with the domain B amino acid sequences that correspond to the sequences of human apo A-I helices 4 and/or 6 (see e.g. Liu, et al. J Biol Chem 2002, 277:21576-21584, herein incorporated by reference in its entirety).
In certain embodiments, these capabilities of the peptides and compositions of the present invention can be used to diagnose, treat and/or prevent cancers (including brain cancer), diabetic retinopathy and retinopathy of prematurity, neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases and other diseases and conditions where delivery of the peptides and compositions of the invention to the brain, retina and/or tumor is needed.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical self-assembled particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-sSLP) or an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
A mouse model of ALD mimics the early phase of the human disease, yet mRNA
levels of early fibrosis markers Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of these makers was remarkably attenuated in the vehicle-treated group and, importantly, further decreased by the TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B cc-Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
TREM-1 inhibitor effects were evaluated on hepatocyte damage and steatosis in liver.
Serum ALT levels obtained during week 5 of the alcohol feeding showed significant increases in alcohol-fed mice compared to PF controls. This ALT increase was attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig. 21A).
Surprisingly, vehicle .. treatment (HDL) also showed a similar protective effect (Fig. 21A).
Consistent with steatosis, we found a significant increase in Oil Red 0 staining in livers of alcohol-fed mice compared to PF controls (Fig. 21C). Oil Red 0 (Fig. 21B-D) and H& (Fig.
21D) staining revealed attenuation of steatosis in the alcohol-fed TREM-1 inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig.
21B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
WooP < 0.01;*"/#"P <0.001; ****P < 0 .0001. *** , 0.001 > P > 0.0001; ##, 0.01 > P > 0.001.
B. TCR-Related Trifunctional Peptides The T-cell receptor (TCR)-CD3 complex plays a role in T-cell differentiation, in .. protecting the organism from infectious agents, and in the function of T-cells. The TCR is a complex of a heterodimer of TCRa and TCRb chains, which are responsible for antigen recognition and interaction with the major histocompatibility complex (MHC) molecules of antigen-presenting cells, and CD3d, CD3g, CD3e and CD3z chains, which are responsible for transmembrane signal transduction (see e.g., Manolios, et al. Cell Adh Migr 2010, 4:273-283;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 20130039948;
Manolios.
US 6,057,294; Manolios. US 7,192,928; Manolios. US 20100267651; and Manolios, et al. US
20120077732, each of which is herein incorporated by reference in its entirety).
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic alpha helical peptide fragment. As described above, the inclusion of an amphipathic amino acid sequences aids the assistance in the ability to interact with native lipoproteins in a bloodstream in vivo and to form naturally long half-life lipopeptide/lipoprotein particles LP. It further aids the ability to provide targeted delivery to the sites of interest. It further aids the ability to cross the BBB, BRB and BTB.
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP
and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides:
(MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 11), TCR/TRIOPEP
ME32 (MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 12), TCR/TRIOPEP GA36 (GARSMTLTVQARQLPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO 13), TCR/TRIOPEP GE36 (GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 14), TCR/TRIOPEP GA32 (GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO 15), and TCR/TRIOPEP
(GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 16). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRa chain with the CD3ed heterodimer and CD3zz homodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
(LGKATLYAVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 17) and TCR/TRIOPEP
LE32 (LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 18). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRb chain with the CD3eg heterodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
(YLLDGILFIYPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 19) and TCR/TRIOPEP
YE32 (YLLDGILFIYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 20). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3zz homodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32 (IIVTDVIATLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 21) and TCR/TRIOPEP
1E32 (IIVTDVIATLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 22). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3ed heterodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
(FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 23) and TCR/TRIOPEP
FE32 (FLFAEIVSIFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 24). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg heterodimer with TCRb chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
IA32e (IVIVDICITGPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 25) and TCR/TRIOPEP
IE32e (IVIVDICITGPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 26). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg and CD3ed heterodimers with TCRb and TCRa chains, respectively, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TCR can be used to treat and/or prevent TCR-related diseases and conditions including but not limiting to, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis; autoimmune disease e.g. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel disease, Crohn's disease, primary biliary cirrhosis, chronic active hepatitis;
skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g.
allergic alveolitis; cardiovascular problems e.g. autoimmune pericarditis;
organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; and any other disorder where T
cells are involved/recruited In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TCR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In 64cut one embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to the TCR/TRIOPEP sequences In one embodiment, imaging (visualization) of TCR
levels using PET and/or other imaging techniques can be used to diagnose TCR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
C. NKG2D-Related Trifunctional Peptides NKG2D is an activating receptor expressed by natural killer (NK) and T cells.
The NKG2D is a complex of an NKG2D chain, which is responsible for ligand recognition, and DAP10 homodimer, which is responsible for transmembrane signal transduction (see e.g.
Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589;
Sigalov. Self Nonself 2010, 1:4-39; and Sigalov. Self Nonself 2010, 1:192-224, each of which is herein incorporated by reference in its entirety). NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease (see e.g. Gonzalez, et al. Trends Immunol 2008, 29:397-403; Ito, et al. Am J Physiol Gastrointest Liver Physiol 2008, 294:G199-207; Vilarinho, et al. Proc Natl Acad Sci U S A
2007, 104:18187-18192; Van Belle, et al. J Autoimmun 2013, 40:66-73; Lopez-Soto, et al. Int J
Cancer 2015, 136:1741-1750; and Urso, et al. US 9,127,064, each of which is herein incorporated by reference in its entirety).
The preferred NKG2D-related peptides and compositions of this class comprise the domain A comprising the NKG2D modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating NKG2D (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative NKG2D-related trifunctional peptides: NKG2D/TRIOPEP IA36 (IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 27) and NKG2D/TRIOPEP 1E36 (IAVAMGIRFIIMVAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 28). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with NKG2D in the cell membrane and selectively disrupt intramembrane interactions of NKG2D chain with the DNAX-activation protein 10 (DAP-10) signaling homodimer, resulting to specific ligand-independent inhibition of NKG2D upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
.. and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of NKG2D/TRIOPEP peptides are modified.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit NKG2D can be used to treat and/or prevent NKG2D-related diseases and conditions including but not limiting to, celiac disease, type I diabetes, hepatitis, and rheumatoid arthritis, and any other disorder where NKG2D cells are involved/recruited. In one embodiment, the present invention provides methods and compositions for preventing NK cell-mediated graft rejection.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with NKG2D can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the NKG2D/TRIOPEP sequences In one embodiment, imaging (visualization) of NKG2D levels using PET and/or other imaging techniques can be used to diagnose NKG2D-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
D. GPVI-Related Trifunctional Peptides.
In recent years, the central activating platelet collagen receptor, glycoprotein (GP) VI, has emerged as a promising antithrombotic target because its blockade or antibody-mediated depletion in circulating platelets was shown to effectively inhibit experimental thrombosis and thromboinflammatory disease states, such as stroke, without affecting hemostatic plug formation.
GPVI is a complex of an GPVI chain, which is responsible for ligand recognition, and FcRg homodimer, which is responsible for transmembrane signal transduction (see e.g. Sigalov.
Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov.
Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. J
Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692;
Dutting, et al. Trends Pharmacol Sci 2012, 33:583-590; Ungerer, etal. PLoS One 2013, 8:e71193;
Sigalov, US
8,278,271; Sigalov, US 8,614,188, each of which is herein incorporated by reference in its entirety). The binding of GPVI to collagen or other antagonists ligands induces platelet adhesion, activation and aggregation. Platelet activation is a step in the pathogenesis of ischemic cardio-.. and cerebrovascular diseases, which represent the leading causes of death and severe disability worldwide. Although existing antiplatelet drugs have proved beneficial in the clinic, their use is limited by their inherent effect on primary hemostasis, making the identification of novel pharmacological targets for platelet inhibition a goal of cardiovascular research.
The preferred GPVI-related peptides and compositions of this class comprise the domain .. A comprising the GPVI modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating GPVI (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative GPVI-related trifunctional peptides:
(GNLVRICLGAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 29) and GPVI/TRIOPEP
GE32 (GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 30). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with GPVI in the cell membrane and selectively disrupt intramembrane interactions of GPVI chain with the FcRg signaling homodimer, resulting to specific ligand-independent inhibition of GPVI upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of GPVI/TRIOPEP peptides are modified.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit GPVI can be used to treat and/or prevent GPVI-related diseases and conditions including but not limiting to, ischemic and thromboinflammatory diseases, and any other disorder where platelets are involved/recruited.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with GPVI can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the GPVI/TRIOPEP sequences In one embodiment, imaging (visualization) of GPVI levels using PET and/or other imaging techniques can be used to diagnose GPVI-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
E. DAP-10- and DAP-12-Related Trifunctional Peptides The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. DAP-10- and DAP-12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play a role in innate immune responses. See e.g. Lanier. Immunol Rev 2009, 227:150-160; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224, each of which is herein incorporated by reference in its entirety.
The preferred DAP-10 and DAP-12-related peptides and compositions of this class comprise the domain A comprising the DAP-10 or DAP-12 modulatory peptide sequences, respectively, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating DAP-10- and associated receptors (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP
.. structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim .. Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I
sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-10-related trifunctional peptides: DAP-10/TRIOPEP LA32 (LVAADAVASLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 33) and DAP-10/TRIOPEP LE32 (LVAADAVASLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 34).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-10-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-10 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of DAP-10/TRIOPEP
peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-12-related trifunctional peptides: DAP-12/TRIOPEP VA32 (VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 31) and DAP-12/TRIOPEP VE32 (VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 32).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-12-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-12 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of DAP-12/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the DAP-10- and DAP-12-associated receptors, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the DAP-10- and DAP-12-associated receptors, respectively, can be used to visualize (image) these receptors and evaluate their expression in the areas of interest. In one 64cut embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to the DAP-10/TRIOPEP and DAP-12/TRIOPEP sequences. In one embodiment, imaging (visualization) of levels of the DAP-10- and DAP-12-associated receptors using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
F. EGFR-Related Trifunctional Peptides.
The epidermal growth factor (EGF) receptor (EGFR) family, or ErbB family, is the best studied example of oncogenic receptor tyrosine kinases (RTKs). HER2/ErbB2 is overexpressed on the surface of 25-30% of breast cancer cells, and it has been associated with a high risk of relapse and death. EGFR amplification and mutations have been associated with many carcinomas. In particular, the EGFR pathway appears to play a role in pancreatic carcinoma. See e.g. Overholser, et al. Cancer 2000, 89:74-82; Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224. Short hydrophobic peptides corresponding to the transmembrane domains of EGFR, ErB2 and insulin receptors inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor (see Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474).
The preferred EGFR-related peptides and compositions of this class comprise the domain A comprising the EGFR modulatory peptide sequences, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating EGFR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I
sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative EGFR-related trifunctional peptides: EGFR/TRIOPEP SA47 (SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO.
35) and EGFR/TRIOPEP 5E47 (SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO.
36). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with EGFR in the cell membrane and selectively disrupts intramembrane interactions between the receptors, resulting to specific ligand-independent inhibition of the receptor (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of EGFR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the EGFR and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the receptors of the EGFR and/or ErB receptor families, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the EGFR- and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the receptors of the EGFR and/or ErB receptor families can be used to visualize (image) these receptors and evaluate their expression in the areas of interest. In one embodiment, 64co for this purpose, an imaging probe (e.g. [ can be conjugated to the EGFR/TRIOPEP
sequence. In one embodiment, imaging (visualization) of levels of the receptors of the EGFR
and/or ErB receptor families using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
G. Additional Trifunctional Peptides Additional therapeutic peptide sequences and/or other therapeutic agents can comprise the domain A of the peptides and compositions of the present invention.
Additional examples are provided in, for e.gs., Vlieghe, et al. Drug Discov Today 2010, 15:40-56;
Tsung, et al. Shock 2007, 27:364-369; Chang, et al. PLoS One 2009, 4:e4171; Tjin Tham Sjin, et al.
Cancer Res 2005, 65:3656-3663; Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336;
Khan, et al. Hum Immunol 2002, 63:1-7; Banga. Therapeutic peptides and proteins: formulation, processing, and delivery systems. 2nd ed. Boca Raton, FL: Taylor & Francis Group; 2006;
Stevenson. Curr Pharm Biotechnol 2009, 10:122-137; Wu and Chi, US 9,387,257; Wu, et al., US
8,415,453;
Faure, et al., US 8,013,116; Faure, et al., US 9,273,111; Eggink and Hoober, US 7,811,995;
Eggink and Hoober, US 8,496,942; Morgan and Pandha. US 2012/0177672 Al;
Broersma, et al., US 5,681,925), each of which is herein incorporated by reference in its entirety.
In one embodiment, this domain comprises the Toll Like Receptor (TLR) modulatory sequence (see e.g. Tsung, et al. Shock 2007, 27:364-369). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TLR-related trifunctional peptides: TLR/TRIOPEP DA32 (DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 37) and TLR/TRIOPEP DE32 (DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 38). In one embodiment, methionine residues of TLR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TLR can be used to treat and/or prevent TLR-related diseases and conditions including but not limiting to, sepsis and other infectious diseases, and any other disorder where TLR receptors are involved.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TLR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the TLR/TRIOPEP sequences In one embodiment, imaging (visualization) of TLR levels using PET
and/or other imaging techniques can be used to diagnose TLR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In one embodiment, the domain A of the peptides and compositions of the invention comprises the Atrial Natriuretic Peptide (ANP) receptor (ANPR)-modulatory sequence (see e.g.
Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative ANPR-related trifunctional peptides: ANPR/TRIOPEP SA50 (SLRRS SCFGGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO. 39) and ANPR/TRIOPEP SE50 (SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 40). In one embodiment, methionine residues of ANPR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit ANPRs can be used to treat and/or prevent ANPR-related diseases and conditions including but not limiting to, cardiovascular and inflammatory diseases, and any other disorder where ANP
receptors are involved.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with ANPR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the ANPR/TRIOPEP sequences In one embodiment, imaging (visualization) of ANPR levels using PET and/or other imaging techniques can be used to diagnose ANPR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In certain embodiments, other therapeutic agents including but not limiting to, to those described in Page and Takimoto. Principles of chemotherapy. In: Pazdur R, Wagman LD, Camphausen KA, editors. Cancer Management: A Multidisciplinary Approach. 11th ed.
Manhasset, NY: Cmp United Business Media; 2009. p. 21-37; Sipsas, et al., Therapy of Mucormycosis, J Fungi (Basel) 2018, 4; Lin, et al. Chem Commun (Camb) 2013, 49:4968-4970;
and in Turner, et al. Peptide Conjugates of Oligonucleotide Analogs and siRNA
for Gene Expression Modulation. In: Langel U, ed, editor. Handbook of Cell-Penetrating Peptides. 2nd edition ed. Boca Raton: CRC Press; 2007. p. 313-328 and disclosed in Schiffman and Altman, US 4,427,660; Castaigne, et al., US 9,161,988; Castaigne, et al., US
8,921,314; and in Castaigne, et al., US 9,173,891, each of which is herein incorporated by reference in its entirety (see also TABLE 2) can comprise the domain A of the peptides and compositions of the present invention.
III. Lipoproteins And rHDLS.
Lipoproteins, inlcuding circulating lipoproteins in blood plasma, are natural complexes that contain both proteins (apolipoproteins, apo) and lipids bound to the proteins, which allow water-insoluble molecules such as fats to move through the water inside and outside cells.
Lipoproteins serve to emulsify the lipid molecules. Examples include the plasma lipoprotein particles classified under high-density lipoproteins (HDL), which enable cholesterol and other hydrophobic lipid molecules to be carried in the bloodstream. In particular, HDL transport cholesterol and other water insoluble or poorly soluble lipids from the peripheral tissues to the liver.
The use of HDLs as delivery vehicles was proposed however in order to properly function in vivo for delivery of drugs or imaging agents to sites of interest, HDLs should mimic native lipoproteins as close as possible. In a human body, HDL exists in two forms: nascent or discoidal HDL and spherical HDL. The use of isolated plasma lipoproteins, including isolated HDLs, as delivery vehicles is impractical.
However in vitro, long half-life lipoprotein particles that mimic native HDL
(as synthetic sHDL or recombinant HDL, rHDL) can be readily reconstituted (synthesized) from lipid formulations and apolipoproteins (apo) resulting in, for example, sub 30 nm-sized particles of discoidal or spherical morphology. Morphology of rHDLs is determined by the composition of lipid and apo mixtures and preparation procedures.
Many types of rHDLs were evaluated both clinically and experimentally as a delivery system for administering hydrophobic agents and for mitigating the toxic effects associated with administration of imaging probes such as Gd-containing contrast agents (GBCAs) for magnetic resonance imaging (MM).
As delivery vehicles, rHDL have several competitive advantages as compared with other delivery platforms: 1) apo A-I, a major HDL protein, is used for rHDL
preparation as it's recombinant or synthesized peptide/protein represents an endogenous protein that does not trigger immunoreactions; 2) apo A-I's small size allows rHDL to pass through blood vessel walls, enter and then accumulate in the places of interest, including for treatment and/or detection, such as tumor sites, areas of disease, such as liver tissue, etc., or atherosclerotic plaques; 3) rHDL's small particle size also allows for intravenous, intramuscular and subcutaneous applications; 4) rHDL's naturally long half-life extends the half-life of incorporated drugs and/or imaging agents in a bloodstream; and 5) a variety of drugs and imaging agents can be incorporated into this platform.
However, in order to properly function in vivo and as a result, to realize all the advantages mentioned above, rHDL should mimic native lipoproteins including but not limited to HDL as close as possible. This is a complicated task because two functions, assistance in the self-assembly of rHDL and therapeutic and/or imaging action in vivo, have to be executed by at least, two separate rHDL ingredients such as human apolipoprotein and therapeutic agent and/or imaging probe. In addition, in contrast to, for example, native HDL that are normally target the liver, rHDL have to be able to target other sites of interest such as, for example, macrophages which results in the need of targeting moieties thus adding the third function of rHDL ingredients ¨ targeting. This hampers wider use of rHDL by difficulties in industrializing the manufacture of rHDL, along with rHDL' lack of stability and reproducibility. In addition, the use of native or recombinant human apolipoproteins significantly complicates development of the commercial product, drastically increases its cost and possesses potential clinical and regulatory pitfalls.
An alternative, fully synthetic lipopeptide system for targeted treatment and/or imaging that closely mimics native lipoproteins and exhibits the advantageous properties of rHDL as well as superior stability, uniformity, ease of use, and reproducibility of preparation is needed for administration and targeted delivery of therapeutic agents (e.g. anti-cancer and anti-sepsis agents, other anti-inflammatory drugs) and/or imaging probes. The invention provides such a system and a method of using the system (e.g., for delivery of anti-cancer, anti-arthritic, anti-sepsis, anti-angiogenic and other therapeutic agents and/or imaging probes to a subject).
These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Additional contemplative advantages of a lipoprotein delivery platform includes increasing activity due to specific targeting, sequestration of the drug at the target site, protection of the drug from rapid metabolism, amplified therapeutic effect due to packaging of numerous drug molecules in each particle, and decreased toxicity due to altered pharmacokinetics. Due to the naturally long half-life of native discoidal and spherical HDL in normal subjects being 12-20 hrs and 3-5 days, respectively, rHDL represent a promising versatile delivery platform in particular for therapeutic peptides that have a bloodstream half-life of minutes.
For example, it would be desirable to combine in one molecule therapeutic (and/or diagnostic), particle forming and targeting functions. The invention addresses these needs, among others, and provides such a system/molecule and a method of using the system (e.g., for delivery of anti-cancer, anti-arthritic, anti-sepsis, anti-angiogenic, anti-inflammatory and other therapeutic agents and/or imaging probes to a subject). These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
IV. Trifunctional Peptides In rHDL Formulations.
A. TREM-1-related Trifunctional peptides: TREM-1 Signaling Pathway and Its Blockade.
TREM-1 is expressed on the majority of innate immune cells and to a lesser extent on parenchymal cells. Upon activation, TREM-1 can directly amplify an inflammatory response.
Although it was initially demonstrated that TREM-1 was predominantly associated with infectious diseases, recent evidences demonstrate that TREM-1 receptor and its signaling pathways contribute to the pathology of non-infectious acute and chronic inflammatory diseases, .. including but not limiting to, rheumatoid arthritis, atherosclerosis, ischemia reperfusion-induced tissue injury, colitis, fibrosis, neurodegenerative diseases, liver diseases, retinopathies, and cancer (see e.g., Tammaro, et al. Pharmacol Ther 2017, 177:81-95; Saadipour.
Neurotox Res 2017, 32:14-16; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov.
US 8,513,185;
Sigalov. US 9,981,004; Rojas, et al. Biochim Biophys Acta 2018, 1864: 2761-2768, and Kuai, et al. US 2008/0247955, each of which is herein incorporated by reference in its entirety).
In some preferred embodiments, TREM-1-related peptides and associated compositions of the present invention have a domain A conjugated to a domain B. See, Fig.
1. Domain A
comprises a TREM-1 modulatory peptide sequence designed using a known model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TREM-1 receptor expressed on myeloid cells (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 8,513,185; and Sigalov. US 9,981,004), all of which are herein incorporated by reference in their entirety. In some preferred embodiments, peptides and compositions of the present invention comprise the TREM-1 modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model.
In some preferred embodiments, peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic alpha helical peptide fragment, such as a apo A-I and/or A-II peptide fragment, to form upon interaction with lipid and/or lipid mixtures. In certain embodiments, exemplary trifunctional peptides comprise the domain B comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In one embodiment, preferred peptides and compositions of the invention further comprise at least one modified or unmodified amphipathic apo A-I and/or A-II
peptide fragment capable upon interaction with lipid and/or lipid mixtures, to form synthetic lipopeptide particles (SLP) structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US 20130039948;
Shen, et al.
PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J
Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). The inclusion of an amphipathic apo A-I
sequences in the peptides and compositions of the invention further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
FIG. 1 presents an exemplary schematic representation of one embodiment of a trifunctional peptide of the present invention comprising amino acid domains A and B where amino acid domain A represents a therapeutic peptide sequence with or without an attached drug compound and/or imaging probe that functions to treat, prevent and/or detect a disease or condition, whereas amino acid domain B represents an amphipathic alpha helical peptide sequence, with or without an additional targeting peptide sequence, and functions to 1) assist in the self-assembly of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with lipids or lipid mixtures in vitro, for use in transporting these trifunctional peptides as lipoprotien nanoparticles to sites of interest in vitro or in vivo and/or 2) form long half-life lipopeptide/lipoprotein particles upon interaction with endogenous lipoproteins for transporting these trifunctional peptides to the sites of interest. Endogenous lipoproteins may be lipoproteins added to or found in cell cultures, or lipoproteins in a mammalian body.
In certain embodiments, FIG. 2 shows the structures of representative TREM-1-related trifunctional peptides, TREM-1/TRIOPEP GE31 (GFL5K5LVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and TREM-1/TRIOPEP GA31 (GFL5K5LVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ
ID NO. 2) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. GFLSKSLVF, is found within isoform 1 of human (UniProtKB - Q9NP99 (TREM1 HUMAN), and in human TREM-1 isoform CRA a (UniProtKB - Q38L15 (Q38L15 HUMAN), both downloaded 10-24-2018)).
Q9NP991TREM1 HUMAN Isoform 1 Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
Q38L15938L15 HUMAN Triggering receptor expressed on myeloid cells 1, Homo sapiens isoform CRA a:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
GFLSKSLVF, is not found within human TREM-1 isoforms 2 or 3.
Q9NP99-21TREM1 HUMAN Isoform 2 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFRCSTLSFSWLVDS
Q9NP99-31TREM1 HUMAN Isoform 3 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KST.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP). GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) comprising amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22 amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
In certain embodiments, other preferred TREM-1-related trifunctional peptides and compositions of this class comprise the domain A comprising the TREM-1 inhibitory peptide sequences LR12 and LP17 (described in Gibot, et al. Infect Immun 2006, 74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J Immunol 2007, 37:456-466;
Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J Clin Pharmacol 2018, in press; Zhou, et al.
Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et al., US
8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US 9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136, each of which is herein incorporated by reference in it's entirety) while the domain B comprises at least one modified or unmodified amphipathic apo A-I
and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal. In some embodiments, resulting trifunctional peptide sequences may be radiolabeled and/or contain unmodified or modified methionine residues (TABLE 2) including but not limiting to, the following sequences:
LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide (SEQ ID NO 7), LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO 8), LQVTDSGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA (M(0), methinone sulfoxide (SEQ ID NO 9), LQVTDSGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO 10).
SLP (rHDL) structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US
20130039948;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorpoated by referene in it's entirety). The inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 2) and TREM-1/TRIOPEP
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. In one embodiment, interaction of TREM-1/TRIOPEP GA31 with lipids results in self-assembly of nanosized SLP of discoidal or spherical morphology (dSLP and sSLP, respectively) (see FIG. 3).
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising amino acid domains A and B. Depending on lipid mixture compositions added to the peptides, sub 50 nm-sized SLP particles of discoidal (TREM-1/TRIOPEP-d5LP) or spherical (TREM-s5LP) morphology are self-assembled upon binding of the trifunctional peptide to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to intraplaque macrophages in vivo (FIG. 4A). In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to tumor-associated macrophages (TAMs) in vivo (FIG. 4B).
FIG. 4A illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for atherosclerosis), whereas domain B represents a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of atherosclerosis. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP
of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by intraplaque macrophages and return to the circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 5), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in it's entirety FIG. 5 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the trifunctional peptide of the present invention comprising two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence, whereas domain B is a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
In one embodiment, FIG. 6 shows that the fluorescently labeled TREM-1/TRIOPEP
peptide GE31 delivered to macrophages by the SLP particles of the present invention colocalizes with TREM-1 expressed on these cells (see also Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). In certain embodiments, the capability of the TREM-1-related trifunctional peptides and compounds of the present invention including but not limiting to, TREM-1/TRIOPEP GA31 and TREM-1/TRIOPEP GE31, to colocalize with TREM-1 can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64Cu], see TABLE 2) can be conjugated to the TREM-1/TRIOPEP sequences, GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, imaging (visualization) of TREM-1 levels using PET and/or other imaging techniques can be used to diagnose glioblastoma multiforme (GBM) and/or to select and monitor novel GBM therapies (see e.g., Johnson, et al.
Neuro Oncol 2017, 19:vi249 and James and Andreasson, WO 2017083682A1). In certain embodiments, imaging (visualization) of TREM-1 levels can be used to diagnose other TREM-1-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with TREM-1 in the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and a merged image (FIG. 6C).
As described herein (see FIG. 7A-B), sulfoxidation of methionine residues in the TREM-1/TRIOPEP peptides GE31 and GA31 results in increased macrophage endocytosis of the SLP
containing an equimolar mixture of these peptides (designated as TREM-1/TRIOPEP), TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP. Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, all of which are herein incorporated in their entirety.
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic lipopeptide particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that contain an equimolar mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 (TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The post 4 h incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified methionine residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4 (white bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001 to 0.001 as compared with 4 h incubation time.
In certain embodiments, FIGS. 8 and 10 demonstrate that TREM-1/TRIOPEP in free and SLP-bound forms inhibits TREM-1 function as shown by reduction of TREM-1-mediated release of pro-inflammatory cytokines both in vitro (FIG. 8) and in vivo (FIG.
10). While not being bound to any particular theory, it is believed that this indicates that similarly to TREM-1-inhibitory peptide GF9 (see e.g., Sigalov. Int Immunopharmacol 2014, 21:208-219; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in it's entirety), TREM-1-related trifunctional peptides can reach their site of action from both outside (free TREM-1/TRIOPEP) and inside (SLP-bound TREM-1/TRIOPEP) the cell. It is also believed that upon administration, free TREM-1/TRIOPEP may form LP in vivo and/or interact with native lipoproteins, resulting in formation of HDL-mimicking LP. In one embodiment, these LP may further target the cells of interest delivering their content to the areas of interest in a body.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lb eta production by lipopoly sacchari de (LPS)-stimulated macrophages incubated for 24 h at 37 C with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged macrophages.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post lipopolysaccharide (LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline (PBS), dexamethasone (DEX), control peptide and with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
Control peptide represents an equimolar mixture of two peptides, each of them comprising two amino acid domains A and B where domain A represents a non-functional 9 amino acids-long sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5 is substituted with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as compared with PBS-treated animals.
While not being bound to any particular theory, it is believed that increased uptake described herein, is mediated by macrophage scavenger receptors (SR) including, but not limiting to, SR-A and SR-B1 (see FIG. 9A-C). While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 9A), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in it's entirety.
FIG. 9A1-A2-C shows schematic representations of activation of the TREM-1/DAP12 receptor complex expressed on macrophages and presents the exemplary data showing that scavenger .. receptors SR-A and SR-B1 mediate the macrophage endocytosis of GF9-sSLP
(GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1, right panel).
FIG. 9A2, left panel shows schematic representations of activation of the TREM-receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm;
GF9 self-penetrates the cell membrane and blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and the downstream signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774 macrophages were cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-HDL, cells were treated for 1 hour at 37 C with 401.IM cytochalasin D and either (FIG.
9B) 400 pg/mL
fucoidan or (FIG. 9C) 10 1.1M BLT-1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars) or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence intensities of lysates were measured and normalized to the protein content.
Results are expressed as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of GF9-HDL and GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo-oligomerization.
In certain embodiments, FIGS. 11A-B -14A-C demonstrate that TREM-1/TRIOPEP in free and SLP-bound forms inhibits tumor growth, reduces infiltration of macrophages into the tumor in mouse models of NSCLC and PC and is well-tolerated by cancer mice during the treatment period (see also Shen and Sigalov. Mol Pharm 2017, 14:4572-4582).
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *, p < 0.05; **, p < 0.01, ****, p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP--- bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar = 200 pm.
In embodiment, FIG. 15 demonstrates that TREM-1/TRIOPEP in free and SLP-bound forms significantly prolongs survival in mice with lipopolysaccharide (LPS)-induced septic shock.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
-- FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
In one embodiment, FIG. 16 shows that TREM-1/TRIOPEP is non-toxic in healthy mice at least up to 400 mg/kg.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
In one embodiment, FIG. 17 demonstrates that TREM-1/TRIOPEP in free and SLP-bound form ameliorates arthritis in mice with collagen-induced arthritis (CIA) and is well-tolerated by arthritic mice during the treatment period of 2 weeks (see Shen and Sigalov. J Cell -- Mol Med 2017, 21:2524-2534).
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1--- related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *, p <0.05, **, p <0.01;
***, p <0.001 as compared with vehicle-treated or naive animals.
In certain embodiments, FIG. 18 demonstrates that TREM-1/TRIOPEP-sSLP prevents pathological RNV in mice with oxygen-induced retinopathy and is well-tolerated by these mice during the treatment period (see Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768).
FIG. 18A-D presents the exemplary data showing reduction of pathological retinal neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-1 (FIG. 18C) and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-induced retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP) particles of spherical morphology (TREM-1/TRIOPEP-sSLP). ***,p < 0.001 as compared with vehicle-treated animals.
As described in Stukas, et al. J Am Heart Assoc 2014, 3:e001156, systemically administered human apo A-I accumulates in murine brain. It is also known that transcytosis of HDL in brain microvascular endothelial cells is mediated by SRBI (see Fung, et al. Front Physiol 2017, 8:841). However, until tested, it was not known that a self-assembled SLP of the present invention comprising a trifunctional peptide was capable of crossing the BBB.
In certain embodiments, FIG. 19 shows that the self-assembled SLP of the present invention may cross the BBB, BRB and BTB, thus delivering their constituents including but not limiting to, TREM-1/TRIOPEP to the areas of interest in the brain, retina and tumor. While not being bound to any particular theory, it is believed that the brain-, retina-, and tumor-penetrating capabilities of these SLP can be mediated by interaction of SRBI with the domain B amino acid sequences that correspond to the sequences of human apo A-I helices 4 and/or 6 (see e.g. Liu, et al. J Biol Chem 2002, 277:21576-21584, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, these capabilities of the peptides and compositions of the present invention can be used to diagnose, treat and/or prevent cancers (including brain cancer), diabetic retinopathy and retinopathy of prematurity, neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases and other diseases and conditions where delivery of the peptides and compositions of the invention to the brain, retina and/or tumor is needed.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical self-assembled particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-sSLP) or an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
Mouse model of ALD mimics the early phase of the human disease, yet mRNA
levels of early fibrosis markers Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of these makers -- was remarkably attenuated in the vehicle-treated group and, importantly, further decreased by the TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B a-Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
TREM-1 inhibitor effects were evaluated on hepatocyte damage and steatosis in liver. Serum ALT levels obtained during week 5 of the alcohol feeding showed significant increases in alcohol-fed mice compared to PF controls. This ALT increase was attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig.
4A).
Surprisingly, vehicle treatment (HDL) also showed a similar protective effect (Fig. 4A).
Consistent with steatosis, we found a significant increase in Oil Red 0 staining in livers of alcohol-fed mice compared to PF controls (Fig. 4C). Oil Red 0 (Fig. 4B-D) and H& (Fig.
4D) staining revealed attenuation of steatosis in the alcohol-fed TREM-1 inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig.
4B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
44/ P < 0.01;*/"413 <0001; ****P < 0.0001.***, 0.001 > P> 0.0001; ##, 0.01 >
P> 0.001.
B. TCR-Related Trifunctional Peptides The T-cell receptor (TCR)-CD3 complex plays a role in T-cell differentiation, in protecting the organism from infectious agents, and in the function of T-cells. The TCR is a complex of a heterodimer of TCRa and TCRb chains, which are responsible for antigen recognition and interaction with the major histocompatibility complex (MHC) molecules of antigen-presenting cells, and CD3d, CD3g, CD3e and CD3z chains, which are responsible for transmembrane signal transduction (see e.g., Manolios, et al. Cell Adh Migr 2010, 4:273-283;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 20130039948;
Manolios.
US 6,057,294; Manolios. US 7,192,928; Manolios. US 20100267651; and Manolios, et al. US
20120077732, each of which is herein incorporated by reference in it's entirety).
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or -- unmodified amphipathic alpha helical peptide fragment. As described above, the inclusion of an amphipathic amino acid sequences aids the assistance in the ability to interact with native lipoproteins in a bloodstream in vivo and to form naturally long half-life lipopeptide/lipoprotein particles LP. It further aids the ability to provide targeted delivery to the sites of interest. It further aids the ability to cross the BBB, BRB and BTB.
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids -- and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP MA32 (MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 11), TCR/TRIOPEP
ME32 (MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 12), TCR/TRIOPEP GA36 (GARSMTLTVQARQLPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO 13), TCR/TRIOPEP GE36 (GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 14), TCR/TRIOPEP GA32 (GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO 15), and TCR/TRIOPEP GE32 (GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 16). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRa chain with the CD3ed heterodimer and CD3zz homodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP LA32 (LGKATLYAVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 17) and TCR/TRIOPEP
LE32 (LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 18). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRb chain with the CD3eg heterodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP YA32 (YLLDGILFIYPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 19) and TCR/TRIOPEP
YE32 (YLLDGILFIYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 20). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3zz homodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32 (IIVTDVIATLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 21) and TCR/TRIOPEP
1E32 (IIVTDVIATLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 22). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3ed heterodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP FA32 (FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 23) and TCR/TRIOPEP
FE32 (FLFAEIVSIFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 24). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg heterodimer with TCRb chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32e (IVIVDICITGPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 25) and TCR/TRIOPEP
IE32e (IVIVDICITGPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 26). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg and CD3ed heterodimers with TCRb and TCRa chains, respectively, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein incorporated by reference in their entirety).
In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified as described herein.
In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TCR can be used to treat and/or prevent TCR-related diseases and conditions including but not limiting to, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis; autoimmune disease e.g. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel disease, Crohn's disease, primary biliary cirrhosis, chronic active hepatitis;
skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g.
allergic alveolitis; cardiovascular problems e.g. autoimmune pericarditis;
organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; and any other disorder where T
cells are involved/recruited In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TCR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu-.j, see TABLE 2) can be conjugated to the TCR/TRIOPEP sequences In one embodiment, imaging (visualization) of TCR
levels using PET and/or other imaging techniques can be used to diagnose TCR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
C. NKG2D-Related Trifunctional Peptides NKG2D is an activating receptor expressed by natural killer (NK) and T cells.
The NKG2D is a complex of an NKG2D chain, which is responsible for ligand recognition, and DAP10 homodimer, which is responsible for transmembrane signal transduction (see e.g.
Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589;
Sigalov. Self Nonself 2010, 1:4-39; and Sigalov. Self Nonself 2010, 1:192-224, all of which are herein incorporated by reference in their entirety). NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease (see e.g. Gonzalez, et al. Trends Immunol 2008, 29:397-403; Ito, et al. Am J Physiol Gastrointest Liver Physiol 2008, 294:G199-207; Vilarinho, et al. Proc Natl Acad Sci U S A
2007, 104:18187-18192; Van Belle, et al. J Autoimmun 2013, 40:66-73; Lopez-Soto, et al. Int J
Cancer 2015, 136:1741-1750; and Urso, et al. US 9,127,064, each of which is herein incorporated by reference in it's entirety).
The preferred NKG2D-related peptides and compositions of this class comprise the domain A comprising the NKG2D modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating NKG2D (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, all of which are herein incorporated by reference in their entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative NKG2D-related trifunctional peptides: NKG2D/TRIOPEP IA36 (IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 27) and NKG2D/TRIOPEP 1E36 (IAVAMGIRFIIMVAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 28). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with NKG2D in the cell membrane and selectively disrupt intramembrane interactions of NKG2D chain with the DNAX-activation protein 10 (DAP-10) signaling homodimer, resulting to specific ligand-independent inhibition of NKG2D upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein incorporated by reference in their entirety). In one embodiment, methionine residues of NKG2D/TRIOPEP peptides are modified.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit NKG2D can be used to treat and/or prevent NKG2D-related diseases and conditions including but not limiting to, celiac disease, type I diabetes, hepatitis, and rheumatoid arthritis, and any other disorder where NKG2D cells are involved/recruited. In one embodiment, the present invention provides methods and compositions for preventing NK cell-mediated graft rejection.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with NKG2D can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the NKG2D/TRIOPEP sequences In one embodiment, imaging (visualization) of NKG2D levels using PET and/or other imaging techniques can be used to diagnose NKG2D-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
D. GPVI-Related Trifunctional Peptides In recent years, the central activating platelet collagen receptor, glycoprotein (GP) VI, has emerged as a promising antithrombotic target because its blockade or antibody-mediated depletion in circulating platelets was shown to effectively inhibit experimental thrombosis and thromboinflammatory disease states, such as stroke, without affecting hemostatic plug formation.
GPVI is a complex of a GPVI chain, which is responsible for ligand recognition, and FcRg homodimer, which is responsible for transmembrane signal transduction (see e.g. Sigalov.
Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov.
Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. J
Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692;
Dutting, et al. Trends Pharmacol Sci 2012, 33:583-590; Ungerer, et al. PLoS One 2013, 8:e71193;
Sigalov, US
8,278,271; Sigalov, US 8,614,188, all of which are herein incorporated by reference in their entirety). The binding of GPVI to collagen or other antagonists ligands induces platelet adhesion, activation and aggregation. Platelet activation is a step in the pathogenesis of ischemic cardio-and cerebrovascular diseases, which represent the leading causes of death and severe disability worldwide. Although existing antiplatelet drugs have proved beneficial in the clinic, their use is limited by their inherent effect on primary hemostasis, making the identification of novel pharmacological targets for platelet inhibition a goal of cardiovascular research.
The preferred GPVI-related peptides and compositions of this class comprise the domain A comprising the GPVI modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating GPVI (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative GPVI-related trifunctional peptides: GPVI/TRIOPEP GA32 (GNLVRICLGAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 29) and GPVI/TRIOPEP
GE32 (GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 30). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with GPVI in the cell membrane and selectively disrupt intramembrane interactions of GPVI chain with the FcRg signaling homodimer, resulting to specific ligand-independent inhibition of GPVI upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of GPVI/TRIOPEP peptides are modified.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit GPVI can be used to treat and/or prevent GPVI-related diseases and conditions including but not limiting to, ischemic and thromboinflammatory diseases, and any other disorder where platelets are involved/recruited.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with GPVI can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the GPVI/TRIOPEP sequences In one embodiment, imaging (visualization) of GPVI levels using PET and/or other imaging techniques can be used to diagnose GPVI-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
E. DAP-10- and DAP-12-Related Trifunctional Peptides The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. DAP-10- and DAP-12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play a role in innate immune responses. See e.g. Lanier. Immunol Rev 2009, 227:150-160; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224, all of which are herein incorporated by reference in their entirety.
The preferred DAP-10 and DAP-12-related peptides and compositions of this class comprise the domain A comprising the DAP-10 or DAP-12 modulatory peptide sequences, respectively, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating DAP-10- and associated receptors (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP
structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-10-related trifunctional peptides: DAP-10/TRIOPEP LA32 (LVAADAVASLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 33) and DAP-10/TRIOPEP LE32 (LVAADAVASLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 34).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-10-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-10 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of DAP-10/TRIOPEP
peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-12-related trifunctional peptides: DAP-12/TRIOPEP VA32 (VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 31) and DAP-12/TRIOPEP VE32 (VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 32).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-12-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-12 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of DAP-12/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the DAP-10- and DAP-12-associated receptors, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the DAP-10- and DAP-12-associated receptors, respectively, can be used to .. visualize (image) these receptors and evaluate their expression in the areas of interest. In one 64cut embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to the DAP-10/TRIOPEP and DAP-12/TRIOPEP sequences. In one embodiment, imaging (visualization) of levels of the DAP-10- and DAP-12-associated receptors using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
6. EGFR-Related Trifunctional Peptides The epidermal growth factor (EGF) receptor (EGFR) family, or ErbB family, is the best studied example of oncogenic receptor tyrosine kinases (RTKs). HER2/ErbB2 is overexpressed on the surface of 25-30% of breast cancer cells, and it has been associated with a high risk of relapse and death. EGFR amplification and mutations have been associated with many carcinomas. In particular, the EGFR pathway appears to play a role in pancreatic carcinoma. See e.g. Overholser, et al. Cancer 2000, 89:74-82; Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224, each of which is herein incorporated by reference in it's entiretyy. Short hydrophobic peptides corresponding to the transmembrane domains of EGFR, ErB2 and insulin receptors inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor (see Bennasroune, et al.
Mol Biol Cell 2004, 15:3464-3474, all of which are herein incorporated by reference in their entirety).
The preferred EGFR-related peptides and compositions of this class comprise the domain A comprising the EGFR modulatory peptide sequences, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating EGFR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, all of which are herein incorporated by reference in their entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al.
PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In one embodiment, TREM-1 inhibitory SCHOOL peptide GF9 described herein is incorporated into SLP that contain apo A-I peptide fragments comprising 22 amino acid residue-long peptide sequences of the apo A-I helix 4 and/or helix 6. In one embodiment, the inclusion of an amphipathic apo A-I sequences in the peptides and compositions of the invention further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form lipopeptide particles (LP) that mimic native lipoproteins. It further aids the ability to cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB).
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative EGFR-related trifunctional peptides: EGFR/TRIOPEP SA47 (SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO.
35) and EGFR/TRIOPEP 5E47 (SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO.
36). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with EGFR in the cell membrane and selectively disrupts intramembrane interactions between the receptors, resulting to specific ligand-independent inhibition of the receptor (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein incorporated by reference in their entirety). In one embodiment, methionine residues of EGFR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the EGFR and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the receptors of the EGFR and/or ErB receptor families, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the EGFR- and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the receptors of the EGFR and/or ErB receptor families can be used to visualize (image) these receptors and evaluate their expression in the areas of interest. In one embodiment, 64co for this purpose, an imaging probe (e.g. [ can be conjugated to the EGFR/TRIOPEP
sequence. In one embodiment, imaging (visualization) of levels of the receptors of the EGFR
and/or ErB receptor families using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
F. Additional Trifunctional Peptides Additional therapeutic peptide sequences (see e.g., Vlieghe, et al. Drug Discov Today 2010, 15:40-56; Tsung, et al. Shock 2007, 27:364-369; Chang, et al. PLoS One 2009, 4:e4171;
Tjin Tham Sjin, et al. Cancer Res 2005, 65:3656-3663; Ladetzki-Baehs, et al.
Endocrinology 2007, 148:332-336; Khan, et al. Hum Immunol 2002, 63:1-7; Banga. Therapeutic peptides and proteins: formulation, processing, and delivery systems. 2nd ed. Boca Raton, FL: Taylor &
Francis Group; 2006; Stevenson. Curr Pharm Biotechnol 2009, 10:122-137; Wu and Chi, US
9,387,257; Wu, et al., US 8,415,453; Faure, et al., US 8,013,116; Faure, et al., US 9,273,111;
Eggink and Hoober, US 7,811,995; Eggink and Hoober, US 8,496,942; Morgan and Pandha. US
2012/0177672 Al; Broersma, et al., US 5,681,925, each of which is herein incorporated by reference in it's entirety) and/or other therapeutic agents can comprise the domain A of the peptides and compositions of the present invention.
In one embodiment, this domain comprises the Toll Like Receptor (TLR) modulatory sequence (see e.g. Tsung, et al. Shock 2007, 27:364-369, herein incorpoated by referene in it's entirety). The preferred peptides and compositions of this class further comprise the domain B
comprising at least one modified or unmodified amphipathic apo A-I and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP
structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US
20110256224;
Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov.
Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP
and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB
and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TLR-related trifunctional peptides: TLR/TRIOPEP DA32 (DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 37) and TLR/TRIOPEP DE32 (DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 38). In one embodiment, methionine residues of TLR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TLR can be used .. to treat and/or prevent TLR-related diseases and conditions including but not limiting to, sepsis and other infectious diseases, and any other disorder where TLR receptors are involved.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TLR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the TLR/TRIOPEP sequences In one embodiment, imaging (visualization) of TLR levels using PET
and/or other imaging techniques can be used to diagnose TLR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In one embodiment, the domain A of the peptides and compositions of the invention comprises the Atrial Natriuretic Peptide (ANP) receptor (ANPR)-modulatory sequence (see e.g.
Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein .. and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov.
Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, all of which are herein incorporated by reference in their entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative ANPR-related trifunctional peptides: ANPR/TRIOPEP SA50 (SLRRS SCFGGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO. 39) and ANPR/TRIOPEP SE50 (SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 40). In one embodiment, methionine residues of ANPR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit ANPRs can be used to treat and/or prevent ANPR-related diseases and conditions including but not limiting to, cardiovascular and inflammatory diseases, and any other disorder where ANP
receptors are involved.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with ANPR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the ANPR/TRIOPEP sequences In one embodiment, imaging (visualization) of ANPR levels using PET and/or other imaging techniques can be used to diagnose ANPR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In certain embodiments, other therapeutic agents including but not limiting to, to those described in Page and Takimoto. Principles of chemotherapy. In: Pazdur R, Wagman LD, Camphausen KA, editors. Cancer Management: A Multidisciplinary Approach. 11th ed.
Manhasset, NY: Cmp United Business Media; 2009. p. 21-37; Sipsas, et al., Therapy of Mucormycosis, J Fungi (Basel) 2018, 4; Lin, et al. Chem Commun (Camb) 2013, 49:4968-4970;
and in Turner, et al. Peptide Conjugates of Oligonucleotide Analogs and siRNA
for Gene Expression Modulation. In: Langel U, ed, editor. Handbook of Cell-Penetrating Peptides. 2nd edition ed. Boca Raton: CRC Press; 2007. p. 313-328 and disclosed in Schiffman and Altman, US 4,427,660; Castaigne, et al., US 9,161,988; Castaigne, et al., US
8,921,314; and in Castaigne, et al., US 9,173,891, all of which are herein incorporated by reference in their entirety, (see also TABLE 2) can comprise the domain A of the peptides and compositions of the present invention.
V. Diseases Contemplated For Treatment Using Peptides and Compositions Described Herein.
A. Overview.
The present invention encompasses the recognition that it is possible to produce compositions that possess the advantages typically associated with a fully synthetic material and yet also possess certain desirable features of materials derived from natural sources.
In some embodiments, peptides and compounds of the present invention, e.g.
trifunctional peptides, with rHDLs (including discoidal and/or spherical HDLs) or without rHDLs (such as in therapeitic compositions as free trifunctional peptides), are contemplated for use in preventative treatments for diseases associated with activated macrophages and/or T-cells, in particular for preventing one or more symptoms associated with the disease.
In some embodiments, peptides and compounds of the present invention are contemplated for use preventative treatments for diseases associated with activated macrophages and/or T-cells, in particular for reducing one or more symptoms associated with the disease. In some embodiments, peptides and compounds of the present invention are contemplated for use diagnostic applications for detecting/identifying; determining disease progression; determining results of disease treatment, for diseases associated with activated macrophages and/or T-cells.
Such diseases associated with activated macrophages and/or T-cells include but are not limited to including but not limited to lung cancer, such as non small-cell lung cancer (NSCLC); pancreatic cancer (PC); glioblastoma multiforme (GBM, or brain cancer), with or without radiation therapy;
breast cancer with or without radiation therapy; sepsis; retinopathy;
rheumatoid arthritis (RA);
sepsis; and alcoholic liver disease (ALD). Furthermore, diseases associated with activated macrophages and/or T-cells include but are not limited to 1) Alcohol-induced neuroinflammation .. and brain damage; 2) Radiation-induced multiple organ dysfunction syndrome;
3) Scleroderma;
4) Atopic dermatitis; 5) Atherosclerosis; 6) Alzheimer's, Parkinson's and/or Huntington's diseases. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, liver diseases, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other rheumatic diseases, sepsis and other inflammatory diseases or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, including but not limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases.
Thus, in some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a disease symptom. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for delaying onset of a disease symptom. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for preventing a disease symptom. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject receiving therapy for a disease. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject receiving anti-cancer therapy.
In some embodiments, trifunctonal peptides as described herein, are contemplated for administration to a subject as anti-cancer therapy. In some embodiments, trifunctonal peptides as described herein, further comprising a drug compound are contemplated for administration to a subject as anti-cancer therapy. In some embodiments, trifunctonal peptides as described herein, further comprising a Paclitaxel compound are contemplated for administration to a subject as anti-cancer therapy.
As disease progression of a liver in a subject proceeds from epatosteatosis, steatohepatitis, and fibrosis to cirrhosis, it is contemplated that a trifunctonal peptide as described herein, is administered to said subject at any point along the disease progression for reducing disease progression, in part as described herein. Thus, in some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a liver disease symptom, including but not limited to reducing one or more of ALT, procollegen I-alpha and alpha-SMA.
In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a liver disease symptom, in combination with one or more of steroid drugs, ursodiol, etc., in order to delay or prevent further progression of liver .. degeneration to cirrhosis. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a liver disease symptom in combination with one or more of steroid drugs, ursodiol, etc., in order to improve function of a diseased liver.
In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for treating severe hemorrhagic shock. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for treating colitis and colitis-associated tumorigenesis.
In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for decreasing neovascularization.
In some embodiments, trifunctonal peptides as described herein are selected from the group consisting of G-KV21, G-HV21, G-TE21, M-VE32 and M-TK32, and mixtures thereof. In some embodiments, a trifunctonal peptide as described herein is GE31. In some embodiments, a trifunctonal peptide as described herein is GA31. In some embodiments, a trifunctonal peptide as described herein is a mixture of GE31 and GA31.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A
and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (shown for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
B. Cancer.
Approximately 8.8 million people are dying each year of cancer, amounting to one out of six deaths globally, and cancer incidence is estimated to double by 2035 (Prager et al.
2018). Combination-therapy treatments for cancer have become more common, in part due to the perceived advantage of attacking the disease via multiple avenues. Although many effective combination-therapy treatments have been identified over the past few decades;
in view of the continuing high number of deaths each year resulting from cancer, a continuing need exists to identify effective therapeutic regimens for use in anticancer treatment.
The present invention encompasses the recognition that it is possible to prevent and treat different types of cancer including but not limited to, pancreatic cancer, multiple myeloma, leukemia, prostate cancer, breast cancer, liver cancer, bladder cancer, colorectal cancer, lung cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, or osteosarcoma and other cancers, and cancer cachexia by blocking the TREM-1 signaling pathway using the peptide variants and compositions that possess the advantages typically associated with a fully synthetic material and yet possess certain desirable features of materials derived from natural sources. The invention further encompasses the recognition that it is possible to use imaging techniques and the peptide variants and compositions of the invention conjugated to an imaging probe for detecting the labeled probe in an individual with cancer, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. The invention further encompasses that it is possible to predict the efficacy of the peptides and compositions of the invention by determining the number of myeloid cells in the biological sample from the individual with cancer and determining the expression levels of TREM-1 in the cells contained within the biological sample.
Cancer continues to have a huge Social and economic impact. In 2011, 571,950 Americans died of cancer (-25% of all deaths), with US cancer-associated costs of S263.8 billion: S102.8 billion for direct medical costs (total health expenditures);
$20.9 billion for indirect morbidity costs (lost productivity); and S140.1 billion for indirect mortality costs (lost productivity from premature death).
Inflammatory responses play decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis (Grivennikov et al. 2010). Inflammation also affects immune surveillance and responses to therapy (Grivennikov et al. 2010). Many solid tumors are characterized by a marked infiltration of macrophages, inflammatory cells, into the stromal compartment (Shih et al. 2006, Solinas et al. 2009), a process which is mediated by cancer-associated fibroblasts (CAFs) and plays a key role in disease progression and its response to therapy (see FIG. 49). These tumor-associated macrophages (TAMs) secrete a variety of growth factors, cytokines, chemokines, and enzymes .. that regulate tumor growth, angiogenesis, invasion, and metastasis (Shih et al. 2006). See FIG.
49. High macrophage infiltration correlates with the promotion of tumor growth and metastasis development (Solinas et al. 2009, Grivennikov et al. 2010). In patients with PC, macrophage infiltration begins during the preinvasive stage of the disease and increases progressively (Clark et al. 2007). The number of TAMs is significantly higher in patients with metastases (Gardian et al. 2012). TREM-1 is upregulated in cancer and its overexpression correlates with survival of cancer patients. In NSCLC, TREM-1 expression in TAMs is associated with cancer recurrence and poor survival of patients with NSCLC: patients with low TREM-1 expression have a 4-year survival rate of over 60%, compared with less than 20% in patients with high expression (Ho et al. 2008). Activation of the TREM-1/DAP-12 signaling pathway results in release of multiple cytokines, chemokines and growth factors most of which are increased in cancer patients and their upregulation correlates with poor prognosis (See FIG. 1).
The present invention encompasses the recognition that it is possible to prevent and treat different types of cancer in which myeloid cells are involved or recruited and cancer cachexia by combining cancer therapies with a therapeutically effective amount of at least one compound and/or composition ("modulator") which affects myeloid cells by action on the signaling pathway.
The infiltrate of most solid tumors contains tumor-associated macrophages (TAMs) that are attracted by chemokines including CCL2 and represent attractive treatment targets in oncology (Shih et al. 2006, Mantovani et al. 2017). The increased TAM content in NSCLC
(Yusen et al. 2018) is associated with poor prognosis in NSCLC (Welsh et al.
2005). TAM
recruitment, activation, growth and differentiation are regulated by CSF-1 (Elgert et al. 1998, Laoui et al. 2014). Many tumor cells or tumor stromal cells have been found to produce CSF-1, which activates monocyte/macrophage cells through CSF-1 receptor (CSF-1R). The level of CSF-1 in tumors has been shown to correlate with the level of TAMs in the tumor. Higher levels of TAMs have been found to correlate with poorer patient prognoses in the majority of cancers.
Increased pretreatment serum CSF-1 is a strong independent predictor of poor survival in NSCLC (Baghdadi et al. 2018). In addition, CSF-1 has been found to promote tumor growth and progression to metastasis in, for example, human breast cancer xenografts in mice (Paulus et al.
2006). Further, CSF-1R plays a role in osteolytic bone destruction in bone metastasis (Ohno et al. 2006). TAMs promote tumor growth, in part, by suppressing anti-tumor T
cell effector function through the release of immunosuppressive cytokines and the expression of T cell inhibitory surface proteins. Blockade of CSF-1 or CSF-1R not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota et al. 2009) but also improves response to T-cell checkpoint immunotherapies that target programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu et al. 2014).
Importantly, continuous CSF-1 inhibition affects pathological angiogenesis but not healthy vascular and lymphatic systems outside tumors (Kubota et al. 2009). In contrast to blockade of vascular endothelial growth factor (VEGF), interruption of CSF-1 inhibition does not promote rapid vascular regrowth (Kubota et al. 2009).
The present invention provides a method of treating these and other types of cancers by using modulators of the TREM-1/DAP-12 signaling pathway that are capable of binding TREM-1 and modulating TREM-1/DAP-12 receptor complex activity in combination-therapy treatments together with other cancer therapies. The invention further provides the methods for predicting response of a cancer patient to the treatment by using these modulators in combination-therapy regimen. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The invention further encompasses the recognition that it is possible to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regimen by: (a) obtaining a biological sample from the subject; (b) determining the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of CD68-positive .. TAMs or a combination thereof, wherein the higher is the expression of CSF-1, CSF-1R, IL-6, TREM-1 or the higher is number of CD68-positive TAMs or a combination thereof, the better the patient is predicted to respond to a therapy that involves the modulators.
The invention further encompasses the recognition that it is possible to use imaging techniques and the modulators conjugated to an imaging probe for detecting the labeled probe in an individual with cancer in which myeloid cells are involved or recruited, wherein the location and the measured intensity of the labeled probe can diagnose cancer and/or predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway, the higher the measured intensity of the labeled probe, the better the patient is predicted to respond to a therapy that involves the modulators.
1. Lung Cancer.
Lung cancer, including NSCLC, is the leading cause of cancer deaths worldwide (Wong et al. 2018) and has a poor prognosis. Despite advances made in chemotherapy, NSCLC is responsible for over 1.1 million deaths annually worldwide, and the 5-year survival rate for patients with NSCLC is reported to be only 15% or less than 18% (Zappa et al.
2016), showing .. an urgent need for new therapies.
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer (NSCLC) H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
2. Pancreatic cancer.
Pancreatic cancer (PC, 85% of which are pancreatic ductal adenocarcinomas, PDAC) is the fourth leading cause of cancer-related mortality across the world with very poor clinical outcome. (Ilic et al. 2016). Current treatments of PC marginally prolong survival or relieve symptoms in patients with PC (Ilic and Ilic 2016). There has been no significant progress in the field of targeted therapy for PC (Walker et al. 2014) and despite tremendous efforts, the 5-year survival rate remains less than 5% (Ilic and Ilic 2016).This highlights the urgent need for novel approaches to prevent and treat PC and other types of cancer. However, it should be noted that the techniques and compositions listed and described herein are applicable to a broad range of other types of cancer and cancer cachexia. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Current treatments of PC marginally prolong survival or relieve symptoms in patients with PC (Schneider 2005). There has been no significant progress in the field of targeted therapy for PC (Walker and Ko 2014) and despite tremendous efforts, the 5-year survival rate remains less than 5% (2010).
3. Additional Neoplasms: Giant Cell Tumor and PVNS.
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the inflammatory response (Colonna et al. 2003) and is upregulated under inflammatory conditions including .. cancer (Wang et al. 2004). For downstream signal transduction, TREM-1 is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor, DNAX
activation protein of 12kDa (DAP12). TREM-1/DAP-12 receptor complex activation enhances release of multiple cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNFa), interleukin-la (IL-1a), IL-1I3, IL-6 and colony-stimulating factor 1 (referred to herein as CSF1; also referred to in the art as M-CSF) (Schenk et al. 2007, Lagler et al. 2009, Sigalov 2014).
Binding of CSF1 or the interleukin 34 ligand (referred to herein as IL-34) to receptor (referred to herein as CSF1R) leads to receptor dimerization, upregulation of CSF1R
protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues, and downstream signaling events. CSF1R activation by CSF1 or IL-34 leads to the trafficking, survival, proliferation, and differentiation of monocytes and macrophages, as well as other monocytic cell lineages such as osteoclasts, dendritic cells, and microglia.
Many tumor cells or tumor stromal cells have been found to produce CSF1, which activates monocyte/macrophage cells through CSF1R. The level of CSF1 in tumors has been shown to correlate with the level of tumor-associated macrophages (TAMs) in the tumor. Higher levels of TAMs have been found to correlate with poorer patient prognoses in the majority of cancers. In addition, CSF1 has been found to promote tumor growth and progression to metastasis in, for example, human breast cancer xenografts in mice (Paulus et al. 2006). Further, CSF1R plays a role in osteolytic bone destruction in bone metastasis (Ohno et al. 2006). TAMs promote tumor growth, in part, by suppressing anti-tumor T cell effector function through the release of immunosuppressive cytokines and the expression of T cell inhibitory surface proteins.
Blockade of CSF1 or CSF1R not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota et al. 2009) but also improves response to T-cell checkpoint immunotherapies that target programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu et al. 2014). Importantly, continuous CSF1 inhibition affects pathological angiogenesis but not healthy vascular and lymphatic systems outside tumors (Kubota et al. 2009). In contrast to blockade of vascular endothelial growth factor (VEGF), interruption of CSF1 inhibition does not promote rapid vascular regrowth (Kubota et al. 2009).
Giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor (TGCT;
also referred to in the art as TSGCT), and pigmented villonodular synovitis (PVNS) are the common names for a group of rare proliferative disorders that involve synovial joints and tendon sheaths. PVNS is a solid tumor of the synovium with features of both reactive inflammation and clonal neoplastic proliferation in which CSF1 is over expressed. A common translocation of the CSF1 gene (1p13) to the COL6A3 promoter (2q35) is present in approximately 60%
of PVNS
patients. The translocation is accompanied by CSF1 overexpression in the synovium. In addition, approximately 40% of PVNS patients have CSF1 overexpression in the absence of an identified CSF1 translocation. The consistent presence of CSF1 overexpression in all cases of PVNS and reactive synovitis suggests both an important role for CSF1 in the spectrum of synovial pathologies and the utility of targeting the CSF1/CSF1R signaling pathway therapeutically (West et al. 2006). In PVNS, CSF1 overexpression is present in a minority of synovial cells, whereas the majority of the cellular infiltrate expresses CSF1R but not CSF1. This has been characterized as a tumor-landscaping effect with aberrant CSF1 expression in the tumor cells, leading to the abnormal accumulation of non-neoplastic cells that form a mass.
Surgery is the treatment of choice for patients with localized PVNS.
Recurrences occur in 8-20% of patients and are often managed by re-excision. Diffuse tenosynovial giant cell tumor (TGCT/PVNS or PVNS/dtTGCT) tends to recur more often (33-50%) and has a much more aggressive clinical course. Patients are often symptomatic and require multiple surgical procedures during their lifetime and even amputation. For patients with unresectable disease or multiple recurrences, systemic therapy using CSF1R inhibitors may help delay or avoid surgical procedures and improve functional outcomes (Radi et al. 2011).
Imatinib, a non-specific inhibitor of CSF1R, has undergone evaluation in PVNS
patients (Cassier et al. 2012). Twenty-nine patients from 12 institutions in Europe, Australia, and the United States were included. The median age was 41 years and the most common site of disease was the knee (n=17; 59%). Two patients had metastatic disease to the lung and/or bone. Five of 27 evaluable patients had complete (n=1) or partial (n=4) responses per RECIST
for an overall response rate of 19%. Twenty of 27 patients (74%) had stable disease.
Symptomatic improvement was noted in 16 of 22 patients (73%) who were assessable for symptoms. Despite a high rate of symptomatic improvement and an overall favorable safety profile, 10 patients discontinued treatment for toxicity or other reasons.
Pexidartinib (PLX3397), a potent, selective oral CSF1R inhibitor, that traps the kinase in the autoinhibited conformation, has undergone evaluation in TGCT patients (Tap et al. 2015). A
total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. In the extension study, 12 patients with TGCTs had a partial response and 7 patients had stable disease. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. Despite treatment of TGCTs with PLX3397 resulted in a prolonged regression in tumor volume in most patients of this Phase 2 study, later the Phase 3 study was suspended after two reported cases of nonfatal, serious liver toxicity.
Anti-CSF1R antibodies alone or in combination with antibodies against PD1 or against PDL1, one of the ligands for PD1, were proposed as less toxic alternative treatments for PVNS.
See, e.g., US Pat 10,040,858 B2 and US Pat 10,221,224. As with most combination therapies, the promise of increased clinical activity is accompanied by the risk of additive toxicity and therefore requires careful assessment.
Liver enzyme elevations can be considered a class effect of CSF1R-targeting compounds (Cannarile et al. 2017). In addition, the oversuppression of the CSF1/CSF1R
signaling pathway may result in potential serious long term adverse effects (AEs). In animals, CSF1 deficiency results in a range of developmental abnormalities, including skeletal, neurological, growth and fertility defects (Michaelson et al. 1996, Hume et al. 2012, Jones et al.
2013).
Thus, PVNS is a rare, locally aggressive neoplasm of the joint or tendon sheath with features of both reactive inflammation and clonal neoplastic proliferation in which CSF-1 is over expressed (Tap et al. 2015). Surgical resection is the primary treatment;
however, diffuse TGCT
is more difficult to resect and often involves total synovectomy, joint replacement, or amputation (Tap et al. 2015). There are no approved systemic therapies. Therefore, alternative, less toxic and more targeted treatments for PVNS are needed.
Inhibition of TREM-1 lowers levels of proinflammatory cytokines including CSF1 and is a promising approach in a variety of inflammation-associated disorders including cancer (Colonna and Facchetti 2003, Schenk et al. 2007, Pelham et al. 2014, Sigalov 2014, Shen et al.
2017, Shen et al. 2017, Rojas et al. 2018). In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Treml-/- mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines.
Treml-/- mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major (Weber et al. 2014). Furthermore, reduced morbidity was observed for influenza virus-infected Treml-/- mice (Weber et al. 2014).
Importantly, while immune-associated pathologies were significantly reduced, Treml-/- mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Treml+/+ controls (Weber et al. 2014). Humans lacking DAP-12 do not have problems resolving infections with viruses or bacteria (Lanier 2009). Collectively, these findings suggest that in contrast to single cytokine blockers including CSF1 and CSF1R
blockers, therapeutic blocking of TREM-1/DAP-12 signaling in distinct inflammatory disorders including CSF1-dependend TGCTs holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
The present invention provides a method of using the well-tolerable TREM-1/DAP-modulatory peptides and compositions for treatment of PVNS. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Methods of treating tenosynovial giant cell tumor (TGCT) or pigmented villonodular synovitis (PVNS) with peptide variants and compositions that modulate activity of the receptor complex formed by triggering receptor expressed on myeloid cells 1 (TREM-1) and DNAX
activation protein of 12kDa (DAP12) are provided.
Inhibition of TREM-1 lowers levels of proinflammatory cytokines including CSF1 and is a promising approach in a variety of inflammation-associated disorders including cancer (Colonna and Facchetti 2003, Schenk et al. 2007, Pelham et al. 2014, Sigalov 2014, Shen et al.
2017, Shen et al. 2017, Rojas et al. 2018). In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Treml-/- mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines.
Treml-/- mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major (Weber et al. 2014). Furthermore, reduced morbidity was observed for influenza virus-infected Treml-/- mice (Weber et al. 2014).
Importantly, while immune-associated pathologies were significantly reduced, Treml-/- mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Treml+/+ controls (Weber et al. 2014). Humans lacking DAP-12 do not have problems resolving infections with viruses or bacteria (Lanier 2009). Collectively, these findings suggest that in contrast to single cytokine blockers including CSF1 and CSF1R
blockers, therapeutic blocking of TREM-1/DAP-12 signaling in distinct inflammatory disorders including CSF1-dependend TGCTs holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
The present invention provides a method of using the well-tolerable TREM-1/DAP-modulatory peptides and compositions for treatment of PVNS. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
4. Liver Cancer.
Globally, liver cancer is the fifth commonest cancer in 2012, accounting for 9.1% of all cancer deaths worldwide with the overall 5-year relative survival rate for patients with liver cancer of 17%. Owing to its extremely aggressive nature and poor survival rate, it remains an important public health issue worldwide (Wong et al. 2017) 5. Breast Cancer.
Breast cancer is the most common malignancy in women around the world (Ghoncheh et al. 2016). Ii is the most common cancer in women, accounting for 25.1% of all cancers. Breast cancer incidence in developed countries is higher, while relative mortality is greatest in less developed countries (Ghoncheh et al. 2016). Despite significant improvements in clinical outcomes within the field of breast cancer in the last 50 years, the triple-negative breast cancer (TNBC) subtype remains an area of huge unmet clinical need (Partridge et al.
2017).
6. Glioblastoma.
Glioblastoma Multiforme (GBM) is the most common and lethal type of brain cancer (Shergalis et al. 2018). For adults with GBM, treated with standard first-line therapy ¨
concurrent radiation and temozolomide (TMZ) therapy followed by TMZ
monotherapy, the median survival is about 14.6 months (Grossman et al. 2010, Shergalis et al.
2018). Little progress has been made over the past several decades in the treatment of GBM, highlighting an urgent need for new therapies.
7. Colorectal Cancer.
Colorectal cancer (CRC) has a considerable impact on patients and healthcare systems in developed countries and around 25% of patients present with metastatic disease that significantly impacts on prognosis (Van Cutsem et al. 2013). For those with localized CRC of stages I and II, the 5-year survival rate is as high as 93%, declining to 60%, 42% and 25% for patients with stages IIIA, IIIB and IIIC, respectively. However, most patients with metastatic CRC (stage IV) are not curable, with the 5-year survival rate falling to less than 10%. While early diagnosis of CRC in recent years combined with advances in treatment has considerably improved survival, management of the disease remains challenging and further progress is needed (Van Cutsem et al. 2013).
Scleroderma, related autoimmune conditions and fibrotic conditions.
It is estimated that scleroderma or systemic sclerosis (SSc) affects 100,000-300,000 Americans, predominantly young to middle aged women. Systemic sclerosis is a progressive and untreatable disease of unknown cause and high mortality. Fibrosis in SSc resembles uncontrolled .. wound healing, where healing occurs by intractable fibrosis rather than normal tissue regeneration.
It is believed that SSc is associated with the highest case-fatality rates among the rheumatic diseases or connective tissue diseases. Currently, there are no validated biomarkers for diagnosis. Furthermore, no effective disease-modifying therapies are currently available. In fact, .. while some treatment can alleviate the pain associated with SSc, to date no therapy has been shown to significantly alter survival. The pathogenesis of SSc is characterized by early vascular injury, with inflammation followed by progressive tissue damage and fibrosis.
Excessive production of collagen and ECM and accumulation of myofibroblasts in lesional tissues are believed to be responsible for progressive organ failure. Pathological fibrosis resembles a normal wound healing response that has become deregulated. It is estimated that fibrosis accounts for>25% of all deaths in the U.S. Thus, fibrosis represents one of the major unmet medical needs.
Accordingly, there is a need for an effective anti-fibrotic therapy.
Project Summary/Abstract Scleroderma that includes localized scleroderma (LS) and systemic sclerosis (SSc) is a rare but devastating autoimmune disorder. Current therapies all have side effects, are limited and associate with 10 year survival of 55%, showing the need for novel approaches.
The long-term goal of this project is to develop a new mechanism-based, efficient and well tolerable scleroderma therapy.
Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, contributes to the development of fibrosis in SSc. In patients, number of activated macrophages in the fibrotic areas is increased and associates with fibrosis severity.
Activation of TREM-1 leads to overproduction of MCP-1/CCL2 and M-CSF/CSF-1, resulting in macrophage recruitment to an injured area and the sclerotic lesion formation in rats with scleroderma. In animal models, TREM-1 blockade inhibits inflammation and ameliorates a variety of autoimmune diseases. The hypothesis of the "proof-of-concept" Phase I is that blockade can prevent and treat scleroderma.
Current TREM-1 inhibitors all attempt to block binding of TREM-1 to its still uncertain ligand(s). To minimize risk of failure in clinical development, we developed a first-in-class ligand-independent TREM-1 inhibitory peptide GF9 that is well-tolerated and can be formulated into SignaBlok's long half-life macrophage-specific lipopeptide complexes (LPC) to improve its half-life and targeting to the inflammation areas. The major goal of the Phase I study is to show that TREM-1 blockade by GF9-LPC alleviates the disease in a bleomycin (BLM)-induced mouse model of scleroderma.
Phase I specific aims are to: 1) optimize TREM-1 inhibitory compositions for their functionality in vitro and pharmacokinetics in vivo and select the lead, 2) test two doses of the lead selected in a BLM-induced mouse model of scleroderma. We will generate, optimize and select the lead based upon its functionality in vitro and its PK profile in vivo. We will test two doses of the lead for its ability to prevent and treat lung, heart, muscle and skin fibrosis in a mouse model of multiorgan fibrosis in vivo. Histology/IHC studies will be performed. Serum and tissue cytokines will be evlauated, nonlmiting examples including MCP-1, CSF-1, VEGF, TGF-beta, TNF-alpha, IL-6, and IL-1-beta, will be analyzed.
It is anticipated that the Phase I study will identify a novel, first-in-class, well tolerable agent as a powerful platform for development of an effective and well-tolerable systemic scleroderma therapy, thereby improving treatment and survival of patients. Its anticipated safety is supported by good tolerability of SignaBlok's GF9-based formulations by long term-treated mice. Prototypes of SignaBlok's LPC are well tolerated in humans. TREM-1 blockade by SignaBlok competitor's inhibitory peptide LR12 (Inotrem, France) was safe in healthy and septic subjects. If successful, Phase I will be followed in Phase II by toxicology, ADME, pharmacology and CMC studies, filing an IND and subsequent evaluation in humans.
Project Narrative.
Scleroderma (also known as systemic sclerosis) is a rare autoimmune disorder that affects about 20 to 24 people per million population in the US each year, with the majority being women of childbearing age. There is no approved drug for scleroderma. Current therapies all have side effects, are limited and associated with 10 year survival of 55%, highlighting the urgent need for novel approaches The proposed research is anticipated to result in the development of novel mechanism-based first-in-class therapeutics that could substantially improve treatment of scleroderma and patient survival.
SPECIFIC AIMS.
The Product. The final product will represent a new mechanism-based, efficient, stable, well tolerable systemic immunomodulatory therapy for scleroderma in order to significantly decrease long-term disability, morbidity and mortality of the patients with scleroderma and improve the quality of their life.
Scleroderma is a rare but devastating autoimmune disorder (Lawrence et al.
1998, Mayes et al. 2003, Helmick et al. 2008) with no approved drug available. Current main treatments all have side effects, are limited and associated with 10 year survival of 55%
(Badea et al. 2009, Kowal-Bielecka et al. 2009, Shah et al. 2013), highlighting an urgent need for new therapies.
Macrophages are associated with fibrosis (Ishikawa et al. 1992, Kraling et al.
1995, Lech et al.
2013, Chia et al. 2015) and are recruited to inflammation sites by MCP-1 which is significantly elevated in patients with systemic sclerosis (SSc) (Hasegawa et al. 1999).
Activated macrophages produce VEGF, IL-lbeta, TNFalpha, IL-6, TGFbeta and PDGF that play a role in scleroderma (Bonner et al. 1991, Clouthier et al. 1997, Yamamoto 2011, Yamamoto et al. 2011, Liu et al. 2013, Manetti 2015). In animals, blockers of IL-6 receptor (IL-6R) (Kitaba et al. 2012), VEGF (Koca et al. 2016), TNFalpha (Koca et al. 2008) and TGFalpha (Varga et al. 2009, Varga et al. 2009) alleviate scleroderma but all may have serious side effects including fatal infections and sepsis (Varga 2004). CSF-1/M-CSF plays a role in pulmonary fibrosis that occurs in 90% of scleroderma patients (Baran et al. 2007). TREM-1 mediates release of MCP-1/CCL2, TNFalpha, IL-lbeta, IL-6 and CSF-1 (Schenk et al. 2007, Dower et al. 2008, Lagler et al.
2009, Sigalov 2014, Shen et al. 2015). TREM-1 expression is increased in the lungs of mice with BLM-induced pulmonary fibrosis (Peng et al. 2016). Together, this implicates TREM-1 as a new target to develop a first-in-class therapy for scleroderma.
Innovation. At least two aspects: /. This is the first project to study TREM-1 blockade in an animal model of scleroderma. 2. To block TREM-1, we use a proprietary peptide formulated into macrophage-specific LipoPeptide Complexes (LPC) to extend its half-life and increase targeting (Sigalov 2014, Shen et al. 2017, Shen et al. 2017). Other TREM-1 blockers (e.g., LR12 peptide by Inotrem, France (Cuvier et al. 2018)) all attempt to block binding of currently uncertain ligands of TREM-1 and have a risk of failure in clinics, while GF9 is an advantageously ligand-independent.
Previously ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai et al.
2019), Preliminary Data), we found that TREM-1 blockade using GF9: ameliorates disease in mice with collagen-induced arthritis (CIA); reduces serum CSF-1, TNFalpha, IL-lalpha, IL-6 in mice with CIA, cancer, and liver disease; and inhibits expression of MCP-1/CCL2, TNFalpha, Pro-Colll-alpha and alpha -SMA in mice with liver disease.
The goal of this project is to develop TREM-1-targeting drug for the treatment of scleroderma.
Aim 1: Optimize TREM-1 inhibitory compositions for their functionality in vitro and pharmacokinetics in vivo and select the lead. GF9-LPC will be generated using GF9, lipids and two modified peptides that mediate macrophage uptake of GF9-LPC and affect their half-life in vivo. We will vary lipid/peptide composition and peptide ratios to prepare long half-life GF9-LPC with fast and high uptake by J774 cells and high inhibitory effect on cytokine release by LPS-stimulated J774 cells. Three most promising GF9-LPC injectables selected based on their functionality in vitro will be tested in rats for their pharmacokinetic (PK) profiles. To analyze GF9 in animal serum, we will develop and validate an LC-MS assay with ZATA
Pharmaceuticals. Milestone 1 includes development of the long half-life lead, which is efficient in inhibiting cytokine release in vitro. Completion of the Aim 1 will answer the question on the possibility of generating of the lead optimized to provide fast, efficient and long-lasting therapeutic effect.
Aim 2: Test two doses of the GF9-LPC lead in a bleomycin-induced mouse model of scleroderma. We have shown that chronic subcutaneous injection of BLM in mice results in the development of progressive multiple organ fibrosis with histological changes in the skin, muscle and lungs that resemble those seen in patients with SSc (Bhattacharyya et al.
2018, Bhattacharyya et al. 2018). Two doses of the GF9-LPC lead generated in the Aim 1 will be tested for its effect on lung, heart, muscle and skin fibrosis in this mouse model. Studies will be performed at Northwestern Scleroderma by lab of Dr. John Varga, a world-renowned expert in autoimmune diseases with special emphasis on scleroderma. Histology/IHC
studies will be performed. Serum and tissue CCL2, CSF-1, VEGF, TGFbeta, TNFalpha, IL-6, and IL-lbeta will be analyzed. Milestone 2 includes in vivo testing of suitability of TREM-1 blockade to prevent and treat scleroderma. Completion of the Aim 2 will answer a question about feasibility of using GF9-LPC as a first-in-class therapy for scleroderma.
The project is anticipated to identify the lead that will set the stage for development of first-in-class, safe and effective scleroderma therapies. If successful, Phase I will be followed in Phase II by toxicology, pharmacology ADME, PK/PD, and CMC studies, filing an IND and subsequent evaluation in humans.
Anticipated low toxicity of GF9 therapy is supported by the safety and well tolerability of 300 mg/kg GF9 in healthy mice (Sigalov 2014) (while its therapeutic dose varies from 2.5 mg/kg for GF9-LPC to 25 mg/kg for free GF9 (Sigalov 2014, Shen and Sigalov 2015, Rojas et al. 2017, .. Shen and Sigalov 2017, Tornai et al. 2019)) and lack of body weight changes in cancer and arthritic mice long-term treated with GF9-LPC (Sigalov 2014, Shen and Sigalov 2017).
Prototypes of SignaBlok's LPC are safe in humans (Newton et al. 2002, Kingwell et al. 2013).
TREM-1 blockade using peptide LR12 developed by SignaBlok's top competitor (Inotrem, France) is safe in humans (Cuvier et al. 2018, Francois et al. 2018).
Successful completion of Phase I will provide the animal proof of concept that might be applicable not only to scleroderma but also to other rare musculoskeletal, rheumatic or skin diseases.
Research Strategy Scleroderma: An unmet need for an effective and low toxic treatment options Scleroderma is a rare but devastating autoimmune disorder (Lawrence et al.
1998, Mayes et al.
2003, Helmick et al. 2008) with no approved drug available. Current main treatments all have side effects, are limited and associated with 10 year survival of 55% (Badea et al. 2009, Kowal-Bielecka et al. 2009, Shah and Wigley 2013), highlighting an urgent need for new therapies. The long-term goal of the proposed project is to develop a novel, first-in-class, efficient and well tolerable systemic therapy for scleroderma.
Macrophages and scleroderma.
Macrophages are the predominant infiltrating cells in skin lesions of patients with scleroderma and are associated with fibrosis (Ishikawa and Ishikawa 1992, Kraling et al. 1995, Lech and Anders 2013, Chia and Lu 2015). MCP-1 recruits macrophages to inflammation sites and is significantly elevated in patients with systemic sclerosis (S Sc) (Hasegawa et al. 1999).
Activated macrophages produce VEGF, IL-1-beta, TNFalpha, IL-6, TGF-beta and PDGF, which are of crucial importance in the profibrogenic role of fibroblasts in scleroderma (Bonner et al. 1991, Clouthier et al. 1997, Yamamoto 2011, Yamamoto and Katayama 2011, Liu et al. 2013, Manetti 2015). In animals, blockers of IL-6 receptor (IL-6R) (Kitaba et al.
2012), VEGF (Koca et al. 2016), TNF-alpha (Koca et al. 2008) and TGF-beta (Varga and Pasche 2009, Varga and Whitfield 2009) alleviate scleroderma but all may have serious side effects including fatal infections and sepsis (Varga 2004). M-CSF plays a role in pulmonary fibrosis that occurs in 90%
of scleroderma patients (Baran et al. 2007). In rats, elevated MCP-1 and M-CSF
lead to macrophage recruitment in an injured area and to the lesion formation (Juniantito et al. 2013).
Inhibition of TRE11-1 signaling: A new approach to disorders associated with systemic inflammation Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), an inflammation amplifier, plays a role in immune response (Bouchon et al. 2000, Bouchon et al. 2001, Bleharski et al. 2003, Colonna et al. 2003, Klesney-Tait et al. 2006, Tessarz et al.
2008) and is upregulated upon inflammation (Wang et al. 2004, Gonzalez-Roldan et al. 2005, Koussoulas et al. 2006, Schenk et al. 2007). TREM-1 mediates release of multiple cytokines including MCP-1, TNF 0, IL-1 0, IL-6 and M-CSF (Schenk et al. 2007, Dower et al. 2008, Lagler et al.
2009, Sigalov 2014, Shen and Sigalov 2015). TREM-1 blockade is a new approach to inflammatory disorders (Bouchon et al. 2001, Colonna and Facchetti 2003, Schenk et al. 2007, Gibot et al. 2008, Ho et al. 2008, Ford et al. 2009, Gibot et al. 2009, Murakami et al. 2009, Luo et al. 2010, Pelham et al.
2014, Pelham et al. 2014, Bosco et al. 2016), In mice, TREM-1 blockade inhibits M-CSF, TNFalpha, IL-lbeta and IL-6, suppresses tumor growth and ameliorates autoimmune arthritis (Sigalov 2014, Shen and Sigalov 2017).
TREM-1 blockade blunts excessive inflammation but in contrast to single cytokine blockers, preserves the capacity for microbial control (Weber et al. 2014).
TREM-1 blockade was suggested as a treatment of neonatal infection (Qian et al. 2014).
Endotoxic and septic mice lacking DAP12, a signaling adapter of TREM-1, have improved survival (Turnbull et al. 2005).
Humans lacking DAP12 do not have problems resolving infections (Lanier 2009).
Inhibition of TRE11-1 signaling: A new approach to preventing and treating scleroderma TREM-1 is overexpressed in the lungs of mice with BLM-induced pulmonary fibrosis (Peng et al. 2016). In experimental autoimmune arthritis, cancer and retinopathy, TREM-1 blockade reduces inflammation and inhibits the macrophage infiltration / activation (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) (Section 3.3.3.1). In mice with alcohol-induced liver disease (ALD), TREM-1 blockade inhibits expression of TREM-1, MCP-1/CCL2, TNF
0, Pro-Colll 0 and 0 -SMA (Tornai et al. 2019). Collectively, these findings implicate TREM-1 as a target for development of new therapy for scleroderma.
The main concepts of the proposed project: Silencing the scleroderma-related specific inflammatory response can be superior to anti-single cytokine strategies in the treatment of scleroderma in terms of safety and efficacy; Delivery of systemically administered TREM-1 blockers to macrophages may have several advantages: (a) striking the target cell population, (b) sparing other cells that have no (or marginal) effects on scleroderma, (c) minimizing off-target effects, and (d) reducing the therapeutic dose; and Rate and efficiency of intracellular delivery of TREM-1 blockers to macrophages may be important to provide a prompt and effective therapeutic response during scleroderma progression.
INNOVATION
TREM-1 Blockade Major challenge. Current approaches (eg Inotrem's LR12) that all attempt to block TREM-1 binding to its ligand(s) (Fig. 97A) have a risk of failure since exact nature of TREM-1 ligand(s) is still uncertain (Tammaro et al. 2017).
SignaBlok's solution. Using our new model of signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model (Sigalov 2006, Sigalov 2010), we developed a first-in-class ligand-independent TREM-1 inhibitory peptide GF9 (US 8,513,185) that disrupts recognition and signaling functions of TREM-1 in the membrane (Fig. 97B) (Sigalov 2010, Sigalov 2013, Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017).
As other peptides (Graff et al. 2003, Lien et al. 2003, Gotthardt et al. 2004, Ladner et al.
2004, Prive et al. 2006, Sato et al. 2006, Antosova et al. 2009, Koskimaki et al. 2010), GF9 is advantageous compared to large protein molecules. Mechanistically, GF9 self-penetrates into the cell membrane and can reach its site of action from both inside and outside the cell (Fig. 97B
and Fig. 97C). GF9 is well-tolerated by healthy mice (up to 300 mg/kg; Fig.
98A). GF9 at 25 mg/kg in a free form or at 2.5 mg/kg when formulated into LipoPeptide Complexes (LPC, below), reduces tissue TREM-1 and M-CSF upon inflammation (shown on the example of the retina of mice with oxygen-induced retinopathy, OIR) (Fig. 98B), and ameliorates diseases in mouse models of cancer (Sigalov 2014, Shen and Sigalov 2017), retinopathy (Rojas et al. 2018), ALD (Tornai et al. 2019), sepsis and autoimmune arthritis (Sigalov 2014, Shen and Sigalov 2017).
LPC mimic human High Density Lipoproteins (HDL) and consist of lipids and peptides of human apolipoprotein (apo) A-I, the major protein of HDL. In contrast to native HDL, these peptides contain naturally occurring modifications that target LPC to macrophages. SignaBlok's LPC can deliver GF9 to macrophages in vitro and in vivo (Fig. 99A-C) and increase its therapeutic efficacy (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017). NOTE:
GF9-LPC describes GF9 formulated into either discoidal (GF9-dLPC, short 612:
hrs) or spherical (GF9-sLPC; long 612: days) LPC.
Epitope-based rational design of long half-life GF9-LPC fast and effective in delivery of GF9.
GF9-LPC tested to date, all contained a fixed amount of GF9 and an equimolar mixture of oxidized (MetS0) 22-mer peptides with sequences from either helix 4 (PE22) or 6 (PA22) of human apo A-I. Although these modifications increase macrophage uptake of LPC
in vitro and in vivo (Sigalov 2014, Sigalov 2014, Shen et al. 2015) (Fig. 99A), the uptake can be optimized to make it faster and more efficient. Oxidized PE22 and PA22 contain different MetS0 epitopes for binding to Scavenger Receptor (SR) SR-A (Apo A-I peptides contain putative epitopes for binding with SR-A (italics-M(0)) and SR-BI (bold). PE22:
PYLDDFQKKWQEEM(0)ELYRQKVE. PA22: PLGEEM(0)RDRARAHVDALRTHLA) (Neyen et al. 2009). In addition, PA22 contains an epitope for binding to macrophage and hepatocyte SR-BI (Liadaki et al. 2000, Cai et al. 2012). Its exposure affects binding to SR-BI (de .. Beer et al. 2001) and can determine the LPC half life.
APPROACH
Overall strategy, methodology, and analyses to be used to accomplish the specific aims Towards the overall goal of the proposed Phase I research, we will:
Aim 1. Optimize TREM-1 inhibitory compositions for their functionality in vitro and pharmacokinetics in vivo and select the lead.
1) generate and characterize GF9-LPC of different GF9/1pid/PE22/PA22 compositions; 2) use J774 cells and the relevant antibodies to explore mechanisms of SR-mediated uptake of GF9-LPC; 3) use the mechanistic data to optimize GF9/1pid/PE22/PA22 compositions and generate .. long half-life GF9-LPC with high GF9 load and high rate and efficiency of macrophage delivery of GF9; 4) functionally test the generated GF9-LPC for inhibition of cytokine release in LPS-stimulated J774 cells; 5) develop an LC-MS assay for analysis of GF9 in rat serum;
6) test three most promising formulations in PK studies in Sprague-Dawley (SD) rats;
7) analyze the data obtained and select the lead GF9-LPC formulation for further animal testing.
Aim 2: Test two doses of the GF9-LPC lead in a bleomycin-induced mouse model of scleroderma 1) test two doses of the GF9-LPC lead generated in the Aim 1 in preventative and established BLM-induced mouse models of scleroderma for its efficacy in preventing and treating the disease;
2) perform comprehensive histology / immunohistochemistry studies;
3) analyze serum and tissue GF9 and cytokines (LC-MS; ELISA).
Preliminary data Preliminary data, rationale, methodology, and analyses to be used to accomplish the Aim 1. Previously (Sigalov 2014), we showed that oxidation of PE22 and PA22 results in increased in vitro J774 cell uptake of GF9-LPC (Fig. 99A-C) and that GF9 (but not a control peptide) either in a free form (not shown) or formulated into LPC of discoidal (GF9-dLPC) or spherical (GF9-sLPC) shape inhibits cytokine release both in vitro and in vivo and protects mice from LPS-induced sepsis-related death (ig. 100A-D). GF9-dLPC and GF9-sLPC
both contained the same amount of GF9 and an equimolar mixture of oxidized PE22 and PA22.
Rationale GF9-dLPC and GF9-sLPC both inhibit LPS-stimulated cytokine release in vitro and in vivo to about the same degree (Fig. 100A, Fig. 100B) but their protective effect at the dose of 5 mg/kg in LPS-induced septic mice differs: GF9-sLPC provide less effective but longer-lasting protection as compared with GF9-dLPC (Fig. 100C). Further, despite the same GF9 load and 1:1 PE22:PA22 molar ratio, these GF9-LPC differ in rate and efficiency of the macrophage uptake in vitro (Fig. 101) (Sigalov 2014). Stronger protection by GF9-dLPC may result from higher efficiency and rate of their uptake (Fig. 101), while longer protection by GF9-sLPC ¨ from their longer half-life. Thus, uptake of GF9-LPC may depend on exposure of SR-binding apo A-I
epitopes (Liu et al. 2002, Horiuchi et al. 2003) (Apo A-I peptides contain putative epitopes for binding with SR-A (italics-M(0)) and SR-BI (bold). PE22:
PYLDDFQKKWQEEM(0)ELYRQKVE.
PA22: PLGEEM(0)RDRARAHVDALRTHLA) that affect both rate and efficiency of the uptake.
In Phase I Aim 1, we will optimize exposure of SR-A- and SR-BI-binding epitopes and GF9 content of long half-life GF9-LPC by varying of GF9/lipid/PE22/PA22 ratios to increase GF9 load and rate and efficiency of its delivery in vivo and thus to provide prompt, effective and long-.. term therapeutic response.
Methodology and analyses Peptides. GF9 and two oxidized 22-mer peptides PE22 and PA22 will be ordered from Bachem, Inc. and characterized as described previously (Sigalov et al. 1998, Sigalov et al. 2001, Sigalov et al. 2002, Sigalov 2014, Shen et al. 2015, Shen and Sigalov 2017, Shen and Sigalov 2017).
Long half-life GF9-LPC (spherical). Previously used non-optimized GF9-LPC be synthesized as described (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) and used as a reference in all in vitro studies. In some studies, GF9 and/or PE22 will be Dylight (Dy) 488-labeled. In some studies, GF9-LPC will be Rhodamine B (Rho B)-labeled.
Optimization. The following parameters will be varied: a) phospholipid chain length; 2) lipid composition; 3) lipid/PE22/PA22 composition/ ratio; and 4) GF9 content. The obtained GF9-LPC will be purified and their integrity, stability, and GF9 content will be analyzed as reported (Sigalov 2014, Shen and Sigalov 2017). As analyzed by Dynamic Light Scattering (DLS), GF9-LPC are stable at 4 C for at least, up to 6 months (Fig. 102).
In vitro macrophage uptake assay. GF9-LPC will be characterized by in vitro macrophage uptake assay as reported (Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai et al. 2019). To explore the mechanisms of GF9-LPC uptake, cells will be incubated with either anti-SR-BI, anti-SR-A, or isotype controls for 15 min on ice before adding Rho B-labeled GF9-LPC with Dy 488-labeled GF9 and/or PE22. After incubation, cells will be washed, lysed and fluorescence and protein concentrations in the lysates will be measured.
In vitro cytokine release. The assay will be performed in LPS-stimulated J774 macrophages (Fig. 100B) as previously reported (Sigalov 2014).
Confocal analysis. J774A.1 cells will be grown at 37 C in 6 well tissue culture plates containing glass coverslips. After reaching target confluency of ¨ 50%, cells will be incubated for 6 h at 37 C with Rho B-GF9-LPC. In subsets of experiments, Rho B- GF9-LPC
that contain Dylight 488-PE22 or Dylight 488-GF9 will be used. TREM-1 staining will be performed as described (Shen and Sigalov 2017). The slides will be imaged as reported (Shen and Sigalov 2017).
Integrity and stability studies. RP-HPLC, SEC, and DLS will be used as described (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) to study structural integrity and stability of GF9-LPC.
LC-MS for GF9 analysis in animal serum. LC-MS assay for analysis of GF9 in rat serum in PK studies in rats will be developed and validated (with ZATA). The assay will include ultracentrifugation step followed by LC-MS. The snap-frozen samples of rat serum will be ordered from BioreclamationIVT (Westbury, NY) and processed as reported (Walther et al.
2011, Yanachkov et al. 2011, Yanachkova et al. 2015, Yanachkov et al. 2016).
GF9-LPC will be added to serum and GF9 will be assayed by LC-MS. The assay will be validated using the FDA
guidelines (https://www.fda.gov/downloads/drugs/guidances/ucm368107.pdf).
PK studies in SD rats. Animal studies will be provided by WBI. SignaBlok will perform LC-MS/histology/IHC. Sex as a biological variable. To exclude differences in PK
in male and female rats (Shelnutt et al. 1999), we propose to use both sexes. 3 most promising GF9-LPC
selected based on their TREM-1 inhibitory activity in vitro will be tested in 8 wk-old SD rats (200-250 g) (3 groups; 3 males+3 females / group, 18 SD rats). Briefly, SD
rats will be IV
administered with 2.5 mg/kg GF9-sLPC. Serum samples will be collected at 8 post-injection timepoints within 24 hrs, frozen and shipped to SignaBlok for LC-MS analysis of GF9.
Ultracentrifugation of serum to float lipoproteins and GF9-LPC will be performed as reported (Sigalov et al. 1991, Sigalov 1993, Sigalov et al. 1997, Sigalov and Stern 1998, Sigalov and Stern 2001). Briefly, 50 OL serum, 50 OL saline, 0.5 mM EDTA, and 130 OL KBr (d = 1.37 g/mL) will be mixed (final d=1.21 g/mL) and centrifuged in a 42.2 TI rotor at 42,000 rpm for 12 h at 10 C. 50 OL will be taken from top, dialyzed for 4 h at 4 C and analyzed for GF9 by LC-MS.
Statistical analysis. GraphPad Prism will be used for statistical testing. In in vitro uptake assay and cytokine assay data, statistical significances will be determined by two-tailed Student's t test as described (Sigalov 2014). Results will be considered significant atp<0.05.
PK data will be analyzed using PKSolver, a menu-driven add-in Microsoft Excel software (Zhang et al. 2010).
Outcome measures Stability of GF9-LPC will be tested by DLS (Fig. 102). GF9 in GF9-LPC will be analyzed as reported (Sigalov 2014, Shen and Sigalov 2017) and by LC-MS. In vitro J774 cell uptake will be measured by Rho B fluorescence in cell lysates (Sigalov 2014). Activity of GF9-LPC in reduction of cytokine release by LPS-stimulated cells will be tested as reported (Sigalov 2014).
In PK studies, half-life, Cmax, Tmax and the area under the AUC will be analyzed.
Anticipated results and interpretations Native dHDL and sHDL have half-lives of 12-20 hrs and 3-5 days, respectively (Scanu et al.
1962, Furman et al. 1964). We expect that formulation of GF9 into spherical LPC will extend its half-life closer to that for sHDL. Based on our preliminary data and (Sigalov 2014), we predict that: 1) GF9-sLPC of different compositions will have different exposure of SR-A and SR-BI
epitopes, and 2) use of SR inhibitors will allow to find the preferential receptor involved in cell uptake. We predict that PK profiles of GF9-LPC formulations with different exposure of SR-A
and SR-BI epitopes will differ. Thus, we anticipate to optimize SR-A/SR-BI
epitope exposure and prepare GF9-LPC with high in vitro efficacy and favorable PK in vivo.
Completion of Aim 1 will show the feasibility of using of epitope-based design to optimize GF9-LPC
for effective and long-term inhibition of TREM-1 in vitro and in vivo. Milestone 1 includes selection of the lead based on its stability, in vitro activity and PK profile.
Anticipated problems, alternative strategies and future directions.
We do not expect technical problems as we at SignaBlok, Drs. Tabatadze and Yanachkov at ZATA, and the WBI's staff have expertise in all methods (Yanachkov et al.
2011, Sigalov 2014, Sigalov 2014, Shen et al. 2015, Yanachkova et al. 2015, Shen et al. 2016, Yanachkov et al. 2016, Shen and Sigalov 2017, Shen and Sigalov 2017, Yanachkov et al. 2017).
Preliminary data Previously, using non-optimized GF9-LPC, we demonstrated that:
1) in mice with ALD, systemic 2.5 mg/kg GF9-LPC reduces TREM-1, MCP-1/CCL2, early fibrosis markers (alpha-smooth muscle actin [alpha-SMA] and procollagenl-alpha [Pro-Colll-alpha]) at the mRNA level (Tornai et al. 2019) Fig. 103A-D);
2) in cancer mice, systemic 25 mg/kg GF9 and 2.5 mg/kg GF9-LPC are well-tolerated (Fig.
Fig. 104A), reduce macrophage infiltration into the tumor (Fig. 103B, Fig.
103C) and inhibit CSF-1/M-CSF (Fig. 104D) (Shen and Sigalov 2017);
3) in mice with CIA, systemic 25 mg/kg GF9 and 2.5 mg/kg GF9-LPC are well-tolerated (Fig. 105AA), ameliorate arthritis (Fig. 104B) and inhibit IL-1-beta, IL-6, TNF-alpha and CSF-1/M-CSF (Fig. 105AC) (Shen and Sigalov 2017).
Rationale TREM-1 blockade by GF9-LPC suppress macrophage infiltration and activation, reduce cytokine, CSF-1/ M-CSF and early fibrosis markers and ameliorate disease in ALD, cancer, septic and arthritic mice ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai et al. 2019), Fig. 103A-D-Fig. 105A- C). This suggests that GF9-LPC will be effective in the treatment of scleroderma (Ishikawa and Ishikawa 1992, Kraling et al. 1995, Lech and Anders 2013, Chia and Lu 2015). BLM mouse model is a valuable tool for drug development for scleroderma (Yamamoto et al. 1999, Yamamoto et al. 1999, Huber et al. 2007, Beyer et al. 2010, Kitaba et al. 2012, Avci et al. 2013, Artlett 2014, Toyama et al. 2016). We have shown that chronic subcutaneous (s.c.) injection of BLM in mice results in development of progressive multiple organ fibrosis (Bhattacharyya et al. 2018, Bhattacharyya et al.
2018). In Aim 2, we will use this model to test GF9- LPC ability to prevent and treat organ fibrosis.
Serum and tissue CCL2, CSF-1/M-CSF, VEGF, TGF 0, TNF 0, IL-6, and IL-10 will be analyzed.
Methodology and analyses We will design perform and analyze animal studies with Dr. John Varga, M.D.
(Director, Northwestern Scleroderma, Northwestern University Feinberg School of Medicine (Chicago, IL).
Sex (gender) as a biological variable. In a BLM-induced scleroderma mouse model, while a more pronounced fibrosis phenotype was reported for male compared with female mice (Ruzehaji et al. 2015), other data show no histologic differences between male and female mice (Yamamoto et al. 1999, Yamamoto et al. 1999) We suggest to use both sexes of mice in this project.
Mouse model of scleroderma. S.c. BLM delivery leads to slowly-progressive fibrosis in multiple organs with no mortality, and histological changes in the skin, muscle and lungs that resemble those seen in patients with SSc (Bhattacharyya et al. 2018, Bhattacharyya et al. 2018).
8-12 wk-old C57BL6 mice (288 in total) will be randomized and divided into 3 arms by 12 groups of 8 mice per group (6 male and 6 female groups). In preventative model (arms 1 and 2), mice will receive s.c. injections of 10 mg/kg BLM or PBS daily for 10 days (5 days/week), along with 2.5 or 5 mg/kg GF9-LPC by daily i.p. injections starting concurrently with BLM, and will be sacrificed on day 7 (arm 1) or 22 (arm 2). In established model (arm 3), mice will receive 2.5 or 5 mg/kg GF9-LPC daily starting at day 15, and continue until sacrifice at day 28. In all arms, control groups of mice will receive BLM or PBS alone daily for 10 (7, arm 1) days or 2.5 or 5 mg/kg GF9-LPC alone daily until sacrifice at days 7, 22 or 28.
Statistical analysis. Statistical significance of differences in parameters of fibrosis and inflammation between control and treated mice will be determined by F-test.
Comparison among three or more groups will be executed with one-way ANOVA followed by a post hoc Tukey's test. Based on our previous studies using this model (Bhattacharyya et al.
2018, Bhattacharyya et al. 2018), a sample size of 8 mice in each group is chosen to give a power of 80% to detect 10%
difference in mean values between experimental and control groups, with a significance level of 0.05.
Outcome measures Experiments will test efficacy of treatment given as prevention, as well as treatment, to determine if TREM-1 inhibition can promote regression of established skin, lung and heart fibrosis and resolution of tissue damage. Clinical observations (daily) and body weights (weekly) will be made until termination. DRAIZE scoring will be recorded once weekly for all groups.
Effect of TREM-1 blockade will be tested on early (day 3-7) inflammatory changes and monocyte/macrophage influx in the lungs and skin by IHC; subsequent development of fibrotic parenchymal changes (at day 10-20) by histology/IHC, biochemical and functional assays.
Tissues will be collected, prepared, stained with H&E and Trichrome and evaluated by board-certified pathologist. Part of collected tissues will be homogenized and along with blood and FFPE tissue samples shipped to SignaBlok for GF9, cytokine and IHC analysis.
Tissue collagen content will be determined by hydroxyproline assays (Bhattacharyya et al.
2016). Lung fibrosis will be quantitated in histological lung sections using the modified Ashcroft score determined from 5 h.p.f. per mice (Hubner score) (Hubner et al. 2008). Skin hardness will be measured using a Vesmeter three times at the injection area. Dermal thickness will be determined at three randomly selected sites in each animal. a-SMA-positive cells will be counted.
Macrophage infiltration will be evaluated by IHC. Serum and tissue CCL2, VEGF, CSF-1, TGF
0, TNF 0, IL-6 and IL-1 0 will be analyzed by ELISA. Tissue TREM-1 expression will be tested by Western Blot.
Anticipated results and interpretations These studies are expected to demonstrate if TREM-1 blockade using GF9-LPC
can, by attenuating TLR4 activity in target organs, prevent, slow the progression, and promote the recovery from, fibrotic injury in the skin, lungs, muscle and heart. Further, the results are expected to indicate whether observed beneficial effects are primarily due to attenuated early inflammation, reduced fibrosis due to attenuated activation of (myo)fibroblasts, or a combination of both of these mechanisms.
Based on our previous data ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017); Fig. 100A-D, Fig. 103A-D -Fig. 105A-C), we predict that treatment with GF9-LPC will be well-tolerated and associated with reductions in levels of CCL2, CSF-1, TNF-beta, TGF-alpha, IL-6 and IL-lbeta. We expect that GF9-LPC will be effective in a dose-dependent manner and that LPC (no GF9) will be without effect. Completion of Aim 2 will answer a question about the feasibility of using GF9-LPC as a first-in-class therapy for scleroderma.
Anticipated problems, alternative strategies and future directions.
We do not expect technical problems as we at SignaBlok and the Varga laboratory's and animal facility' staff have extensive expertise in all methods (Varga and Whitfield 2009, Sigalov 2014, Shen et al.
2015, Shen and Sigalov 2016, Shen and Sigalov 2017, Shen and Sigalov 2017, Bhattacharyya et al.
2018, Bhattacharyya et al. 2018, Yamashita et al. 2018, Lakota et al. 2019, Tornai et al. 2019).
Final product. SignaBlok's GF9-LPC consist of phospholipids widely used in pharmacology and synthetic peptides, all derived from human sequences, suggesting the lack of potential immunogenicity. Lipoprotein- and peptide-based drug formulations are currently on the market (Chang et al. 2012, Adler-Moore et al. 2016) or in clinical trials (Tricoci et al.
2015), which makes SignaBlok's efficient and well tolerable systemic therapy for scleroderma commercially viable.
Future directions. If successful, Phase I will be followed in Phase II where to evaluate the efficacy of TREM-1 blockade for mitigating organ fibrosis, we will use complementary mouse models of SSc, including the Tsk1/+ mouse, which (spontaneously) develop skin fibrosis in the absence of inflammation. Other administration schedules and regimen will be tested. The lead and its manufacturing technology will be further optimized and the more detailed safety, TOX, ADME, CMC and other IND-enabling studies will be performed. Upon completion, an IND will be filed for subsequent testing in humans.
Anticipated low toxicity of GF9-LPC is supported by the safety of 300 mg/kg GF9 in healthy mice (Sigalov 2014) (therapeutic doses are 25 mg/kg for GF9 or 2.5 mg/kg for GF9-LPC), lack of body weight changes in mice long-term treated with GF9-LPC (Sigalov 2014, Shen et al.
2017, Tornai et al. 2019), and by the fact that prototypes of SignaBlok's LPC
were well tolerated in humans (Newton and Krause 2002, Kingwell and Chapman 2013). TREM-1 blockade using inhibitory peptide LR12 which is in development by SignaBlok's top competitor (Inotrem, France) was well tolerated in healthy and septic subjects (Cuvier et al. 2018, Francois et al.
2018).
The decision to go to Phase II will be made if the significant (more than 50%) decrease in fibrosis is shown in treated mice as compared with those shown in control mice.
Bibliography and References Cited.
1. Helmick CG, Felson DT, Lawrence RC, Gabriel S, Hirsch R, Kwoh CK, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part I. Arthritis Rheum 2008; 58:15-25. PMID: 18163481.
2. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini EH, et al.
Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum 1998; 41:778-99. PMID: 9588729.
3. Mayes MD, Lacey JV, Jr., Beebe-Dimmer J, Gillespie BW, Cooper B, Laing TJ, et al.
Prevalence, incidence, survival, and disease characteristics of systemic sclerosis in a large US
population. Arthritis Rheum 2003; 48:2246-55. PMID: 12905479.
4. Kowal-Bielecka 0, Landewe R, Avouac J, Chwiesko S, Miniati I, Czirjak L, et al.
EULAR recommendations for the treatment of systemic sclerosis: a report from the EULAR
Scleroderma Trials and Research group (EUSTAR). Ann Rheum Dis 2009; 68:620-8.
PMID:
19147617.
5. Badea I, Taylor M, Rosenberg A, Foldvari M. Pathogenesis and therapeutic approaches for improved topical treatment in localized scleroderma and systemic sclerosis. Rheumatology (Oxford) 2009; 48:213-21. PMID: 19022832.
6. Shah AA, Wigley FM. My approach to the treatment of scleroderma.
Mayo Clin Proc 2013; 88:377-93. PMID: 23541012.
7. Ishikawa 0, Ishikawa H. Macrophage infiltration in the skin of patients with systemic sclerosis. J Rheumatol 1992; 19:1202-6. PMID: 1404154.
8. Chia JJ, Lu TT. Update on macrophages and innate immunity in scleroderma. Curr Opin Rheumatol 2015; 27:530-6. PMID: 26352734.
9. Lech M, Anders HJ. Macrophages and fibrosis: How resident and infiltrating mononuclear phagocytes orchestrate all phases of tissue injury and repair.
Biochim Biophys Acta 2013; 1832:989-97. PMID: 23246690.
10. Kraling BM, Maul GG, Jimenez SA. Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages. Pathobiology 1995; 63:48-56. PMID: 7546275.
Fig.77 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue is well tolerable in mice with collagen-induced arthritis (CIA). Mouse body weight (BW) was measured every other day from day 24 to day 38. Mean BW
changes were calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the CIA mice intraperitoneally (i.p.) treated daily for 14 consecutive days with vehicle, dexamethasone, GF9, GF9-LPC and GA/E31-LPC at indicated doses. All results are expressed as the mean SEM (n = 10 mice per group).
Fig.78 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) prevents pathological appearances from collagen-induced arthritis (CIA) in mice. As described herein, toluidine blue staining of the joints from mice with CIA treated with TREM-1 inhibitory GF9 sequences or control peptide GF9-G
(GFLSGSLVF) was performed. Photomicrographs of fore paws, hind paws, knees and ankles from representative mice are shown for each treatment group. For paws (original magnification 16x) and ankles (original magnification 40x), arrows identify affected joints. For knees (original magnification 100x), large arrow identifies cartilage damage, small arrow identifies pannus and arrowhead identifies bone resorption. W, wrist; S, synovium.
Fig.79 presents the exemplary data of one embodiment showing that treatment with free TREM-1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) reduces plasma cytokines in mice with collagen-induced arthritis (CIA). Plasma was collected on days 24, 30 and 38 from arthritic mice treated with vehicle (black diamonds), GF9 (white circles), GF9-LPC (black circles) and GA/E31-LPC (half black half white circles). Plasma samples were analyzed for concentrations of interleukin-lb (IL-lb), IL-6, and colony-stimulating factor 1 (CSF1). Results are expressed as the mean SEM (n =
mice per group).
Fig. 80 presents the exemplary data of one embodiment showing that treatment with free TREM-5 1 modulatory peptide GF9 (GFLSKSLVF) or GF9 incorporated into lipopeptide complex (GF9-LPC) or LPC comprising lipids and an equimolar mixture of the 31 amino acids-long TREM-1 modulatory peptide GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA) where M(0) is a methionine sulfoxide residue and the 31 amino acids-long TREM-1 modulatory peptide GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where M(0) is a methionine sulfoxide residue (GA/E31-LPC) significantly reduces tissue expression of colony-stimulating factor 1 (CSF1) and TREM-1 in the retina of mice with oxygen-induced retinopathy (OIR) at postnatal day 17 (P17). Representative Western blots of retinal lysates from OIR mice are shown. The membrane was probed for TREM-1, reprobed for CSF1 and then for 13-actin. Values in the bar graphs represent the mean SEM, n=6. *, p < 0.05, **, p < 0.01 vs.
vehicle-treated mice.
Fig. 81 shows exemplary illustrations of peptide GF9 blocking TREM-1 signaling by disruption of intramembrane interactions with its signaling partner, DAP-12. One example of a comparision of current approaches (upper) with a SCHOOL approach (lower), e.g. Route 1.
Fig. 82 shows exemplary illustrations of LPC delivering of peptide GF9 to macrophages, as two exemplary embodiments, e.g. each as Route 2.
Fig. 83 shows exemplary results using Pancreas Cancer: PANC-1 Xenografts demonstrating GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM
+ nab-PTX, ABX) and Adding GF9 treatment sensitizes the tumor to chemotherapy and at least triples survival rate.
Fig. 84 shows exemplary results using Pancreas Cancer: AsPC-1 Xenografts demonstrating GF9 treatment alone does not inhibit tumor growth.Adding of the GF9 treatment sensitizes the tumor to chemotherapy. NOTE: Most tumors ¨ abscessed.
Fig. 85 shows exemplary results using Pancreas Cancer: MiaPaca-2 Xenografts demonstrating GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM + nab-PTX, ABX) and Adding of the GF9 treatment to chemo does not affect.
Fig. 86 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts demonstrating GF9 treatment inhibits tumor growth as effective as chemotherapy (Gemcitabine, GEM
+ nab-PTX, ABX) and Adding of the GF9 treatment to chemo does not significantly affect survival rate.
Fig. 87 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts demonstrating GF9 treatment reduces macrophage content in the tumor, Vehicle, 2.5 mg/kg GF9-LPC
(5 qw, 4 wk).
Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 88 shows exemplary results using Pancreas Cancer: BxPC-3 Xenografts demonstrating GF9 treatment reduces serum cytokine levels, Vehicle, 2.5 mg/kg GF9-LPC. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 89 shows exemplary results using Pancreas Cancer: Xenografts demonstrating GF9 Treatment is Non-Toxic. Free GF9 tolerability (upper). GF9-LPC* tolerability (lower). * Shown for PANC-1 xenograft Fig. 90 shows exemplary results demonstrating that GF9 peptide is well-tolerable by healthy mice up to at least, 300 mg/kg.
Fig. 91 shows exemplary results demonstrating that in mice with collagen-induced arthritis (CIA), GF9 suppresses arthritis as effectively as dexamethasone (DEX). Study Day (Treatment:
Days 24-38). I, inflammation; P, pannus; CD, cartilage damage; BR, bone resorption; PBF, periosteal new bone formation. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 92 shows exemplary results demonstrating that in mice with collagen-induced arthritis (CIA), GF9 treatment reduces serum IL-lb\ TNFal, IL-6 and CSF-1. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 93 shows exemplary results demonstrating that in mice with collagen-induced arthritis (CIA), GF9 treatment is well-tolerable: no body weight changes or other clinical symptoms are observed. Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 94 shows exemplary results demonstrating that in NSCLC: A549 Xenografts, GF9 inhibits tumor growth as effectively aschemo (20 mg/kg Paclitaxel, PTX). Sigalov, 2014, 21:208.
Fig. 95 shows exemplary results demonstrating that in Capan-1 xenografts, GF9 inhibits tumor growth and reduces serum cytokines, including CSF-1 (but not VEGF). Shen and Sigalov, Mol Pharm 2017,14:4572, 2017.
Fig. 96 shows exemplary results demonstrating that GF9 is well-tolerated by long term treated cancer mice inCapan-1 Xenografts and A549 Xenografts, GF9 inhibits tumor growth as effectively aschemo (20 mg/kg Paclitaxel, PTX). Sigalov, 2014, 21:208.
Fig. 97A-C shows exemplary current approaches for blocking TREM-1 binding to its uncertain ligand (Bouchon et al. 2001, Schenk et al. 2007, Gibot et al. 2008, Gibot et al. 2009, Murakami et al. 2009, Luo et al. 2010, Derive et al. 2013, Derive et al. 2014) Fig. 97A
In contrast, GF9 self-penetrates into the membrane and disrupts TREM-1 / DAP12 interactions Fig. 97B when colocalizes with TREM-1 Fig. 97C. Fig. 97A. CURRENT. Fig. 97B. SCHOOL Fig.
97C.
CONFOCAL.
Fig. 98A-B shows exemplary results demonstrating that GF9 is non-toxic in healthy mice Fig.
98A and reduces TREM-1 and M-CSF overexpression in the retina of mice with oxygen-induced retinopathy Fig. 98B. Fig. 98A Graph. Fig. 98B Blot.
Fig. 99A-C shows exemplary results demonstrating that Oxidized apo A-I
peptides in LPC
increase J774 intracellular uptake of GF9-LPC in vitro Fig. 99A, 99B and enable in vivo delivery to macrophages Fig. 99C (as shown using magnetic resonance imaging (Mill) and confocal microscopy (Sigalov 2014, Sigalov 2014, Shen and Sigalov 2017)). Fig. 99A. IN
VITRO. Fig.
.. 99B. CONFOCAL red: Rho B-PE; green: 488-GF9; blue: 405-apo A-I PE22. Fig.
99C. MOUSE
AORTA.
Fig. 100A-D shows exemplary results demonstrating that GF9-dLPC (disks) and GF9-sLPC
(spheres) reduce LPS-induced cytokine release in vitro Fig. 100A and in vivo Fig. 100B and prolong survival Fig. 100C (Sigalov 2014). In cancer mice, GF9 and GF9-LPC
treatments inhibit production of CSF-1/M-CSF but not VEGF Fig. 100D (Shen and Sigalov 2017). Fig.
100A.
CYTOKINES IN VITRO. Fig. 100B. CYTOKINES IN VIVO. Fig. 100C. SURVIVAL IN LPS-INDUCED SEPTIC MICE. Fig. 100D. M-CSF / VEGF RELEASE IN CANCER MICE.
Fig. 101 shows exemplary results demonstrating that Different rate and efficiency of GF9-dLPC
and GF9-sLPC in vitro uptake by J774 macrophages (Sigalov 2014).
Fig. 102 shows exemplary results demonstrating that Stability of GF9-LPC. GF9-LPC AT 4 C.
Fig. 103A-D shows exemplary results demonstrating that GF9-LPC daily i.p.
administered at 2.5 mg/kg suppress the expression of TREM-1, MCP-1/CCL2 and early fibrosis marker molecules in mice with ALD. Indicates significance level compared to nontreated pair-fed group; # indicates significance level compared to nontreated alcohol-fed group. Significance levels are as follows:
<0.05; **/#4, p < 0.01; ***,p <0.001; ****/ p <0.0001. Fig. 102A. TREM-1.
Fig.
102B. MCP-1/CCL2. Fig. 102C. Pro-Colllalpha. Fig. 102D. alpha-SMA.
Fig. 104A-D shows exemplary results demonstrating that GF9 and GF9-LPC daily i.p.
administered are well-tolerated Fig. 104A, suppress macrophage infiltration into the tumor Fig.
104B, 104C and inhibit release of CSF-1/M-CSF but not VEGF Fig. 104D. Scale bar = 200 pm.
*,p< 0.05; **,p < 0.01; ***,p < 0.001; ****,p < 0.0001 (vs vehicle). Fig.
104A. BODY
WEIGHT. Fig. 104B. INTRATITMORAL MACROPHAGE INFILTRATION ¨ INHIBITION
BY GF9 AND GF9-LPC. Fig. 104C. MACROPHAGE INFILTRATION. Fig. 104D. M-CSF /
VEGF RELEASE IN CANCER MICE.
Fig. 105A- C shows exemplary results demonstrating that in mice with autoimmune arthritis, GF9, discoidal GF9-LPC (GF9-dHDL) and spherical GF9-LPC (GF9-sHDL) i.p.
administered daily are well-tolerated Fig. A, ameliorate the disease Fig. 105B and inhibit production of cytokines and M-CSF Fig. C (Shen and Sigalov 2017). Fig. 105A. BODY WEIGHT
CHANGES.
Fig. 105B. ARTHRITIS AMELIORATION. Fig. 105C. CYTOKINE RELEASE IN
ARTHRITIC MICE.
DEFINITIONS
The term, "composition", as used herein, refers to any mixture of substances comprising a peptide and/or compound contemplated by the present invention. Such a composition may include the substances individually or in any combination.
As used herein the term "lipoprotein" such as VLDL (very low density lipoproteins), LDL (low density lipoproteins) and HDL (high density lipoproteins), refers to lipoproteins found in the serum, plasma and lymph, in vivo, related to lipid transport. The chemical composition of each lipoprotein differs, for examples, HDL has a higher proportion of protein versus lipid, whereas the VLDL has a lower proportion of protein versus lipid. When referring to lipoproteins, the term "native" refers to naturally-occurring (e.g., a "wild-type") lipoproteins.
The terms "AP0A1 HUMAN", "Apolipoprotein A-I", "Apolipoprotein A-1", "AP0A1", "ApoA-I", "Apo-AI", "ApoA-1", "apo-Al", "apoA-1" and "Apo-Al" refer to the naturally occurring human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name "AP0A1 HUMAN". The protein amino acid sequence can be found under the entry UniProt KB/Swiss-Prot P02647 (www.uniprot.org/uniprot/P02647). The terms "AP0A2 HUMAN", "Apolipoprotein A-II", Apolipoprotein A-2", "AP0A2", "ApoA-II", "Apo-All", "ApoA-2", "apo-A2", "apoA-2" and "Apo-A2" refer to the naturally occurring human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name "AP0A2 HUMAN". The protein amino acid sequence can be found under the entry .. UniProt KB/Swiss-Prot P02652 (http://www.uniprot.org/uniprot/P02652).
The term "TREM receptor", as used herein, refers to a member of TREM receptor family including: TREM-1, TREM-2, TREM-3 and TREM-4. The terms "TREM1 HUMAN", "TREM-1 receptor", "TREM-1 receptor subunit", "TREM-1 subunit", and "TREM-1 recognition subunit" refer to the naturally occurring human protein listed in the UniProt Knowledgebase (UniProtKB, www.uniprot.org) under the name "TREM1 HUMAN". The protein amino acid sequence can be found under the entry UniProt KB/Swiss-Prot Q9NP99.
The term "TREM receptor", as used herein, refers to a member of TREM receptor family: TREM-1, TREM-2, TREM-3 and TREM-4.
As used herein, the term "T cell receptor" or "TCR" refers to a complex of membrane proteins that participate in the activation of T cells in response to the presentation of antigen. The TCR is responsible for recognizing antigens bound to major histocompatibility complex molecules. TCR is composed of a heterodimer of an alpha (a) and beta (0) chain, although in some cells the TCR consists of gamma and delta (y/6) chains. TCRs may exist in alpha/beta and gamma/delta forms, which are structurally similar but have distinct anatomical locations and functions. Each chain is composed of two extracellular domains, a variable and constant domain, in some embodiments, the TCR may be modified on any ceil comprising a TCR, including, for example, a helper T cell, a cytotoxic T cell, a memory T ceil, regulatory T
cell, natural killer T
cell, and gamma delta T cell.
As employed herein and understood by the ordinary skill in the art, "amino acid domain"
is a contiguous polymer of at least 2 amino acids joined by peptide bond(s).
The domain may be joined to another amino acid or amino acid domain by one or more peptide bonds. An amino acid domain can constitute at least two amino acids at the N-terminus or C-terminus of a peptide or can constitute at least two amino acids in the middle of a peptide.
The term "antibody" herein refers to a protein, derived from a germline immunoglobulin sequence, which is capable of specifically binding to an antigen (TREM-1) or a portion thereof.
The term includes full length antibodies of any class or isotype (that is, IgA, IgE, IgG, IgM
and/or IgY) and any single chain or fragment thereof. An antibody that specifically binds to an antigen, or portion thereof, may bind exclusively to that antigen, or portion thereof, or it may bind to a limited number of homologous antigens, or portions thereof.
As used herein, a "peptide" and "polypeptide" comprises a string of at least two amino acids linked together by peptide bonds. A peptide generally represents a string of between approximately 2 and 200 amino acids, more typically between approximately 6 and 64 amino acids. Peptide may refer to an individual peptide or a collection of peptides.
Inventive peptides typically contain natural amino acids, although non-natural amino acids (i.e., compounds that do not occur in nature but that can be incorporated into a polypeptide chain and/
or amino acid analogs as are known in the art may alternatively be employed. In particular, D-amino acids may be used.
As employed herein and understood by the ordinary skill in the art, "peptide sequence", or "amino acid sequence", is the order in which amino acid residues, connected by peptide bonds, lie in the chain in peptides. The sequence is generally reported from the N-terminal end containing free amino group to the C-terminal end containing free carboxyl group. "Peptide sequence" is often called "protein sequence" if it represents the primary structure of a protein (http://en.wikipedia.org/wiki/Peptide sequence).
Peptides and compositions of the present invention made synthetically may include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or .. unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH2(CH2)õCOOH
wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;
citrulline and .. methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
Naturally occurring residues are divided into groups based on common side chain properties: as described herein. Analogues may be generated by substitutional mutagenesis and retain the biological activity of the original trifunctional peptides.
Examples of substitutions identified as "conservative substitutions" are shown in TABLE 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in TABLE 1, or as further described herein in reference to amino acid classes, are introduced and the products screened for their capability of executing three functions.
The term "amphipathic" is used herein to describe a molecule that has both polar and .. non-polar parts and as such, has two different affinities, as a polar end that is attracted to water and a nonpolar end that is repelled by it. An amphipathic helix is defined as an alpha helix with opposing polar and nonpolar faces oriented along the long axis of the helix.
As well known in the art, amino acid sequences can be screened for amphipathic helixes and an amphipathicity score can be calculated using a variety of computer programs available online (see, for example, http ://www.tcdb . org/progs/?tool=pepwheel, http ://lbqp.unb . br/NetWhe el s/, https ://np s a-prabi.ibcp.fr/cgi-bin/npsa automat.pl?page=/NPSA/npsa amphipaseek.html, http ://rzlab .ucr. e du/scripts/wheel/whe el .cgi, http ://heli quest. ipm c.
cnrs.fr/cgi-bin/ComputParams.py) or other techniques including but not limiting to those described in Jones, et al. J Lipid Res 1992, 33:287-296.
As used herein, the term "aptamer" or "specifically binding oligonucleotide"
refers to an oligonucleotide that is capable of forming a complex with an intended target substance.
In the present disclosure, the term "modified peptide" is used to describe chemically or enzymatically, or chemically and enzymatically modified oligopeptides, oligopseudopeptides, polypeptides, and pseudopolypeptides (synthetic or otherwise derived), regardless of the nature of the chemical and/or enzymatic modification. The term "pseudopeptide" refers to a peptide where one or more peptide bonds are replaced by non-amido bonds such as ester or one or more amino acids are replaced by amino acid analogs. The term "peptides" refers not only to those comprised of all natural amino acids, but also to those which contain unnatural amino acids or other non-coded structural units. The terms "peptides", when used alone, include pseudopeptides.
It is worth mentioning that "modified peptides" have utility in many biomedical applications because of their increased stability against in vivo degradation, superior pharmacokinetics, and altered immunogenicity compared to their native counterparts.
The term "modified peptides," as employed herein, also includes oxidized peptides.
The term "oxidized peptide" refers to a peptide in which at least one amino acid residue is oxidized.
The term "analog", as used herein, includes any peptide having an amino acid sequence substantially identical to one of the sequences specifically shown herein in which one or more residues have been conservatively substituted with a functionally similar residue and which displays the abilities as described herein. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as isoleucine, valine, leucine or methionine for another, the substitution of one polar (hydrophilic) residue for another such as between arginine and lysine, between glutamine and asparagine, between glycine and serine, the substitution of one basic residue such as lysine, arginine or histidine for another, or the substitution of one acidic residue, such as aspartic acid or glutamic acid for another.
The term "conservative substitution", as used herein, also includes the use of a chemically derivatized residue in place of a non-derivatized residue provided that such peptide displays the requisite inhibitory function on myeloid cells as specified herein. The term derivative includes any chemical derivative of the peptide of the invention having one or more residues chemically derivatized by reaction of side chains or functional groups.
The term "homolog" or "homologous" when used in reference to a polypeptide refers to a high degree of sequence identity between two polypeptides, or to a high degree of similarity between the three-dimensional structures or to a high degree of similarity between the active site and the mechanism of action. In a preferred embodiment, a homolog has a greater than 60%
sequence identity, and more preferably greater than 75% sequence identity, and still more preferably greater than 90% sequence identity, with a reference sequence.
As applied to polypeptides, the term "substantial identity" means that two peptide sequences, when optimally aligned, such as, for example, by the programs KALIGN, DOTLET, LALIGN and DIALIGN (https://www.expasy.org/tools) using default gap weights, share at least 80 percent sequence identity, preferably at least 90 percent sequence identity, more preferably at least 95 percent sequence identity or more (e.g., 99 percent sequence identity). Preferably, residue positions which are not identical differ by conservative amino acid substitutions.
The term "modified peptides," as employed herein, also includes oxidized peptides. The term "oxidized peptide" refers to a peptide in which at least one amino acid residue is oxidized.
The term "oxidation status" refers to a metric of the extent to which specific amino acid residues are replaced by corresponding oxidized amino acid residues in a peptide. The term "extent of oxidation" refers to the degree to which potentially oxidizable amino acids in a peptide have undergone oxidation. For example, if the peptide contains a single tyrosine residue which is potentially oxidized to 3-chlorotyrosine, then an increase in mass of about 34 Dalton (i.e., the approximate difference in mass between chlorine and hydrogen) indicates oxidation of tyrosine to 3-chlorotyrosine. Similarly, if the peptide contains a single methionine residue which is potentially oxidized to methionine sulfoxide, then an increase in mass of 16 Dalton (i.e., the difference in mass between methionine and methionine containing one extra oxygen) indicates oxidation of methionine to methionine sulfoxides.
The term "oxidation status" refers to a metric of the extent to which specific amino acid residues are replaced by corresponding oxidized amino acid residues in a peptide. The term "extent of oxidation" refers to the degree to which potentially oxidizable amino acids in a peptide have undergone oxidation. For example, if the peptide contains a single tyrosine residue which is potentially oxidized to 3-chlorotyrosine, then an increase in mass of about 34 Dalton (i.e., the approximate difference in mass between chlorine and hydrogen) indicates oxidation of tyrosine to 3-chlorotyrosine. Similarly, if the peptide contains a single methionine residue which is potentially oxidized to methionine sulfoxide, then an increase in mass of 16 Dalton (i.e., the difference in mass between methionine and methionine containing one extra oxygen) indicates oxidation of methionine to methionine sulfoxides.
The oxidation status can be measured by metrics known to the arts of protein and peptide chemistry (as disclosed in Caulfield, US 8,114,613 and Hazen, et al., US
8,338,110, herein incorportaed by reference) including, without limitation, assay of the number of oxidized residues, mass spectral peak intensity, mass spectral integrated area, and the like. In some embodiments, oxidation status is reported as a percentage, wherein 0% refers to no oxidation and 100% refers to complete oxidation of potentially oxidizable amino acid residues within apo A-I
or apo A-II peptide fragments.
The term "potentially subject to oxidation," "potentially oxidizable amino acid residues", and the like refer to an amino acid which can undergo oxidation, for example by nitration or chlorination.
A "biologically active peptide motif' is a peptide that induces a phenotypic response or change in an appropriate cell type when the cell is contacted with the peptide. The peptide may be present either in isolated form or as part of a larger polypeptide or other molecule. The ability of the peptide to elicit the response may be determined, for example, by comparing the relevant parameter in the absence of the peptide (e.g., by mutating or removing the peptide when normally present within a larger polypeptide). Phenotypic responses or changes include, but are not limited to, enhancement of cell spreading, attachment, adhesion, proliferation, secretion of an extracellular matrix (ECM) molecule, or expression of a phenotype characteristic of a particular differentiated cell type.
As used herein, a "minimal biologically active sequence" refers to the minimum length of a sequence of a peptide that has a specific biological function. In a first example, -IVILLAGGFLSKSLVFSVLFA- (e.g., Domain A, SEQ ID NO. 47) is a biologically active TREM-1 inhibitory sequence corresponding to the human TREM-1 transmembrane domain, wherein -GFLSKSLVF- (e.g. Domain A, SEQ ID NO. 1) has the sole function of inhibition. Thus, in this case, -GFLSKSLVF- (Domain A, SEQ ID NO. 1) is a "minimal biologically active sequence." In a second example, the sequence ¨
PLGEEMRDRARAHVDALRTHLARGD, and an internal sequence -GEEMRDRARAHVRGD-(Domain B, SEQ ID NO. 5) contains the sequence -RGD-; -RGD- has a cell attachment function.
However, -PLGEEMRDRARAHVDALRTHLARGD and -GEEMRDRARAHVRGD- (Domain B, SEQ ID NO. 5) also functions to assist in the formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with native lipoproteins and to promote binding of these particles with scavenger receptor type I (SR-B1). Thus, in this case, both -PLGEEMRDRARAHVDALRTHLARGD- andGEEMRDRARAHVRGD- (Domain B, SEQ ID
NO. 5) in addition to -RGD- are considered a "minimal biologically active sequence." In another example, the sequence -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO.
...) contains the sequence -RGD-; -RGD-has a cell attachment function.
However, -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO. ...) also has the functions of inhibition of TREM-1, assistance in the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures particle and of interaction with scavenger receptor type I (SRBI). Thus, in this case, both -GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD- (SEQ ID NO. ...) and -RGD- are considered a "minimal biologically active sequence." As is understood from the present invention, the first and second amino acid domains of a resulting peptide contain at least one minimal biologically active sequence. This minimal biologically active sequence is any length of sequence from an original peptide sequence. Moreover, with the exception of the amino acids of the minimal biologically active sequence, the amino acids of any or both amino acid domain can be exchanged, added or removed according to the design of the molecule to adjust its overall hydrophilicity and/or net charge. In certain embodiments, the minimal biologically active sequence refers to any one of the sequences provided in TABLE 2.
The term "imaging agent" or "imaging probe" as used herein refers to contrast agents used in imaging techniques such as computed tomography (CT), gamma-scintigraphy, positron emission tomography (PET), single photon emission computed tomography (SPECT), magnetic resonance imaging (MM), and combined imaging techniques in order to improve diagnostic performance of medical imaging.
The term "labeling substance or label or labeled probe" refers to a substance that can image whether there is a binding between the modulator and the cellular component (e.g., TREM-1/DAP-12 receptor complex), and can visualize the binding by a pattern.
It may include radioactive materials, fluorescent or emitting materials.
The term "carrier" as used herein, refers to a biocompatible nanoparticle that facilitates administration of a pharmaceutical agent to an individual.
The term "encapsulation" as used herein refers to the enclosure of a molecule, such as trifunctional peptides and compounds of the present invention, inside the nanoparticle. The term "incorporation" as used herein refers to imbibing or adsorbing the trifunctional peptides and compounds onto the nanoparticle. The terms "reconstituted" and "recombinant"
as used herein both refer to synthetic lipopeptide particles that represent both discoidal and spherical nanoparticles and mimic native HDL particles.
As used herein, "naturally occurring" means found in nature. A naturally occurring biomolecule is, in general, synthesized by an organism that is found in nature and is unmodified by the hand of man, or is a degradation product of such a molecule. A molecule that is synthesized by a process that involves the hand of man (e.g., through chemical synthesis not involving a living organism or through a process that involves a living organism that has been manipulated by the hand of man or is descended from such an organism) but that is identical to a molecule that is synthesized by an organism that is found in nature and is unmodified by the hand of man is also considered a naturally occurring molecule.
A "site of interest" on a target as used herein is a site to which modified peptides and compounds of the present invention bind.
The term "target site", as used herein, refers to sites/tissue areas of interest.
As used in this invention, the terms "target cells" or "target tissues" refer to those cells or tissues, respectively that are intended to be targeted using the compositions of the present invention delivered in accord with the invention. Target cells or target tissues take up or link with the modified peptides and compounds of the invention. As used in this invention, the terms "target cells" or "target tissues" refer to those cells or tissues, respectively that are intended to be treated and/or visualized in imaging techniques such as CT, gamma-scintigraphy, PET, SPECT, Mill, and combined imaging techniques, using the compositions of the present invention delivered in accord with the invention. Target cells are cells in target tissue, and the target tissue includes, but is not limited to, atherosclerotic plaques, vascular endothelial tissue, abnormal vascular walls of tumors, solid tumors, tumor-associated macrophages, and other tissues or cells related to cancer, cardiovascular, inflammatory, autoimmune diseases, and the like. Further, target cells include virus-containing cells, and parasite-containing cells.
Also included among target cells are cells undergoing substantially more rapid division as compared to non-target cells.
The term "target cells" also includes, but is not limited to, microorganisms such as bacteria, viruses, fungi, parasites, and infectious agents. Thus, the term "target cell" is not limited to living cells but also includes infectious organic particles such as viruses. "Target compositions" or "target biological components" include, but are not be limited to: toxins, peptides, polymers, and other compounds that may be selectively and specifically identified as an organic target that is intended to be visualized in imaging techniques using the compositions of the present invention.
The term "therapeutic agent" or "drug" as used herein refers to any compound or composition having preventive, therapeutic or diagnostic activity, primarily but not exclusively in the treatment of patients with macrophage (myeloid cell)-related diseases.
The term "myeloid cells" include monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, and megakaryocytes to platelets.
The terms "macrophage-associated", "macrophage-mediated", and "macrophage-related diseases" include diseases associated with macrophages as disclosed in Low and Turk, US
8,916,167, herein incorportaed by reference in its entirety.
The term "plaque" includes, for example, an atherosclerotic plaque.
The term "myeloid cell-mediated pathology" (or "myeloid cell-related pathologies", or "myeloid cell-mediated disorder, or "myeloid cell-related disease"), as used herein, refers to any condition in which an inappropriate myeloid cell response is a component of the pathology. The term is intended to include both diseases directly mediated by myeloid cells, and also diseases in which an inappropriate myeloid cell response contributes to the production of abnormal antibodies, antibodies, as well as graft rejection.
The term "ligand-induced myeloid cell activation", as used herein, refers to myeloid cell activation in response to the stimulation by the specific ligand.
The term "stimulation", as used herein, refers to a primary response induced by ligation of a cell surface moiety. For example, in the context of receptors, such stimulation entails the ligation of a receptor and a subsequent signal transduction event. With respect to stimulation of a myeloid cell, such stimulation refers to the ligation of a myeloid cell surface moiety that in one embodiment subsequently induces a signal transduction event, such as binding the TREM-1/DAP- 12 complex. Further, the stimulation event may activate a cell and up-regulate or down-regulate expression or secretion of a molecule.
The term "ligand", or "antigen", as used herein, refers to a stimulating molecule that binds to a defined population of cells. The ligand may bind any cell surface moiety, such as a receptor, an antigenic determinant, or other binding site present on the target cell population. The ligand may be a protein, peptide, antibody and antibody fragments thereof, fusion proteins, synthetic molecule, an organic molecule (e.g., a small molecule), or the like.
Within the specification and in the context of myeloid cell stimulation, the ligand (or antigen) binds the TREM receptor and this binding activates the myeloid cell.
The term "activation", as used herein, refers to the state of a cell following sufficient cell surface moiety ligation to induce a noticeable biochemical or morphological change. Within the context of myeloid cells, such activation, refers to the state of a myeloid cell that has been sufficiently stimulated to induce production of interleukin (IL) 1, 6 and/or 8 (IL-1, IL-6 and/or IL-8, respectively) and tumor necrosis factor alpha (TNF-alpha), differentiation of primary monocytes into immature dendritic cells, and enhancement of inflammatory responses to microbial products. Within the context of other cells, this term infers either up or down regulation of a particular physico-chemical process.
The term "inhibiting myeloid cell activation" (or "TREM-mediated cell activation"), as used herein, refers to the slowing of myeloid cell activation, as well as completely eliminating and/or preventing myeloid cell activation.
The term, "treating a disease or condition", as used herein, refers to modulating myeloid cell activation including, but not limited to, decreasing cytokine production and differentiation of primary monocytes into immature dendritic cells and/or slowing myeloid cell activation, as well as completely eliminating and/or preventing myeloid cell activation. Myeloid cell-related diseases and/ or conditions treatable by modulating myeloid cell activation include, but are not limited to, cancer including but not limited to lung cancer, pancreatic cancer, multiple myeloma, melanoma, leukemia, prostate cancer, breast cancer, liver cancer, bladder cancer, stomach cancer, prostate cancer, colon cancer, colorectal cancer, CNS cancer, melanoma, ovarian cancer, gastrointestinal cancer, renal cancer, or osteosarcoma and other cancers, brain and skin cancers, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor (TGCT;
also referred to in the art as TSGCT), PVNS and other cancers in which myeloid cells are involved or recruited, cancer cachexia, in addition to ALD, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other rheumatic diseases, sepsis, diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases, and other myeloid cell-related inflammatory conditions eg myositis, tissue/organ rejection, brain and spinal cord injuries. Other exemplary cancers include, but are not limited to, adrenocortical carcinoma, acquired immune deficiency syndrome (AIDS)-related cancers, AIDS-related lymphoma, anal cancer, anorectal cancer, cancer of the anal canal, appendix cancer, childhood cerebellar astrocytoma, childhood cerebral astrocytoma, basal cell carcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bile duct cancer, intrahepatic bile duct cancer, bladder cancer, uringary bladder cancer, bone and joint cancer, osteosarcoma and malignant fibrous histiocytoma, brain cancer, brain tumor, brain stem glioma, cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodeimal tumors, visual pathway and hypothalamic glioma, bronchial adenomas/carcinoids, carcinoid tumor, nervous system cancer, nervous system lymphoma, central nervous system lymphoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, cutaneous T-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome, esophageal cancer, extracranial germ cell tumor, extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor (GIST), germ cell tumor, ovarian germ cell tumor, gestational trophoblastic tumor glioma, Hodgkin lymphoma, hypopharyngeal cancer, intraocular melanoma, ocular cancer, islet cell tumors (endocrine pancreas), Kaposi Sarcoma, renal cancer, laryngeal cancer, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer, lung cancer, small cell lung cancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma, melanoma, intraocular (eye) melanoma, merkel cell carcinoma, mesothelioma malignant, mesothelioma, metastatic squamous neck cancer, mouth cancer, cancer of the tongue, multiple endocrine neoplasia syndrome, mycosis fungoides, myelodysplastic syndromes, myelodysplastic/myeloproliferative diseases, chronic myelogenous leukemia, acute myeloid leukemia, multiple myeloma, chronic myeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oral cancer, oral cavity cancer, oropharyngeal cancer, ovarian epithelial cancer, ovarian low malignant potential tumor, islet cell pancreatic cancer, paranasal sinus and nasal .. cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineoblastoma and supratentorial primitive neuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiple myeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renal pelvis and ureter, transitional cell cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors, Kaposi Sarcoma, soft tissue sarcoma, uterine cancer, .. uterine sarcoma, skin cancer (non-melanoma), skin cancer (melanoma), merkel cell skin carcinoma, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, supratentorial primitive neuroectodermal tumors, testicular cancer, throat cancer, thymoma, thymoma and thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter and other urinary organs, gestational trophoblastic tumor, urethral cancer, endometrial uterine cancer, .. uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer, and Wilm's Tumor.
The term "detectable" refers to the ability to detect a signal over the background signal.
In accordance with the present disclosure, "a detectably effective amount" of the labeled probe of the present disclosure is defined as an amount sufficient to yield an acceptable image using equipment that is available for clinical use. A detectably effective amount of the labeled probe of the present disclosure may be administered in more than one injection. The detectably effective amount of the labeled probe of the present disclosure can vary according to factors such as the degree of susceptibility of the individual, the age, sex, and weight of the individual, idiosyncratic responses of the individual, and the like.
Detectably effective amounts of the probe of the present disclosure can also vary according to instrument and film-related factors. Optimization of such factors is well within the level of skill in the art.
The term "in vivo imaging" as used herein refers to methods or processes in which the structural, functional, molecular, or physiological state of a living being is examinable without the need for a life-ending sacrifice.
The term "inhibiting T cell activation", as used herein, refers to the slowing of T cell activation, as well as completely eliminating and/or preventing T cell activation.
The term "T cell-mediated pathology" (or "T cell-related pathologies", or "T
cell-mediated disorder, or "T cell-related disease"), as used herein, refers to any condition in which an inappropriate T cell response is a component of the pathology. The term is intended to include both diseases directly mediated by T cells, and also diseases in which an inappropriate T cell response contributes to the production of abnormal antibodies, as well as graft rejection.
The term "treating a T cell-mediated disease or condition", as used herein, refers to modulating T cell activation including, but not limited to, decreasing cellular proliferation, cytokine production and performance of regulatory or cytolytic effector functions and/or slowing T cell activation, as well as completely eliminating and/or preventing T cell activation. T cell-related diseases and/or conditions treatable by modulating T cell activation include, but are not limited to, systemic lupus erythematosus, rheumatoid arthritis, psoriatic arthritis, multiple sclerosis, type I diabetes, gastroenterological conditions e.g. inflammatory bowel disease, Crohn's disease, celiac, Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia, primary biliary cirrhosis, chronic active hepatitis; skin problems e.g. atopic dermatitis, psoriasis, pemphigus vulgaris; cardiovascular problems e.g. autoimmune pericarditis, allergic diathesis e.g.
delayed type hypersensitivity, contact dermatitis, AIDS virus, herpes simplex/zoster, respiratory conditions e.g. allergic alveolitis, inflammatory conditions e.g. myositis, ankylosing spondylitis, tissue/organ rejection.
The term, "subject" or "patient", as used herein, refers to any individual organism. For example, the organism may be a mammal, such as a primate (i.e., for example, a human) or a laboratory animal. Further, the organism may be a domesticated animal (i.e., for example, cats, dogs, etc.), livestock (i.e., for example, cattle, horses, pigs, sheep, goats, etc.), or a laboratory animal (i.e., for example, mouse, rabbit, rat, guinea pig, etc.).
The term, "therapeutically effective amount", "therapeutically effective dose"
or "effective amount", as used herein, refers to an amount needed to achieve a desired clinical result .. or results (e.g. inhibiting receptor-mediated cell activation) based upon trained medical observation and/or quantitative test results. The potency of any administered peptide or compound determines the "effective amount" which can vary for the various compounds that inhibit myeloid cell activation (i.e., for example, compounds inhibiting TREM
ligand-induced myeloid cell activation and/or TCR-mediated T cell activation). Additionally, the "effective amount" of a compound may vary depending on the desired result, for example, the level of myeloid cell activation inhibition desired. The "therapeutically effective amount" necessary for inhibiting differentiation of primary monocytes into immature dendritic cells may differ from the "therapeutically effective amount" necessary for preventing or inhibiting cytokine production.
The term, "agent", as used herein, refers to any natural or synthetic compound (i.e., for example, a peptide, a peptide variant, or a small molecule).
The term, "intrinsic helicity", as used herein, refers to the helicity which is adopted by a peptide in an aqueous solution. The term, "induced helicity", as used herein, refers to the helicity which is adopted by a peptide when in the presence of a helicity inducer, including, but not limited to, trifluoroethanol (TFE), detergents (e.g., sodium dodecyl sulfate, SDS) or lipids.
The term "therapeutic drug", as used herein, refers to any pharmacologically active substance capable of being administered which achieves a desired effect. Drugs or compounds can be synthetic or naturally occurring, non-peptide, proteins or peptides, oligonucleotides or nucleotides, polysaccharides or sugars. Drugs or compounds may have any of a variety of activities, which may be stimulatory or inhibitory, such as antibiotic activity, antiviral activity, antifungal activity, steroidal activity, cytotoxic, cytostatic, anti-proliferative, anti-inflammatory, analgesic or anesthetic activity, or can be useful as contrast or other diagnostic agents.
The term "effective dose" as used herein refers to the concentration of any compound or drug contemplated herein that results in a favorable clinical response. In solution, an effective dose may range between approximately 1 ng/ml and 100 mg/ml, preferably between 100 ng/ml and 10 mg/ml, but more preferably between 500 ng/ml and 1 mg/ml.
The term "effective amount" or "therapeutically effective amount" refers to an amount of a drug effective to treat a disease or disorder in a subject. In certain embodiments, an effective amount refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result. A therapeutically effective amount of the compound or composition of the invention that modulate TREM-1/DAP-12 receptor complex signaling may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the compound or composition to elicit a desired response in the individual. A
therapeutically effective amount encompasses an amount in which any toxic or detrimental effects of the compound or composition are outweighed by the therapeutically beneficial effects.
As one example, in some embodiments, the expression "effective amount" refers to an amount of the compound or composition that is effective for treating cancer.
A "prophylactically effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result.
Typically, but not necessarily, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount would be less than the therapeutically effective amount.
A "induction therapy" refers to the first treatment given for a disease. It is often part of a standard set of treatments, such as surgery followed by chemotherapy and radiation. When used by itself, induction therapy is the one accepted as the best treatment. If it doesn't cure the disease or it causes severe side effects, other treatment may be added or used instead. Also called first-line therapy, primary therapy, and primary treatment.
A "maintenance therapy" refers to a medical therapy that is designed to help a primary treatment succeed. For example, maintenance chemotherapy may be given to people who have a cancer in remission in an attempt to prevent a relapse. In other words, treatment that is given to help keep cancer from coming back after it has disappeared following the initial therapy. It may include treatment with drugs, vaccines, or antibodies that kill cancer cells or keep tumor unfavorable microenvironment, and it may be given for a long time. This form of treatment is also a common approach for the management of many incurable, chronic diseases such as periodontal disease, Crohn's disease or ulcerative colitis.
Administration "in combination with" one or more further therapeutic agents includes .. simultaneous (concurrent) and consecutive (sequential) administration in any order.
A "pharmaceutically acceptable carrier" refers to a non-toxic solid, semisolid, or liquid filler, diluent, encapsulating material, formulation auxiliary, or carrier conventional in the art for use with a therapeutic agent that together comprise a "pharmaceutical composition" for administration to a subject. A pharmaceutically acceptable carrier is non-toxic to recipients at the dosages and concentrations employed and is compatible with other ingredients of the formulation. The pharmaceutically acceptable carrier is appropriate for the formulation employed. For example, if the therapeutic agent is to be administered orally, the carrier may be a gel capsule. If the therapeutic agent is to be administered subcutaneously, the carrier ideally is not irritable to the skin and does not cause injection site reaction.
The term "administered" or "administering" a drug or compound, as used herein, refers to any method of providing a drug or compound to a patient such that the drug or compound has its intended effect on the patient. For example, one method of administering is by an indirect mechanism using a medical device such as, but not limited to a catheter, syringe etc. A second exemplary method of administering is by a direct mechanism such as, local tissue administration (i.e., for example, extravascular placement), oral ingestion, transdermal patch, topical, inhalation, suppository etc.) The term, "agent", as used herein, refers to any natural or synthetic compound (i.e., for example, a peptide, a peptide variant, or a small molecule).
The term, "composition", as used herein, refers to any mixture of substances comprising a peptide and/or compound contemplated by the present invention. Such a composition may include the substances individually or in any combination.
The term "modulator" used in this invention refers to a substance and/or compositions contemplated by the present invention or a combination thereof with capacity to inhibit (e.g., "antagonist" activity) a functional property of biological activity or process (e.g., reducing or blocking TREM-1/DAP-12 activity ¨ signaling and/or activation); such inhibition can be contingent on the occurrence of a specific event, such as reduction or blockade of a signal transduction pathway, and/or can be manifest only in particular cell types.
For instance, small molecules such as drugs, proteins such as antibodies, hormones or growth factors, protein domains, protein motifs, and peptides or a combination thereof can act as a modulator.
The term "tissue sample" refers to a collection of similar cells obtained from a tissue of a subject. The source of the tissue sample may be solid tissue as from a fresh, frozen and/or preserved organ or tissue sample or biopsy or aspirate; blood or any blood constituents; bodily fluids such as cerebral spinal fluid, amniotic fluid, peritoneal fluid, synovial fluid, or interstitial fluid; cells from any time in gestation or development of the subject. In some embodiments, a tissue sample is a synovial biopsy tissue sample and/or a synovial fluid sample. In some .. embodiments, a tissue sample is a synovial fluid sample. The tissue sample may also be primary or cultured cells or cell lines. Optionally, the tissue sample is obtained from a disease tissue/organ. The tissue sample may contain compounds that are not naturally intermixed with the tissue in nature such as preservatives, anticoagulants, buffers, fixatives, nutrients, antibiotics, or the like. A "control sample" or "control tissue", as used herein, refers to a sample, cell, or tissue obtained from a source known, or believed, not to be afflicted with the disease for which the subject is being treated.
For the purposes herein a "section" of a tissue sample means a part or piece of a tissue sample, such as a thin slice of tissue or cells cut from a solid tissue sample.
The term "anti-inflammatory drug" means any compound, composition, or drug useful for preventing or treating inflammatory disease.
The term "medical device", as used herein, refers broadly to any apparatus used in relation to a medical procedure. Specifically, any apparatus that contacts a patient during a medical procedure or therapy is contemplated herein as a medical device.
Similarly, any apparatus that administers a drug or compound to a patient during a medical procedure or therapy is contemplated herein as a medical device. "Direct medical implants"
include, but are not limited to, urinary and intravascular catheters, dialysis catheters, wound drain tubes, skin sutures, vascular grafts and implantable meshes, intraocular devices, implantable drug delivery systems and heart valves, and the like. "Wound care devices" include, but are not limited to, general wound dressings, non-adherent dressings, burn dressings, biological graft materials, tape closures and dressings, surgical drapes, sponges and absorbable hemostats.
"Surgical devices"
include, but are not limited to, surgical instruments, endoscope systems (i.e., catheters, vascular catheters, surgical tools such as scalpels, retractors, and the like) and temporary drug delivery devices such as drug ports, injection needles etc. to administer the medium. A
medical device is "coated" when a medium comprising an anti-inflammatory drug (i.e., for example, the peptides, compositions, and compounds of the present invention) becomes attached to the surface of the medical device. This attachment may be permanent or temporary. When temporary, the attachment may result in a controlled release of an inflammatory drug.
DETAILED DESCRIPTION OF THE INVENTION
The invention disclosed herein provides compositions and methods of treating cancer and other diseases related to activated immune cells using modulators of the TREM-signaling pathway. The compositions, including peptides and peptide variants, modulate TREM-1-mediated immunological response as standalone and combination-therapy treatment regimen.
Further, methods are provided for predicting the efficacy of TREM-1 modulatory therapies in patients. In one embodiment, the present invention relates to targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, cancer cachexia, etc., and other cancers associated with myeloid cell activation and recruitment. Additionally, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST). The invention further relates to personalized medical treatments.
The present disclosure describes novel amphipathic trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating diseases related to activated macrophages. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages.
As described herein, surprisingly it was found that potentially therapeutic trifunctional peptides of the present invention are capable of executing at least, three functions (trifunctional peptides): 1) assistance in the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures in vitro, i.e. incorporation of the trifunctional peptides as part of the lipid portion of synthetic/recombinant HDLs, then after administration; 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. Thus in some embodiments, trifunctional peptides, after mixing with lipids in vitro, may assist in the self-assembly of synthetic lipopeptide particles (SLP) upon binding to a lipid or to lipids in mixtures. In the methods of the present invention, the SLP of interest are synthetic nanoparticles that mimic human lipoproteins as recombinant (r)HDLs. While not being bound to any particular theory, it is believed that this interaction and ability to form lipopeptide/lipoprotein particles is mediated by the amphipathic alpha helical sequences of the trifunctional peptides described herein.
Another surprising discovery was that administration of potentially therapeutic trifunctional peptides of the present invention, that were not in rHDL
formulations, showed: 1) mediation of formation of naturally long half-life lipopeptide/lipoprotein particles (LP) upon interaction with native lipoproteins in vivo, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. Thus in some embodiments, free trifunctional peptides (i.e. not in rHDL formulations) as part of compounds of the present invention, after administration to populations of cells or administration to a mammal, may interact with native lipoproteins and form trifunctional peptide .. containing lipopeptide/lipoprotein particles in vivo.
Thus, potentially therapeutic trifunctional peptides of the present invention were synthesized and used for targeted treatment and imaging in vivo, as either formulations with HDLs or without, i.e. trifunctional peptides in a pharmaceutical formulation without HDLs.
Advantageous of using the trifunctional peptides described herein in order to solve numerous problems administering therapeutic or diagnostic compounds include avoiding high dosages of other TAs (therapeutic agents) and imaging probes required; and the lack of control and reproducibility of formulations, especially in large-scale production. In other words, using trifunctional peptides described herein, including trifunctional peptide formulations including therapeutic drug compounds, would potentially lower the amount of drug needed to reduce symptoms of a disease.
Another advantage is economic. Therapeutic peptides have relatively high synthetic and production costs, For example, the production cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500 Da molecular mass small molecule, which in turn exceeds the production cost of a 500 Da molecular mass small molecule by more than 10-fold up to less than 100-fold for each increase in magnitude of size. By combining three functions in one peptide significantly simplifies the manufacture of these trifunctional peptides as targeted drugs, and as delivery agents for drug compounds and imaging probes.
I. Trifunctional Peptides.
The present invention encompasses the discovery that it is possible to combine multiple functions in one polypeptide amino acid sequence, i.e. a trifunctional peptide, in order to confer a variety of properties on the resulting amphipathic multipeptide.
The present disclosure describes novel amphipathic trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating diseases related to activated immune cells. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In some embodiments, each trifunctional peptide is capable of at least three functions:
1) mediating the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on macrophages. The invention further relates to personalized medical treatments for cancer that involve targeting specific cancers by their tumor environment.
In preferred embodiments, trifunctional peptides of the present invention comprise two amino acid domains (See FIG. 1): domain A that confers therapeutic and/or diagnostic benefits in the context of the treatment, prevention, and/or detection of a disease or condition; and domain B that confers multiple benefits in the context of: 1A) formation of long half-life lipopeptide particles upon binding to lipid or lipid mixtures in vitro 1B) formation of long half-life LP upon interaction with lipoproteins in vivo, and 2) the targeted delivery of the particles formed to cells of interest and/or sites of disease or condition.
In one embodiment, the present invention includes a resulting trifunctional peptide comprising: (a) one amino acid domain that confers therapeutic and/or diagnostic benefits in the context of the treatment, prevention, and/or detection of a disease or condition; and (b) another amino acid domain that confers multiple benefits in the context of the self-assembly of naturally long half-life SLP and LP upon binding to lipid or lipid mixtures and targeted delivery of the particles formed to cells of interest and/or sites of disease or condition. In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence.
In one embodiment, the first amino acid domain comprises a cyclic peptide sequence.
In one embodiment, the first amino acid domain comprises a disulfide-linked dimer. In one embodiment, any or both of the amino acid domains include amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to a drug compound (therapeutic agent:
TA). In one .. embodiment, a therapeutic agent is selected from the group including, but not limited to, anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, and therapeutic peptides. In one embodiment, the therapeutic agent is a hydrophobic therapeutic agent. The therapeutic agent may also be selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to an imaging probe. In one embodiment, the imaging agent is a Gd-based contrast agent (GBCA) for magnetic resonance imaging (MM). In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as a positron emission tomography (PET) imaging systems (and combined PET/computer tomography (CT) and PET/MM systems). In one embodiment, an imaging probe and/or an additional therapeutic agent is conjugated to any or both of the domains. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof Although many examples describe or show results of using trifunctional peptides in formulations with rHDLs, it is not meant to limit the use of these trifunctional peptide sequences in HDL formulations. Conversely, examples describing or showing results of using trifunctional peptides alone, or in formulations without rHDLs is not meant to limit the use of such trifunctional peptides without rHDLs. Thus, in certain embodiments, the trifunctional peptides of the present invention may be administered within rHDLs, or administered in pharmaceutical formulations as part of rHDLs. In other embodiments, the trifunctional peptides of the present invention may be administered without rHDLs, or administered in pharmaceutical formulations without rHDLs.
In one embodiment, the peptides of the present invention form lipopeptide particles in vitro. In one embodiment, the peptides of the present invention form lipopeptide particles in vivo.
In certain embodiments, the present invention relates to peptides consisting of two amino acid domains, wherein upon binding to lipid or lipid mixtures, one amino acid domain assists in the self-assembly of naturally long half-life lipopeptide particles and targets these particles to macrophages, whereas another amino acid domain inhibits TREM-1/DAP-12 receptor complex expressed on macrophages.
In certain embodiments, the present invention relates to peptides comprising at least two amino acid domains, wherein upon binding to lipid or lipid mixtures, the first amino acid domain assists in the self-assembly of naturally long half-life lipopeptide particles and targets these particles to macrophages, whereas the second amino acid domain inhibits TREM-receptor complex expressed on macrophages.
In certain embodiments, the peptides of the present invention self-assemble upon binding to lipid or lipid mixtures in vitro to form synthetic lipopeptide particles (SLP) that mimic human lipoproteins and have a long half-life in a bloodstream. In one embodiment, the peptides and compounds of the present invention interact with endogenous lipoproteins in vivo and form long half-life LP. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof.
The peptides and compounds of the present invention and combinations thereof alone as well as the SLP formed upon their binding to lipid or lipid mixtures have a wide variety of uses, particularly in the areas of oncology, transplantology, dermatology, hepatology, ophthalmology, cardiovascular diseases, sepsis, autoimmune diseases, neurodegenerative diseases and other diseases and conditions. They also are useful in the production of medical devices (for example, medical implants and implantable devices).
The invention disclosed herein provides for methods of treating cancer using inhibitors of the TREM-1 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. The invention also provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using inhibitors of the TREM-1 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. The invention also provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using modulators of the TREM-1/DAP-12 signaling pathway. These compounds and compositions modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer in standalone and combination-therapy treatment regimen. The invention also provides for predicting the efficacy of TREM-1 modulatory therapies in patients with various cancers. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung cancer including non-small cell lung cancer, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, bladder cancer, central nervous system cancer, stomach cancer, prostate cancer, colorectal cancer, colon cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, thyroid cancer, .. neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, and other cancers in which myeloid cells are involved or recruited and cancer cachexia.
The invention disclosed herein provides for methods of treating cancer using inhibitors of the TREM-1 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of cancer. The invention also provides for predicting the efficacy of TREM-1-targeted therapies in various cancers by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to pancreatic cancer, breast cancer, liver cancer, multiple myeloma, leukemia, bladder cancer, CNS cancer, stomach cancer, prostate, colorectal cancer, brain cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma and other cancers and cancer cachexia.
The invention disclosed herein provides for methods of treating scleroderma using modulators of the TREM-1/DAP-12 signaling pathway. These compounds and compositions modulate the TREM-1-m edi ated immunological responses beneficial for the treatment of scleroderma or a related autoimmune or a fibrotic condition in standalone and combination-therapy treatment regimen. The invention also provides for predicting the efficacy of TREM-1 modulatory therapies in patients with scleroderma. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or tel angi ectasi a syndrome (CREST).
In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis and other rheumatic diseases, sepsis and other inflammatory diseases or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, including but not limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases.
The disclosure also provides for a method of treating, preventing and/or detecting an immune-related condition. The method comprises providing a composition comprising peptides and compounds of the present disclosure and/or a synthetic nanoparticle formed upon their binding to lipid or lipid mixtures, a patient having at least one symptom of a disease or condition in which the immune system is involved, and administering the composition to the patient under conditions such that said one symptom is reduced. The immune-related condition of the method may include a heart disease, atherosclerosis, peripheral artery disease, restenosis, stroke, multiple sclerosis, the cancers (e.g., sarcoma, lymphoma, leukemia, carcinoma and melanoma), bacterial infectious diseases, acquired immune deficiency syndrome (AIDS), allergic diseases, autoimmune diseases (e.g., atopic dermatitis, psoriasis, rheumatoid arthritis, Sjogren's syndrome, scleroderma, systemic lupus erythematosus, non-specific vasculitis, Kawasaki's disease, psoriasis, type I diabetes, pemphigus vulgaris), granulomatous diseases (e.g., tuberculosis, sarcoidosis, lymphomatoid granulomatosis, Wegener's granulomatosus), Gaucher's disease, inflammatory diseases (e.g., sepsis, inflammatory lung diseases such as chronic obstructive .. pulmonary disease ( COPD ), interstitial pneumonitis and asthma, retinopathy such as diabetic retinopathy and retinopathy of prematurity, inflammatory bowel disease such as Crohn's disease, and inflammatory arthritis), liver diseases (e.g., alcoholic liver disease and nonalcoholic fatty liver disease), neurodegenerative diseases such as Alzheimer's, Parkinson's and Huntington's diseases, and transplant (e.g., heart/lung transplants) rejection reactions.
The invention relates to personalized medical treatments for scleroderma. More specifically, the invention provides for treatment of scleroderma or a related autoimmune or a fibrotic condition by using inhibitors of the TREM-1/DAP-12 pathway. These inhibitors include peptide variants and compositions that modulate the TREM-1-mediated immunological responses beneficial for the treatment of scleroderma. In addition, the invention provides for predicting the efficacy of TREM-1-targeted therapies in scleroderma by analyzing biological samples for the presence of myeloid cells and for the TREM-1 expression levels. In one embodiment, the peptides and compositions of the present invention modulate receptor complex expressed on macrophages. In one embodiment, the peptides and compositions of the invention are conjugated to an imaging probe. In one embodiment, the invention provides for detecting the TREM-1-expressing cells and tissues in an individual with scleroderma using imaging techniques and the peptides and compositions of the invention containing an imaging probe. In one embodiment, the peptides and compositions of the invention are used in combinations thereof In one embodiment, the peptides and compositions of the invention are used in combinations with other antifibrotic therapeutic agents. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of scleroderma including but not limited to calcinosis, Raynaud's phenomenon, esophageal dysmotility, scleroderma, or telangiectasia syndrome (CREST).
Trifunctional Peptides In rHDL (SLP) Formulations.
In one embodiment, the SLP self-assembled upon binding of the peptides and compounds .. of the present invention and combinations thereof to lipid or lipid mixtures are discoidal or spherical in shape. While the size of the particles is preferably between 5 nm and 50 nm, the diameter may be up to 200 nm. In one embodiment, the lipid of the particles may include cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, or a triacylglycerol. And further, the phospholipid may include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), or phosphatidic acid (PA). And even further, the cationic lipid can be 1,2-dioleoy1-3-trimethylammonium-propane (DOTAP). The lipid of the synthetic nanoparticle may be polyethylene glycol(PEG)ylated. In certain embodiments, the peptides and compounds of the present invention and/or the SLP and LP formed by these peptides and compounds may pass the blood-brain barrier (BBB). In one embodiment, the peptides and compounds of the present invention and/or the SLP and LP formed by these peptides and compounds may pass the blood-retinal barrier (BRB). In one embodiment, the peptides and compounds of the present invention and/or the SLP and LP formed by these peptides and compounds may pass the blood-tumor barrier (BTB).
In certain embodiments, the peptides and compounds of the present invention include an amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E. In one embodiment, the peptides and compounds of the present invention include an amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E and Arginine-glycine-aspartic acid (RGD)-peptide sequence. In certain embodiments, the peptides and compounds of the present invention include an amino acid sequence derived from transmembrane domain sequences of human or animal cell-surface receptors and of signaling subunits thereof In certain embodiments, the peptides and compounds of the present invention include an amino acid sequence derived from virus membrane fusion and structural proteins. In one embodiment, the peptides and compounds of the present invention include an amino acid sequence derived from apo A-I, A-II, A-IV, B, C-I, C-II, C-III, or E conjugated to a targeting moiety to enhance the targeting efficacy of the therapeutic agent. The targeting moiety may include a polypeptide, an antibody, a receptor, a ligand, a peptidomimetic agent, an aptamer or a product of phage display.
In one embodiment, the amino acid domains of the peptides and compounds of the present invention comprise unmodified or modified peptide sequences. The modified peptide sequence may contain at least one amino acid residue which is chemically or enzymatically modified. The modified amino acid residue may be an oxidized amino acid residue. The oxidized amino acid residue may be a methionine residue. The modified peptide sequence may contain at least one amino acid residue, which is oxidized, halogenated, or nitrated. The modified peptide sequence may include an amphipathic amino acid sequence.
In certain embodiments, the present invention relates to the targeted treatment or prevention of inflammatory or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, such as cancer including but not limited to, lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, alcohol-induced liver disease, nonalcoholic fatty liver disease and non-alcoholic steatohepatitis, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, sepsis, diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis and other rheumatic diseases.
In one embodiment, the present invention provides a pharmaceutical composition comprising the peptides and compounds and combinations thereof alone or the SLP
nanoparticles self-assembled upon binding of these peptides and compounds to lipid or lipid mixtures.
A. TREM-1-related Trifunctional peptides.
TREM-1 is expressed on the majority of innate immune cells and to a lesser extent on parenchymal cells. Upon activation, TREM-1 can directly amplify an inflammatory response.
Although it was initially demonstrated that TREM-1 was predominantly associated with infectious diseases, recent evidences demonstrate that TREM-1 receptor and its signaling pathways contribute to the pathology of non-infectious acute and chronic inflammatory diseases, including but not limiting to, rheumatoid arthritis, atherosclerosis, ischemia reperfusion-induced tissue injury, colitis, fibrosis, neurodegenerative diseases, liver diseases, retinopathies, and cancer (see e.g., Tammaro, et al. Pharmacol Ther 2017, 177:81-95; Saadipour.
Neurotox Res 2017, 32:14-16; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov.
US 8,513,185;
Sigalov. US 9,981,004; Rojas, et al. Biochim Biophys Acta 2018, 1864: 2761-2768, Tornai, et al.
Hepatology Communications 2018, in press, and Kuai, et al. US 2008/0247955).
In certain embodiments, a resulting trifunctional peptide of the present invention comprises two amino acid domains, wherein one domain comprises a variant TREM-1 inhibitory amino acid sequence and functions to inhibit TREM-1/DAP-12 receptor complex expressed on myeloid cells (e.g. macrophages), whereas another amino acid domain comprises the chemically and/or enzymatically modified amino acid sequence derived from apolipoprotein amino acid sequences and functions to assist in the self-assembly of SLP upon binding to lipid or lipid mixtures in vitro and/or to form LP in vivo, respectively, and to target these particles to myeloid cells (e.g. macrophages). In one embodiment, the TREM-1 inhibitory amino acid domain is the .. N-terminal domain of a resulting peptide. In one embodiment, the TREM-1 inhibitory amino acid domain is the C-terminal domain of a resulting peptide. In one embodiment, the TREM-1 inhibitory amino acid domain comprises a cyclic peptide sequence. In one embodiment, the TREM-1 inhibitory amino acid domain comprises a disulfide-linked dimer. In one embodiment, the TREM-1 inhibitory amino acid domain includes the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids. In one embodiment, an imaging agent is conjugated to the TREM-1 inhibitory amino acid domain or to the apolipoprotein amino acid sequence-derived domain or to both.
In some preferred embodiments, TREM-1-related peptides and associated compositions of the present invention have a domain A conjugated to a domain B. See, Fig.
1. Domain A
comprises a TREM-1 modulatory peptide sequence designed using a known model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TREM-1 receptor expressed on myeloid cells (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 8,513,185; and Sigalov. US 9,981,004), all of which are herein incorporated by reference in their entirety.
In some preferred embodiments, peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic alpha helical peptide fragment, such as a apo A-I and/or A-II peptide fragment, to form upon interaction with lipid and/or lipid mixtures. In certain embodiments, exemplary trifunctional peptides comprise the domain B comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
FIG. 1 presents an exemplary schematic representation of one embodiment of a trifunctional peptide of the present invention comprising amino acid domains A and B where amino acid domain A represents a therapeutic peptide sequence with or without an attached drug compound and/or imaging probe that functions to treat, prevent and/or detect a disease or condition, whereas amino acid domain B represents an amphipathic alpha helical peptide sequence, with or without an additional targeting peptide sequence, and functions to 1) assist in the self-assembly of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with lipids or lipid mixtures in vitro, for use in transporting these trifunctional peptides as lipoprotien nanoparticles to sites of interest in vitro or in vivo and/or 2) form long half-life lipopeptide/lipoprotein particles upon interaction with endogenous lipoproteins for transporting these trifunctional peptides to the sites of interest. Endogenous lipoproteins may be lipoproteins added to or found in cell cultures, or lipoprotiens in a mammalian body.
In certain embodiments, FIG. 2 shows the structures of representative TREM-1-related trifunctional peptides, TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ
ID NO. 2) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. GFLSKSLVF, is found within isoform 1 of human (UniProtKB - Q9NP99 (TREM1 HUMAN), and in human TREM-1 isoform CRA a (UniProtKB - Q38L15 (Q38L15 HUMAN), both downloaded 10-24-2018)). Peptide GFLSKSLVF is also described without an attached apo I peptide domain, in, for examples, WO
2011/047097 "Inhibition of trem receptor signaling with peptide variants."
Publication Date:
21.04.2011, U59981004B2 "Inhibition of TREM receptor signaling with peptide variants."
Published June 5, 2014, each of which is herein incorporated by reference in its entirety.
Sequence information was downloaded 10-25-, 10-26- or 10-27-2019.
Q9NP991TREM1 HUMAN Isoform 1 Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
Q38L15938L15 HUMAN Triggering receptor expressed on myeloid cells 1, Homo sapiens isoform CRA a:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF S GTP GSNENS T QNVYKIPP TT TKAL CPLYT SPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVF SVLF AVTLR SF VP
GFLSKSLVF, is not found within human TREM-1 isoforms 2 or 3.
Q9NP99-21TREM1 HUMAN Isoform 2 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFRCSTL SF SWLVDS
Q9NP99-31TREM1 HUMAN Isoform 3 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLW GLLWMLF V SELRAATKLTEEKYELKEGQ TLDVKCDYTLEKF A S S QKAWQII
RD GEMPKTLAC TERP SKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF S GTP GSNENS T QNVYKIPP TT TKAL CPLYT SPRTVTQAPP
KST.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP). GE31 (GFL SKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) comprises amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22 amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
Imaging of TREM-1 expression.
Another way to evaluate the TREM-1 expression level is to use imaging (visualization) techniques and procedures. In one embodiment, FIG. 50 shows that the fluorescently labeled TREM-1/TRIOPEP peptide GE31 delivered to macrophages by the SLP particles colocalizes with TREM-1 expressed on these cells. See also (Rojas et al. 2018). As described herein and in (Rojas et al. 2018), TREM-1 inhibitory therapy using the modulators of the signaling pathway results in reduction of tissue TREM-1 expression as measured by Western Blot (See Fig. 13).
In certain embodiments, the capability of the modulators of the TREM-1/DAP-12 signaling pathway described herein, including but not limited to, anti-TREM-1 blocking antibodies and fragments thereof, TREM-1 inhibitory SCHOOL peptides (e.g., GF9) and trifunctional TREM-1 inhibitory peptides including but not limited to, GA31 and GE31, to colocalize with TREM-1 can be used to visualize (image) this receptor and evaluate its expression/level in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu-.j, see TABLE 3) can be conjugated to the peptide sequences, GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 27) and/or GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide) (SEQ ID NO. 26). In one embodiment, methionine residues of the peptides GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 25) and GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 24) are unmodified. In one embodiment, imaging (visualization) of TREM-1 levels using the labeled modulators described herein and the PET and/or other imaging techniques can be used to diagnose GBM
and/or to select and monitor novel GBM therapies as disclosed in WO
2017083682A1 and described in (Johnson et al. 2017, Liu et al. 2019). In certain embodiments, imaging (visualization) of TREM-1 levels can be used to diagnose other TREM-1-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
GF9 immunotherapy targets pathways restricted to pathological conditions and is highly competitive. In some embodiments, safe and effective GF9 therapies are contemplated for use on pancreatic cancer (PC) to be used in combination with standard first-line treatments:
FOLFIRINOX (5-FU, leucovorin, irinotecan and oxaliplatin) or Gemzar +
ABRAXANE .
In some embodiments, advantages for using free GF9 peptide for treating PVNS
include but are not limited to: Low toxicity; Proven efficacy in vivo, including joints; easy formulation development; easy scale-up process; Easy and fast GMP production; Low cost of production;
and Stable and easy to store.
Therapy*
* Shown for Cancer Acute toxicity Risk of side effects Administration Cost Indications Systemic /
GF9 immunotherapy Intranasal /
LOW LOW LOW
(as described herein) Pulmonary /
Oral Cytotoxic drugs (Gemzar, Abraxane, HIGH HIGH Systemic / Oral HIGH
Temozol omi de) Biologies LOW HIGH
Systemic HIGH
(Bevacizumab, Canakinumab) In certain embodiments, other preferred TREM-1-related trifunctional peptides and compositions of this class comprise the domain A comprising the TREM-1 inhibitory peptide sequences LR12 and LP17 (described in Gibot, et al. Infect Immun 2006, 74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J Immunol 2007, 37:456-466;
Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J Clin Pharmacol 2018, in press; Zhou, et al.
Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et al., US
8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US 9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136, each of which is herein incorporated by reference in its entirety) while the domain B comprises at least one modified or unmodified amphipathic apo A-I
and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal. In some embodiments, resulting trifunctional peptide sequences may be radiolabeled and/or contain unmodified or modified methionine residues (TABLE 2) including but not limiting to, the following sequences:
LQEEDAGEYGCNIPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide (SEQ ID NO 7), LQEEDAGEYGCNIPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO
8), LQVTDSGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA (M(0), methinone sulfoxide (SEQ ID NO 9), LQVTDSGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO 10).
SLP (rHDL) structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US
20130039948;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). The inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 2) and TREM-1/TRIOPEP
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. In one embodiment, interaction of TREM-1/TRIOPEP GA31 with lipids results in self-assembly of nanosized SLP of discoidal or spherical morphology (dSLP and sSLP, respectively) (see FIG. 3).
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising amino acid domains A and B. Depending on lipid mixture compositions added to the peptides, sub 50 nm-sized SLP particles of discoidal (TREM-1/TRIOPEP-dSLP) or spherical (TREM-sSLP) morphology are self-assembled upon binding of the trifunctional peptide to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to intraplaque macrophages in vivo (FIG. 4A). In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to tumor-associated macrophages (TAMs) in vivo (FIG. 4B).
FIG. 4A illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for atherosclerosis), whereas domain B represents a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of atherosclerosis. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP
of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by intraplaque macrophages and return to the circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 5), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety.
FIG. 5 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the trifunctional peptide of the present invention comprising two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence, whereas domain B is a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
In one embodiment, FIG. 6 shows that the fluorescently labeled TREM-1/TRIOPEP
peptide GE31 delivered to macrophages by the SLP particles of the present invention colocalizes with TREM-1 expressed on these cells (see also Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). In certain embodiments, the capability of the TREM-1-related trifunctional peptides and compounds of the present invention including but not limiting to, TREM-1/TRIOPEP GA31 and TREM-1/TRIOPEP GE31, to colocalize with TREM-1 can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64Cu], see TABLE 2) can be conjugated to the TREM-1/TRIOPEP
sequences, GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, imaging (visualization) of TREM-1 levels using PET and/or other imaging techniques can be used to diagnose glioblastoma multiforme (GBM) and/or to select and monitor novel GBM therapies (see e.g., Johnson, et al.
Neuro Oncol 2017, 19:vi249 and James and Andreasson, WO 2017083682A1). In certain embodiments, imaging (visualization) of TREM-1 levels can be used to diagnose other TREM-1-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with TREM-1 in the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and a merged image (FIG. 6C).
As described herein (see FIG. 7), sulfoxidation of methionine residues in the TREM-1/TRIOPEP peptides GE31 and GA31 results in increased macrophage endocytosis of the SLP
containing an equimolar mixture of these peptides (designated as TREM-1/TRIOPEP), TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP. Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety.
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic lipopeptide particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that contain an equimolar mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 (TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The post 4 h incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified methionine residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4 (white bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001 to 0.001 as compared with 4 h incubation time.
In certain embodiments, FIGS. 8 and 10 demonstrate that TREM-1/TRIOPEP in free and SLP-bound forms inhibits TREM-1 function as shown by reduction of TREM-1-mediated release of pro-inflammatory cytokines, both in vitro (FIG. 8) and in vivo (in serum) (FIG. 10).
.. While not being bound to any particular theory, it is believed that this indicates that similarly to TREM-1-inhibitory peptide GF9 (see e.g., Sigalov. Int Immunopharmacol 2014, 21:208-219;
Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety), TREM-1-related trifunctional peptides can reach their site of action from both outside (free TREM-1/TRIOPEP) and inside (SLP-bound TREM-1/TRIOPEP) the cell. It is also believed that upon administration, free TREM-1/TRIOPEP may form LP in vivo and/or interact with native lipoproteins, resulting in formation of HDL-mimicking LP. In one embodiment, these LP may further target the cells of interest delivering their content to the areas of interest in a body.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production by lipopolysaccharide (LPS)-stimulated macrophages incubated for 24 h at 37 C with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged macrophages.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post lipopolysaccharide (LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline (PBS), dexamethasone (DEX), control peptide and with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
Control peptide represents an equimolar mixture of two peptides, each of them comprising two amino acid domains A and B where domain A represents a non-functional 9 amino acids-long sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5 is substituted with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as compared with PBS-treated animals.
While not being bound to any particular theory, it is believed that increased uptake described herein, is mediated by macrophage scavenger receptors (SR) including, but not limiting to, SR-A and SR-B1 (see FIG. 9A1,A2-C). While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 9A), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety.
FIG. 9A1-A2-C shows schematic representations of activation of the TREM-1/DAP12 receptor complex expressed on macrophages and presents the exemplary data showing that scavenger receptors SR-A and SR-B1 mediate the macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1, right panel).
FIG. 9A2, left panel shows schematic representations of activation of the TREM-receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm;
GF9 self-penetrates the cell membrane and blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and the downstream signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774 macrophages were cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-HDL, cells were treated for 1 hour at 37 C with 40 [tM cytochalasin D and either (FIG.
9B) 400 g/mL
fucoidan or (FIG. 9C) 10 M BLT-1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars) or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence intensities of lysates were measured and normalized to the protein content.
Results are expressed as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of GF9-HDL and GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo-oligomerization.
In certain embodiments, FIGS. 11A-B -14 demonstrate that TREM-1/TRIOPEP in free .. and SLP-bound forms inhibits tumor growth, reduces infiltration of macrophages into the tumor in mouse models of NSCLC and PC and is well-tolerated by cancer mice during the treatment period (see also Shen and Sigalov. Mol Pharm 2017, 14:4572-4582, each of which is herein incorporated by reference in its entirety).
IG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****, p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *, p < 0.05; **, p < 0.01, ****, p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP-bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar = 200 m.
In embodiment, FIG. 15 demonstrates that TREM-1/TRIOPEP in free and SLP-bound forms significantly prolongs survival in mice with lipopolysaccharide (LPS)-induced septic shock.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
In one embodiment, FIG. 16 shows that TREM-1/TRIOPEP is non-toxic in healthy mice at least up to 400 mg/kg.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
In one embodiment, FIG. 17 demonstrates that TREM-1/TRIOPEP in free and SLP-bound form ameliorates arthritis in mice with collagen-induced arthritis (CIA) and is well-tolerated by arthritic mice during the treatment period of 2 weeks (see Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in its entirety).
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *, p <0.05, **, p <0.01;
***, p <0.001 as compared with vehicle-treated or naive animals.
In certain embodiments, FIG. 18 demonstrates that TREM-1/TRIOPEP-sSLP prevents pathological RNV in mice with oxygen-induced retinopathy and is well-tolerated by these mice during the treatment period (see Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety).
FIG. 18A-D presents the exemplary data showing reduction of pathological retinal neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-1 (FIG. 18C) and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-induced retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP) particles of spherical morphology (TREM-1/TRIOPEP-sSLP). * * *, p < 0.001 as compared with vehicle-treated animals.
As described in Stukas, et al. J Am Heart Assoc 2014, 3:e001156, herein incorporated by reference in its entirety, systemically administered human apo A-I accumulates in murine brain.
It is also known that transcytosis of HDL in brain microvascular endothelial cells is mediated by SRBI (see Fung, et al. Front Physiol 2017, 8:841, herein incorporated by reference in its entirety). However, until tested as described herein, it was not known that a self-assembled SLP
of the present invention comprising a trifunctional peptide was capable of crossing the BBB.
In certain embodiments, FIG. 19 shows that the self-assembled SLP of the present invention may cross the BBB, BRB and BTB, thus delivering their constituents including but not limiting to, TREM-1/TRIOPEP, GF9, GA31 and GE31, to the areas of interest in the brain, retina and tumor. In certain embodiments, FIG. 63 demonstrates that the fluorescently labeled sSLP described herein may cross the BBB, BRB and BTB, thus delivering their constituents including but not limiting to, GBCA imaging probe to the areas of interest in the brain, retina and tumor.
While not being bound to any particular theory, it is believed that the brain-, retina-, and tumor-penetrating capabilities of these SLP can be mediated by interaction of SRBI with the domain B amino acid sequences that correspond to the sequences of human apo A-I helices 4 and/or 6 (see e.g. Liu, et al. J Biol Chem 2002, 277:21576-21584, herein incorporated by reference in its entirety).
In certain embodiments, these capabilities of the peptides and compositions of the present invention can be used to diagnose, treat and/or prevent cancers (including brain cancer), diabetic retinopathy and retinopathy of prematurity, neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases and other diseases and conditions where delivery of the peptides and compositions of the invention to the brain, retina and/or tumor is needed.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical self-assembled particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-sSLP) or an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
A mouse model of ALD mimics the early phase of the human disease, yet mRNA
levels of early fibrosis markers Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of these makers was remarkably attenuated in the vehicle-treated group and, importantly, further decreased by the TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B cc-Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
TREM-1 inhibitor effects were evaluated on hepatocyte damage and steatosis in liver.
Serum ALT levels obtained during week 5 of the alcohol feeding showed significant increases in alcohol-fed mice compared to PF controls. This ALT increase was attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig. 21A).
Surprisingly, vehicle .. treatment (HDL) also showed a similar protective effect (Fig. 21A).
Consistent with steatosis, we found a significant increase in Oil Red 0 staining in livers of alcohol-fed mice compared to PF controls (Fig. 21C). Oil Red 0 (Fig. 21B-D) and H& (Fig.
21D) staining revealed attenuation of steatosis in the alcohol-fed TREM-1 inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig.
21B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
WooP < 0.01;*"/#"P <0.001; ****P < 0 .0001. *** , 0.001 > P > 0.0001; ##, 0.01 > P > 0.001.
B. TCR-Related Trifunctional Peptides The T-cell receptor (TCR)-CD3 complex plays a role in T-cell differentiation, in .. protecting the organism from infectious agents, and in the function of T-cells. The TCR is a complex of a heterodimer of TCRa and TCRb chains, which are responsible for antigen recognition and interaction with the major histocompatibility complex (MHC) molecules of antigen-presenting cells, and CD3d, CD3g, CD3e and CD3z chains, which are responsible for transmembrane signal transduction (see e.g., Manolios, et al. Cell Adh Migr 2010, 4:273-283;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 20130039948;
Manolios.
US 6,057,294; Manolios. US 7,192,928; Manolios. US 20100267651; and Manolios, et al. US
20120077732, each of which is herein incorporated by reference in its entirety).
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic alpha helical peptide fragment. As described above, the inclusion of an amphipathic amino acid sequences aids the assistance in the ability to interact with native lipoproteins in a bloodstream in vivo and to form naturally long half-life lipopeptide/lipoprotein particles LP. It further aids the ability to provide targeted delivery to the sites of interest. It further aids the ability to cross the BBB, BRB and BTB.
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP
and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides:
(MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 11), TCR/TRIOPEP
ME32 (MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 12), TCR/TRIOPEP GA36 (GARSMTLTVQARQLPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO 13), TCR/TRIOPEP GE36 (GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 14), TCR/TRIOPEP GA32 (GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO 15), and TCR/TRIOPEP
(GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 16). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRa chain with the CD3ed heterodimer and CD3zz homodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
(LGKATLYAVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 17) and TCR/TRIOPEP
LE32 (LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 18). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRb chain with the CD3eg heterodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
(YLLDGILFIYPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 19) and TCR/TRIOPEP
YE32 (YLLDGILFIYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 20). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3zz homodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32 (IIVTDVIATLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 21) and TCR/TRIOPEP
1E32 (IIVTDVIATLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 22). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3ed heterodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
(FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 23) and TCR/TRIOPEP
FE32 (FLFAEIVSIFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 24). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg heterodimer with TCRb chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP
IA32e (IVIVDICITGPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 25) and TCR/TRIOPEP
IE32e (IVIVDICITGPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 26). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg and CD3ed heterodimers with TCRb and TCRa chains, respectively, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety).
In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TCR can be used to treat and/or prevent TCR-related diseases and conditions including but not limiting to, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis; autoimmune disease e.g. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel disease, Crohn's disease, primary biliary cirrhosis, chronic active hepatitis;
skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g.
allergic alveolitis; cardiovascular problems e.g. autoimmune pericarditis;
organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; and any other disorder where T
cells are involved/recruited In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TCR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In 64cut one embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to the TCR/TRIOPEP sequences In one embodiment, imaging (visualization) of TCR
levels using PET and/or other imaging techniques can be used to diagnose TCR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
C. NKG2D-Related Trifunctional Peptides NKG2D is an activating receptor expressed by natural killer (NK) and T cells.
The NKG2D is a complex of an NKG2D chain, which is responsible for ligand recognition, and DAP10 homodimer, which is responsible for transmembrane signal transduction (see e.g.
Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589;
Sigalov. Self Nonself 2010, 1:4-39; and Sigalov. Self Nonself 2010, 1:192-224, each of which is herein incorporated by reference in its entirety). NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease (see e.g. Gonzalez, et al. Trends Immunol 2008, 29:397-403; Ito, et al. Am J Physiol Gastrointest Liver Physiol 2008, 294:G199-207; Vilarinho, et al. Proc Natl Acad Sci U S A
2007, 104:18187-18192; Van Belle, et al. J Autoimmun 2013, 40:66-73; Lopez-Soto, et al. Int J
Cancer 2015, 136:1741-1750; and Urso, et al. US 9,127,064, each of which is herein incorporated by reference in its entirety).
The preferred NKG2D-related peptides and compositions of this class comprise the domain A comprising the NKG2D modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating NKG2D (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative NKG2D-related trifunctional peptides: NKG2D/TRIOPEP IA36 (IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 27) and NKG2D/TRIOPEP 1E36 (IAVAMGIRFIIMVAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 28). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with NKG2D in the cell membrane and selectively disrupt intramembrane interactions of NKG2D chain with the DNAX-activation protein 10 (DAP-10) signaling homodimer, resulting to specific ligand-independent inhibition of NKG2D upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
.. and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of NKG2D/TRIOPEP peptides are modified.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit NKG2D can be used to treat and/or prevent NKG2D-related diseases and conditions including but not limiting to, celiac disease, type I diabetes, hepatitis, and rheumatoid arthritis, and any other disorder where NKG2D cells are involved/recruited. In one embodiment, the present invention provides methods and compositions for preventing NK cell-mediated graft rejection.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with NKG2D can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the NKG2D/TRIOPEP sequences In one embodiment, imaging (visualization) of NKG2D levels using PET and/or other imaging techniques can be used to diagnose NKG2D-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
D. GPVI-Related Trifunctional Peptides.
In recent years, the central activating platelet collagen receptor, glycoprotein (GP) VI, has emerged as a promising antithrombotic target because its blockade or antibody-mediated depletion in circulating platelets was shown to effectively inhibit experimental thrombosis and thromboinflammatory disease states, such as stroke, without affecting hemostatic plug formation.
GPVI is a complex of an GPVI chain, which is responsible for ligand recognition, and FcRg homodimer, which is responsible for transmembrane signal transduction (see e.g. Sigalov.
Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov.
Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. J
Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692;
Dutting, et al. Trends Pharmacol Sci 2012, 33:583-590; Ungerer, etal. PLoS One 2013, 8:e71193;
Sigalov, US
8,278,271; Sigalov, US 8,614,188, each of which is herein incorporated by reference in its entirety). The binding of GPVI to collagen or other antagonists ligands induces platelet adhesion, activation and aggregation. Platelet activation is a step in the pathogenesis of ischemic cardio-.. and cerebrovascular diseases, which represent the leading causes of death and severe disability worldwide. Although existing antiplatelet drugs have proved beneficial in the clinic, their use is limited by their inherent effect on primary hemostasis, making the identification of novel pharmacological targets for platelet inhibition a goal of cardiovascular research.
The preferred GPVI-related peptides and compositions of this class comprise the domain .. A comprising the GPVI modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating GPVI (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative GPVI-related trifunctional peptides:
(GNLVRICLGAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 29) and GPVI/TRIOPEP
GE32 (GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 30). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with GPVI in the cell membrane and selectively disrupt intramembrane interactions of GPVI chain with the FcRg signaling homodimer, resulting to specific ligand-independent inhibition of GPVI upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of GPVI/TRIOPEP peptides are modified.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit GPVI can be used to treat and/or prevent GPVI-related diseases and conditions including but not limiting to, ischemic and thromboinflammatory diseases, and any other disorder where platelets are involved/recruited.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with GPVI can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the GPVI/TRIOPEP sequences In one embodiment, imaging (visualization) of GPVI levels using PET and/or other imaging techniques can be used to diagnose GPVI-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
E. DAP-10- and DAP-12-Related Trifunctional Peptides The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. DAP-10- and DAP-12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play a role in innate immune responses. See e.g. Lanier. Immunol Rev 2009, 227:150-160; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224, each of which is herein incorporated by reference in its entirety.
The preferred DAP-10 and DAP-12-related peptides and compositions of this class comprise the domain A comprising the DAP-10 or DAP-12 modulatory peptide sequences, respectively, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating DAP-10- and associated receptors (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP
.. structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim .. Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I
sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-10-related trifunctional peptides: DAP-10/TRIOPEP LA32 (LVAADAVASLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 33) and DAP-10/TRIOPEP LE32 (LVAADAVASLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 34).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-10-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-10 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of DAP-10/TRIOPEP
peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-12-related trifunctional peptides: DAP-12/TRIOPEP VA32 (VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 31) and DAP-12/TRIOPEP VE32 (VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 32).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-12-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-12 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of DAP-12/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the DAP-10- and DAP-12-associated receptors, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the DAP-10- and DAP-12-associated receptors, respectively, can be used to visualize (image) these receptors and evaluate their expression in the areas of interest. In one 64cut embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to the DAP-10/TRIOPEP and DAP-12/TRIOPEP sequences. In one embodiment, imaging (visualization) of levels of the DAP-10- and DAP-12-associated receptors using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
F. EGFR-Related Trifunctional Peptides.
The epidermal growth factor (EGF) receptor (EGFR) family, or ErbB family, is the best studied example of oncogenic receptor tyrosine kinases (RTKs). HER2/ErbB2 is overexpressed on the surface of 25-30% of breast cancer cells, and it has been associated with a high risk of relapse and death. EGFR amplification and mutations have been associated with many carcinomas. In particular, the EGFR pathway appears to play a role in pancreatic carcinoma. See e.g. Overholser, et al. Cancer 2000, 89:74-82; Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224. Short hydrophobic peptides corresponding to the transmembrane domains of EGFR, ErB2 and insulin receptors inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor (see Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474).
The preferred EGFR-related peptides and compositions of this class comprise the domain A comprising the EGFR modulatory peptide sequences, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating EGFR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in its entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I
sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative EGFR-related trifunctional peptides: EGFR/TRIOPEP SA47 (SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO.
35) and EGFR/TRIOPEP 5E47 (SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO.
36). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with EGFR in the cell membrane and selectively disrupts intramembrane interactions between the receptors, resulting to specific ligand-independent inhibition of the receptor (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in its entirety). In one embodiment, methionine residues of EGFR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the EGFR and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the receptors of the EGFR and/or ErB receptor families, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the EGFR- and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the receptors of the EGFR and/or ErB receptor families can be used to visualize (image) these receptors and evaluate their expression in the areas of interest. In one embodiment, 64co for this purpose, an imaging probe (e.g. [ can be conjugated to the EGFR/TRIOPEP
sequence. In one embodiment, imaging (visualization) of levels of the receptors of the EGFR
and/or ErB receptor families using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
G. Additional Trifunctional Peptides Additional therapeutic peptide sequences and/or other therapeutic agents can comprise the domain A of the peptides and compositions of the present invention.
Additional examples are provided in, for e.gs., Vlieghe, et al. Drug Discov Today 2010, 15:40-56;
Tsung, et al. Shock 2007, 27:364-369; Chang, et al. PLoS One 2009, 4:e4171; Tjin Tham Sjin, et al.
Cancer Res 2005, 65:3656-3663; Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336;
Khan, et al. Hum Immunol 2002, 63:1-7; Banga. Therapeutic peptides and proteins: formulation, processing, and delivery systems. 2nd ed. Boca Raton, FL: Taylor & Francis Group; 2006;
Stevenson. Curr Pharm Biotechnol 2009, 10:122-137; Wu and Chi, US 9,387,257; Wu, et al., US
8,415,453;
Faure, et al., US 8,013,116; Faure, et al., US 9,273,111; Eggink and Hoober, US 7,811,995;
Eggink and Hoober, US 8,496,942; Morgan and Pandha. US 2012/0177672 Al;
Broersma, et al., US 5,681,925), each of which is herein incorporated by reference in its entirety.
In one embodiment, this domain comprises the Toll Like Receptor (TLR) modulatory sequence (see e.g. Tsung, et al. Shock 2007, 27:364-369). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TLR-related trifunctional peptides: TLR/TRIOPEP DA32 (DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 37) and TLR/TRIOPEP DE32 (DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 38). In one embodiment, methionine residues of TLR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TLR can be used to treat and/or prevent TLR-related diseases and conditions including but not limiting to, sepsis and other infectious diseases, and any other disorder where TLR receptors are involved.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TLR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the TLR/TRIOPEP sequences In one embodiment, imaging (visualization) of TLR levels using PET
and/or other imaging techniques can be used to diagnose TLR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In one embodiment, the domain A of the peptides and compositions of the invention comprises the Atrial Natriuretic Peptide (ANP) receptor (ANPR)-modulatory sequence (see e.g.
Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in its entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative ANPR-related trifunctional peptides: ANPR/TRIOPEP SA50 (SLRRS SCFGGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO. 39) and ANPR/TRIOPEP SE50 (SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 40). In one embodiment, methionine residues of ANPR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit ANPRs can be used to treat and/or prevent ANPR-related diseases and conditions including but not limiting to, cardiovascular and inflammatory diseases, and any other disorder where ANP
receptors are involved.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with ANPR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the ANPR/TRIOPEP sequences In one embodiment, imaging (visualization) of ANPR levels using PET and/or other imaging techniques can be used to diagnose ANPR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In certain embodiments, other therapeutic agents including but not limiting to, to those described in Page and Takimoto. Principles of chemotherapy. In: Pazdur R, Wagman LD, Camphausen KA, editors. Cancer Management: A Multidisciplinary Approach. 11th ed.
Manhasset, NY: Cmp United Business Media; 2009. p. 21-37; Sipsas, et al., Therapy of Mucormycosis, J Fungi (Basel) 2018, 4; Lin, et al. Chem Commun (Camb) 2013, 49:4968-4970;
and in Turner, et al. Peptide Conjugates of Oligonucleotide Analogs and siRNA
for Gene Expression Modulation. In: Langel U, ed, editor. Handbook of Cell-Penetrating Peptides. 2nd edition ed. Boca Raton: CRC Press; 2007. p. 313-328 and disclosed in Schiffman and Altman, US 4,427,660; Castaigne, et al., US 9,161,988; Castaigne, et al., US
8,921,314; and in Castaigne, et al., US 9,173,891, each of which is herein incorporated by reference in its entirety (see also TABLE 2) can comprise the domain A of the peptides and compositions of the present invention.
III. Lipoproteins And rHDLS.
Lipoproteins, inlcuding circulating lipoproteins in blood plasma, are natural complexes that contain both proteins (apolipoproteins, apo) and lipids bound to the proteins, which allow water-insoluble molecules such as fats to move through the water inside and outside cells.
Lipoproteins serve to emulsify the lipid molecules. Examples include the plasma lipoprotein particles classified under high-density lipoproteins (HDL), which enable cholesterol and other hydrophobic lipid molecules to be carried in the bloodstream. In particular, HDL transport cholesterol and other water insoluble or poorly soluble lipids from the peripheral tissues to the liver.
The use of HDLs as delivery vehicles was proposed however in order to properly function in vivo for delivery of drugs or imaging agents to sites of interest, HDLs should mimic native lipoproteins as close as possible. In a human body, HDL exists in two forms: nascent or discoidal HDL and spherical HDL. The use of isolated plasma lipoproteins, including isolated HDLs, as delivery vehicles is impractical.
However in vitro, long half-life lipoprotein particles that mimic native HDL
(as synthetic sHDL or recombinant HDL, rHDL) can be readily reconstituted (synthesized) from lipid formulations and apolipoproteins (apo) resulting in, for example, sub 30 nm-sized particles of discoidal or spherical morphology. Morphology of rHDLs is determined by the composition of lipid and apo mixtures and preparation procedures.
Many types of rHDLs were evaluated both clinically and experimentally as a delivery system for administering hydrophobic agents and for mitigating the toxic effects associated with administration of imaging probes such as Gd-containing contrast agents (GBCAs) for magnetic resonance imaging (MM).
As delivery vehicles, rHDL have several competitive advantages as compared with other delivery platforms: 1) apo A-I, a major HDL protein, is used for rHDL
preparation as it's recombinant or synthesized peptide/protein represents an endogenous protein that does not trigger immunoreactions; 2) apo A-I's small size allows rHDL to pass through blood vessel walls, enter and then accumulate in the places of interest, including for treatment and/or detection, such as tumor sites, areas of disease, such as liver tissue, etc., or atherosclerotic plaques; 3) rHDL's small particle size also allows for intravenous, intramuscular and subcutaneous applications; 4) rHDL's naturally long half-life extends the half-life of incorporated drugs and/or imaging agents in a bloodstream; and 5) a variety of drugs and imaging agents can be incorporated into this platform.
However, in order to properly function in vivo and as a result, to realize all the advantages mentioned above, rHDL should mimic native lipoproteins including but not limited to HDL as close as possible. This is a complicated task because two functions, assistance in the self-assembly of rHDL and therapeutic and/or imaging action in vivo, have to be executed by at least, two separate rHDL ingredients such as human apolipoprotein and therapeutic agent and/or imaging probe. In addition, in contrast to, for example, native HDL that are normally target the liver, rHDL have to be able to target other sites of interest such as, for example, macrophages which results in the need of targeting moieties thus adding the third function of rHDL ingredients ¨ targeting. This hampers wider use of rHDL by difficulties in industrializing the manufacture of rHDL, along with rHDL' lack of stability and reproducibility. In addition, the use of native or recombinant human apolipoproteins significantly complicates development of the commercial product, drastically increases its cost and possesses potential clinical and regulatory pitfalls.
An alternative, fully synthetic lipopeptide system for targeted treatment and/or imaging that closely mimics native lipoproteins and exhibits the advantageous properties of rHDL as well as superior stability, uniformity, ease of use, and reproducibility of preparation is needed for administration and targeted delivery of therapeutic agents (e.g. anti-cancer and anti-sepsis agents, other anti-inflammatory drugs) and/or imaging probes. The invention provides such a system and a method of using the system (e.g., for delivery of anti-cancer, anti-arthritic, anti-sepsis, anti-angiogenic and other therapeutic agents and/or imaging probes to a subject).
These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Additional contemplative advantages of a lipoprotein delivery platform includes increasing activity due to specific targeting, sequestration of the drug at the target site, protection of the drug from rapid metabolism, amplified therapeutic effect due to packaging of numerous drug molecules in each particle, and decreased toxicity due to altered pharmacokinetics. Due to the naturally long half-life of native discoidal and spherical HDL in normal subjects being 12-20 hrs and 3-5 days, respectively, rHDL represent a promising versatile delivery platform in particular for therapeutic peptides that have a bloodstream half-life of minutes.
For example, it would be desirable to combine in one molecule therapeutic (and/or diagnostic), particle forming and targeting functions. The invention addresses these needs, among others, and provides such a system/molecule and a method of using the system (e.g., for delivery of anti-cancer, anti-arthritic, anti-sepsis, anti-angiogenic, anti-inflammatory and other therapeutic agents and/or imaging probes to a subject). These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
IV. Trifunctional Peptides In rHDL Formulations.
A. TREM-1-related Trifunctional peptides: TREM-1 Signaling Pathway and Its Blockade.
TREM-1 is expressed on the majority of innate immune cells and to a lesser extent on parenchymal cells. Upon activation, TREM-1 can directly amplify an inflammatory response.
Although it was initially demonstrated that TREM-1 was predominantly associated with infectious diseases, recent evidences demonstrate that TREM-1 receptor and its signaling pathways contribute to the pathology of non-infectious acute and chronic inflammatory diseases, .. including but not limiting to, rheumatoid arthritis, atherosclerosis, ischemia reperfusion-induced tissue injury, colitis, fibrosis, neurodegenerative diseases, liver diseases, retinopathies, and cancer (see e.g., Tammaro, et al. Pharmacol Ther 2017, 177:81-95; Saadipour.
Neurotox Res 2017, 32:14-16; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov.
US 8,513,185;
Sigalov. US 9,981,004; Rojas, et al. Biochim Biophys Acta 2018, 1864: 2761-2768, and Kuai, et al. US 2008/0247955, each of which is herein incorporated by reference in its entirety).
In some preferred embodiments, TREM-1-related peptides and associated compositions of the present invention have a domain A conjugated to a domain B. See, Fig.
1. Domain A
comprises a TREM-1 modulatory peptide sequence designed using a known model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TREM-1 receptor expressed on myeloid cells (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 8,513,185; and Sigalov. US 9,981,004), all of which are herein incorporated by reference in their entirety. In some preferred embodiments, peptides and compositions of the present invention comprise the TREM-1 modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model.
In some preferred embodiments, peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic alpha helical peptide fragment, such as a apo A-I and/or A-II peptide fragment, to form upon interaction with lipid and/or lipid mixtures. In certain embodiments, exemplary trifunctional peptides comprise the domain B comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In one embodiment, preferred peptides and compositions of the invention further comprise at least one modified or unmodified amphipathic apo A-I and/or A-II
peptide fragment capable upon interaction with lipid and/or lipid mixtures, to form synthetic lipopeptide particles (SLP) structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US 20130039948;
Shen, et al.
PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J
Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). The inclusion of an amphipathic apo A-I
sequences in the peptides and compositions of the invention further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
FIG. 1 presents an exemplary schematic representation of one embodiment of a trifunctional peptide of the present invention comprising amino acid domains A and B where amino acid domain A represents a therapeutic peptide sequence with or without an attached drug compound and/or imaging probe that functions to treat, prevent and/or detect a disease or condition, whereas amino acid domain B represents an amphipathic alpha helical peptide sequence, with or without an additional targeting peptide sequence, and functions to 1) assist in the self-assembly of synthetic lipoprotein/lipopeptide nanoparticles (SLP) upon interaction with lipids or lipid mixtures in vitro, for use in transporting these trifunctional peptides as lipoprotien nanoparticles to sites of interest in vitro or in vivo and/or 2) form long half-life lipopeptide/lipoprotein particles upon interaction with endogenous lipoproteins for transporting these trifunctional peptides to the sites of interest. Endogenous lipoproteins may be lipoproteins added to or found in cell cultures, or lipoproteins in a mammalian body.
In certain embodiments, FIG. 2 shows the structures of representative TREM-1-related trifunctional peptides, TREM-1/TRIOPEP GE31 (GFL5K5LVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and TREM-1/TRIOPEP GA31 (GFL5K5LVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ
ID NO. 2) and TREM-1/TRIOPEP GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. GFLSKSLVF, is found within isoform 1 of human (UniProtKB - Q9NP99 (TREM1 HUMAN), and in human TREM-1 isoform CRA a (UniProtKB - Q38L15 (Q38L15 HUMAN), both downloaded 10-24-2018)).
Q9NP991TREM1 HUMAN Isoform 1 Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
Q38L15938L15 HUMAN Triggering receptor expressed on myeloid cells 1, Homo sapiens isoform CRA a:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP
GFLSKSLVF, is not found within human TREM-1 isoforms 2 or 3.
Q9NP99-21TREM1 HUMAN Isoform 2 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGFRCSTLSFSWLVDS
Q9NP99-31TREM1 HUMAN Isoform 3 of Triggering receptor expressed on myeloid cells 1, Homo sapiens:
MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQII
RDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIY
QPPKEPHMLFDRIRLVVTKGF SGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPP
KST.
FIG. 2 presents schematic representations of embodiments of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP). GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) comprising amino acid domain A and B
(GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE) where domain A represents a 9 amino acids-long human TREM-1 inhibitory therapeutic SCHOOL peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition, whereas domain B
represents a 22 amino acids-long human apolipoprotein A-I helix 4 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) in vitro for targeting the particles to myeloid cells (e.g.
macrophages). GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide).
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
In certain embodiments, other preferred TREM-1-related trifunctional peptides and compositions of this class comprise the domain A comprising the TREM-1 inhibitory peptide sequences LR12 and LP17 (described in Gibot, et al. Infect Immun 2006, 74:2823-2830; Gibot, et al. Shock 2009, 32:633-637; Gibot, et al. Eur J Immunol 2007, 37:456-466;
Joffre, et al. J Am Coll Cardiol 2016, 68:2776-2793; Cuvier, et al. Br J Clin Pharmacol 2018, in press; Zhou, et al.
Int Immunopharmacol 2013, 17:155-161; and disclosed in Faure, et al., US
8,013,116; Faure, et al., US 9,273,111; Gibot, et al., US 9,657,081; Gibot and Derive, US
9,815,883; and in Gibot and Derive, US 9,255,136, each of which is herein incorporated by reference in it's entirety) while the domain B comprises at least one modified or unmodified amphipathic apo A-I
and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal. In some embodiments, resulting trifunctional peptide sequences may be radiolabeled and/or contain unmodified or modified methionine residues (TABLE 2) including but not limiting to, the following sequences:
LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide (SEQ ID NO 7), LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO 8), LQVTDSGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA (M(0), methinone sulfoxide (SEQ ID NO 9), LQVTDSGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE (M(0), methionine sulfoxide (SEQ ID NO 10).
SLP (rHDL) structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Sigalov. US
20130039948;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorpoated by referene in it's entirety). The inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In one embodiment, methionine residues of the peptides TREM-1/TRIOPEP GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 2) and TREM-1/TRIOPEP
GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 1) are unmodified. In one embodiment, interaction of TREM-1/TRIOPEP GA31 with lipids results in self-assembly of nanosized SLP of discoidal or spherical morphology (dSLP and sSLP, respectively) (see FIG. 3).
FIG. 3 presents a schematic representation of one embodiment of a TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) of the present invention comprising amino acid domains A and B. Depending on lipid mixture compositions added to the peptides, sub 50 nm-sized SLP particles of discoidal (TREM-1/TRIOPEP-d5LP) or spherical (TREM-s5LP) morphology are self-assembled upon binding of the trifunctional peptide to lipids.
Abbreviations: apo, apolipoprotein; SCHOOL, signaling chain homooligomerization; TREM-1, triggering receptor expressed on myeloid cells-1.
In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to intraplaque macrophages in vivo (FIG. 4A). In one embodiment, this provides targeted delivery of the SLP constituents including TREM-1/TRIOPEP to tumor-associated macrophages (TAMs) in vivo (FIG. 4B).
FIG. 4A illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A represents a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for atherosclerosis), whereas domain B represents a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of atherosclerosis. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP
of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in intraplaque macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by intraplaque macrophages and return to the circulation.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (example, for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 5), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in it's entirety FIG. 5 illustrates one embodiment of a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the trifunctional peptide of the present invention comprising two amino acid domains A and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence, whereas domain B is a 22 amino acids-long apolipoprotein A-I
helix 4 or 6 peptide sequence with a sulfoxidized methionine residue. While not being bound to any particular theory, it is believed that this disruption results in "pre-dissociation" of a receptor complex and upon ligand stimulation, leads to inhibition of TREM-1 and silencing the TREM-1 signaling pathway.
In one embodiment, FIG. 6 shows that the fluorescently labeled TREM-1/TRIOPEP
peptide GE31 delivered to macrophages by the SLP particles of the present invention colocalizes with TREM-1 expressed on these cells (see also Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768). In certain embodiments, the capability of the TREM-1-related trifunctional peptides and compounds of the present invention including but not limiting to, TREM-1/TRIOPEP GA31 and TREM-1/TRIOPEP GE31, to colocalize with TREM-1 can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64Cu], see TABLE 2) can be conjugated to the TREM-1/TRIOPEP sequences, GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID
NO. 4) and GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (M(0), methionine sulfoxide) (SEQ ID NO. 3). In one embodiment, imaging (visualization) of TREM-1 levels using PET and/or other imaging techniques can be used to diagnose glioblastoma multiforme (GBM) and/or to select and monitor novel GBM therapies (see e.g., Johnson, et al.
Neuro Oncol 2017, 19:vi249 and James and Andreasson, WO 2017083682A1). In certain embodiments, imaging (visualization) of TREM-1 levels can be used to diagnose other TREM-1-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
FIG. 6A-C shows images depicting colocalization of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptide (TREM-1/TRIOPEP) GE31 with TREM-1 in the cell membrane (FIG. 6A), TREM-1 immunohistochemstry staining (FIG. 6B) and a merged image (FIG. 6C).
As described herein (see FIG. 7A-B), sulfoxidation of methionine residues in the TREM-1/TRIOPEP peptides GE31 and GA31 results in increased macrophage endocytosis of the SLP
containing an equimolar mixture of these peptides (designated as TREM-1/TRIOPEP), TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP. Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, all of which are herein incorporated in their entirety.
FIG. 7A-B presents the exemplary data showing the endocytosis of synthetic lipopeptide particles (SLP) of discoidal (dSLP) and spherical (sSLP) morphology that contain an equimolar mixture of the TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 (TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP, respectively). (FIG. 7A) The post 4 h incubation in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-sSLP with the unmodified (patterned bars) or sulfoxidized TREM-1/TRIOPEP
methionine residues (black bars). ***, P = 0.0001 to 0.001 (sulfoxidized vs. unmodified methionine residues). (FIG. 7B) the in vitro macrophage uptake of TREM-1/TRIOPEP-dSLP and TREM-1/TRIOPEP-sSLP with the sulfoxidized TREM-1/TRIOPEP methionine residues post 4 (white bars), 12 (patterned bars), and 24 h (black bars) incubation. ***, P = 0.0001 to 0.001 as compared with 4 h incubation time.
In certain embodiments, FIGS. 8 and 10 demonstrate that TREM-1/TRIOPEP in free and SLP-bound forms inhibits TREM-1 function as shown by reduction of TREM-1-mediated release of pro-inflammatory cytokines both in vitro (FIG. 8) and in vivo (FIG.
10). While not being bound to any particular theory, it is believed that this indicates that similarly to TREM-1-inhibitory peptide GF9 (see e.g., Sigalov. Int Immunopharmacol 2014, 21:208-219; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in it's entirety), TREM-1-related trifunctional peptides can reach their site of action from both outside (free TREM-1/TRIOPEP) and inside (SLP-bound TREM-1/TRIOPEP) the cell. It is also believed that upon administration, free TREM-1/TRIOPEP may form LP in vivo and/or interact with native lipoproteins, resulting in formation of HDL-mimicking LP. In one embodiment, these LP may further target the cells of interest delivering their content to the areas of interest in a body.
FIG. 8 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lb eta production by lipopoly sacchari de (LPS)-stimulated macrophages incubated for 24 h at 37 C with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
***, P = 0.0001 to 0.001 as compared with medium-treated LPS-challenged macrophages.
FIG. 10 presents the exemplary data showing suppression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-6 and IL-lbeta production in mice at 90 min post lipopolysaccharide (LPS) challenge treated 1 h before LPS challenge with phosphate-buffer saline (PBS), dexamethasone (DEX), control peptide and with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
Control peptide represents an equimolar mixture of two peptides, each of them comprising two amino acid domains A and B where domain A represents a non-functional 9 amino acids-long sequence of the TREM-1 inhibitory therapeutic peptide sequence wherein, Lys5 is substituted with Ala5, whereas domain B is a sulfoxidized methionine residue-containing 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence, respectively. *, P =
0.01 to 0.05 as compared with animals treated with 5 mg/kg TRIOPEP in free form; ***, P =
0.0001 to 0.001 as compared with PBS-treated animals.
While not being bound to any particular theory, it is believed that increased uptake described herein, is mediated by macrophage scavenger receptors (SR) including, but not limiting to, SR-A and SR-B1 (see FIG. 9A-C). While not being bound to any particular theory, it is believed that in one embodiment, this colocalization is accompanied by a specific disruption of intramembrane interactions between TREM-1 and DAP-12 by the TREM-1-related trifunctional peptide of the present invention (see FIG. 9A), resulting in ligand-independent inhibition of TREM-1 upon ligand binding as described in Shen and Sigalov. Mol Pharm 2017, 14:4572-4582 and Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534, each of which is herein incorporated by reference in it's entirety.
FIG. 9A1-A2-C shows schematic representations of activation of the TREM-1/DAP12 receptor complex expressed on macrophages and presents the exemplary data showing that scavenger .. receptors SR-A and SR-B1 mediate the macrophage endocytosis of GF9-sSLP
(GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP). (FIG. 9A) Schematic representation of TREM-1 signaling and the SCHOOL mechanism of TREM-1 blockade. (FIG. 9A1, left panel) Activation of the TREM-1/DAP12 receptor complex expressed on macrophages leads to phosphorylation of the DAP12 cytoplasmic signaling domain and subsequent downstream inflammatory cytokine response (left panel). SR-mediated endocytosis of sSLP-bound GF9, GA31 and GE31 peptide inhibitors by macrophages results in the release of GF9 or GA31 and GE31 into the cytoplasm, which self-penetrate into the cell membrane and block intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and downstream signaling cascade (FIG. 9A1, right panel).
FIG. 9A2, left panel shows schematic representations of activation of the TREM-receptor complex expressed on Kupffer cells leads to phosphorylation of the DAP12 cytoplasmic signaling domain, subsequent SYK recruitment, and the downstream inflammatory cytokine response. (FIG. 9A2, right panel) SR-mediated endocytosis of HDL-bound GF9 peptide inhibitors by Kupffer cells results in the release of GF9 (GA31 or GE31) into the cytoplasm;
GF9 self-penetrates the cell membrane and blocks intramembrane interactions between TREM-1 and DAP12, thereby preventing DAP12 phosphorylation and the downstream signaling cascade.
FIG. 9B-9C Macrophage endocytosis of GF9-sSLP (GF9-HDL) and GA/E31-HDL (TREM-1/TRIOPEP-sSLP) in vitro is SR-mediated in a time-dependent manner and is largely driven by SR-A (FIG. 9B, FIG. 9C). As described in the Materials and Methods, J774 macrophages were cultured at 37 C overnight with medium. Prior to uptake of GF9-HDL and GA/E31-HDL, cells were treated for 1 hour at 37 C with 401.IM cytochalasin D and either (FIG.
9B) 400 pg/mL
fucoidan or (FIG. 9C) 10 1.1M BLT-1, as indicated. Cells were then incubated for either 4 hours or 22 hours with medium containing 2 i.tM rhodamine B (rho B)-labeled GF9-sSLP
(gray bars) or TREM-1/TRIOPEP-sSLP (black bars), respectively. Cells were lysed, and rho B
fluorescence intensities of lysates were measured and normalized to the protein content.
Results are expressed as mean SEM (n = 3); *P < 0.05; **P < 0.01; ****P < 0.0001 versus uptake of GF9-HDL and GA/E31-HDL in the absence of inhibitor. Abbreviations: D, DAP12; DAP12, DNAX
activation protein of 12 kDa; K, Kupffer cell; RFU, relative fluorescence units; SCHOOL, signaling chain homo-oligomerization.
In certain embodiments, FIGS. 11A-B -14A-C demonstrate that TREM-1/TRIOPEP in free and SLP-bound forms inhibits tumor growth, reduces infiltration of macrophages into the tumor in mouse models of NSCLC and PC and is well-tolerated by cancer mice during the treatment period (see also Shen and Sigalov. Mol Pharm 2017, 14:4572-4582).
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *, p < 0.05; **, p < 0.01, ****, p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP--- bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar = 200 pm.
In embodiment, FIG. 15 demonstrates that TREM-1/TRIOPEP in free and SLP-bound forms significantly prolongs survival in mice with lipopolysaccharide (LPS)-induced septic shock.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
-- FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
In one embodiment, FIG. 16 shows that TREM-1/TRIOPEP is non-toxic in healthy mice at least up to 400 mg/kg.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
In one embodiment, FIG. 17 demonstrates that TREM-1/TRIOPEP in free and SLP-bound form ameliorates arthritis in mice with collagen-induced arthritis (CIA) and is well-tolerated by arthritic mice during the treatment period of 2 weeks (see Shen and Sigalov. J Cell -- Mol Med 2017, 21:2524-2534).
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1--- related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *, p <0.05, **, p <0.01;
***, p <0.001 as compared with vehicle-treated or naive animals.
In certain embodiments, FIG. 18 demonstrates that TREM-1/TRIOPEP-sSLP prevents pathological RNV in mice with oxygen-induced retinopathy and is well-tolerated by these mice during the treatment period (see Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768).
FIG. 18A-D presents the exemplary data showing reduction of pathological retinal neovascularization area (FIG. 18A), avascular area (FIG. 18B) and retinal TREM-1 (FIG. 18C) and M-CSF/CSF-1 (FIG. 18D) expression in the retina of the mice with oxygen-induced retinopathy (OIR) treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
incorporated into synthetic lipopeptide particles (TREM-1/TRIOPEP-SLP) particles of spherical morphology (TREM-1/TRIOPEP-sSLP). ***,p < 0.001 as compared with vehicle-treated animals.
As described in Stukas, et al. J Am Heart Assoc 2014, 3:e001156, systemically administered human apo A-I accumulates in murine brain. It is also known that transcytosis of HDL in brain microvascular endothelial cells is mediated by SRBI (see Fung, et al. Front Physiol 2017, 8:841). However, until tested, it was not known that a self-assembled SLP of the present invention comprising a trifunctional peptide was capable of crossing the BBB.
In certain embodiments, FIG. 19 shows that the self-assembled SLP of the present invention may cross the BBB, BRB and BTB, thus delivering their constituents including but not limiting to, TREM-1/TRIOPEP to the areas of interest in the brain, retina and tumor. While not being bound to any particular theory, it is believed that the brain-, retina-, and tumor-penetrating capabilities of these SLP can be mediated by interaction of SRBI with the domain B amino acid sequences that correspond to the sequences of human apo A-I helices 4 and/or 6 (see e.g. Liu, et al. J Biol Chem 2002, 277:21576-21584, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, these capabilities of the peptides and compositions of the present invention can be used to diagnose, treat and/or prevent cancers (including brain cancer), diabetic retinopathy and retinopathy of prematurity, neurodegenerative diseases including Alzheimer's, Parkinson's and Huntington's diseases and other diseases and conditions where delivery of the peptides and compositions of the invention to the brain, retina and/or tumor is needed.
FIG. 19 presents exemplary data showing penetration of the blood-brain barrier (BBB) and blood-retinal barrier (BRB) by systemically (intraperitoneally) administered rhodamine B-labeled spherical self-assembled particles (sSLP) that contain Gd-containing contrast agent (Gd-sSLP) for magnetic resonance imaging (MRI), TREM-1 inhibitory peptide GF9 (GF9-sSLP) or an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides GA 31 and GE 31 (TREM-1/TRIOPEP-sSLP) .
Mouse model of ALD mimics the early phase of the human disease, yet mRNA
levels of early fibrosis markers Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of these makers -- was remarkably attenuated in the vehicle-treated group and, importantly, further decreased by the TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B a-Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
TREM-1 inhibitor effects were evaluated on hepatocyte damage and steatosis in liver. Serum ALT levels obtained during week 5 of the alcohol feeding showed significant increases in alcohol-fed mice compared to PF controls. This ALT increase was attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig.
4A).
Surprisingly, vehicle treatment (HDL) also showed a similar protective effect (Fig. 4A).
Consistent with steatosis, we found a significant increase in Oil Red 0 staining in livers of alcohol-fed mice compared to PF controls (Fig. 4C). Oil Red 0 (Fig. 4B-D) and H& (Fig.
4D) staining revealed attenuation of steatosis in the alcohol-fed TREM-1 inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig.
4B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
44/ P < 0.01;*/"413 <0001; ****P < 0.0001.***, 0.001 > P> 0.0001; ##, 0.01 >
P> 0.001.
B. TCR-Related Trifunctional Peptides The T-cell receptor (TCR)-CD3 complex plays a role in T-cell differentiation, in protecting the organism from infectious agents, and in the function of T-cells. The TCR is a complex of a heterodimer of TCRa and TCRb chains, which are responsible for antigen recognition and interaction with the major histocompatibility complex (MHC) molecules of antigen-presenting cells, and CD3d, CD3g, CD3e and CD3z chains, which are responsible for transmembrane signal transduction (see e.g., Manolios, et al. Cell Adh Migr 2010, 4:273-283;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9; Sigalov. US 20130039948;
Manolios.
US 6,057,294; Manolios. US 7,192,928; Manolios. US 20100267651; and Manolios, et al. US
20120077732, each of which is herein incorporated by reference in it's entirety).
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or -- unmodified amphipathic alpha helical peptide fragment. As described above, the inclusion of an amphipathic amino acid sequences aids the assistance in the ability to interact with native lipoproteins in a bloodstream in vivo and to form naturally long half-life lipopeptide/lipoprotein particles LP. It further aids the ability to provide targeted delivery to the sites of interest. It further aids the ability to cross the BBB, BRB and BTB.
The preferred TCR-related peptides and compositions of this class comprise the domain A comprising the TCR modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating TCR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. PLoS
Pathog 2009, 5:e1000404; Shen and Sigalov. Sci Rep 2016, 6:28672; Sigalov. US
20130039948, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids -- and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP MA32 (MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 11), TCR/TRIOPEP
ME32 (MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 12), TCR/TRIOPEP GA36 (GARSMTLTVQARQLPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO 13), TCR/TRIOPEP GE36 (GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 14), TCR/TRIOPEP GA32 (GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO 15), and TCR/TRIOPEP GE32 (GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO 16). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRa chain with the CD3ed heterodimer and CD3zz homodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP LA32 (LGKATLYAVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 17) and TCR/TRIOPEP
LE32 (LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 18). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of TCRb chain with the CD3eg heterodimer, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP YA32 (YLLDGILFIYPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 19) and TCR/TRIOPEP
YE32 (YLLDGILFIYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 20). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3zz homodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32 (IIVTDVIATLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 21) and TCR/TRIOPEP
1E32 (IIVTDVIATLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 22). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3ed heterodimer with TCRa chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP FA32 (FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 23) and TCR/TRIOPEP
FE32 (FLFAEIVSIFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 24). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg heterodimer with TCRb chain, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety).
In certain embodiments, TABLE 2 demonstrates the following structures of representative TCR-related trifunctional peptides: TCR/TRIOPEP IA32e (IVIVDICITGPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 25) and TCR/TRIOPEP
IE32e (IVIVDICITGPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 26). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with TCR in the cell membrane and selectively disrupt intramembrane interactions of CD3eg and CD3ed heterodimers with TCRb and TCRa chains, respectively, resulting to specific ligand-independent inhibition of TCR upon antigen stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein incorporated by reference in their entirety).
In one embodiment, methionine residues of TCR/TRIOPEP peptides are modified as described herein.
In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TCR can be used to treat and/or prevent TCR-related diseases and conditions including but not limiting to, allergic diathesis e.g. delayed type hypersensitivity, contact dermatitis; autoimmune disease e.g. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, diabetes, Guillain-Barre syndrome, Hashimotos disease, pernicious anaemia; gastroenterological conditions e.g.
inflammatory bowel disease, Crohn's disease, primary biliary cirrhosis, chronic active hepatitis;
skin problems e.g.
atopic dermatitis, psoriasis, pemphigus vulgaris; infective disease;
respiratory conditions e.g.
allergic alveolitis; cardiovascular problems e.g. autoimmune pericarditis;
organ transplantation;
inflammatory conditions e.g. myositis, ankylosing spondylitis; and any other disorder where T
cells are involved/recruited In certain embodiments, the capability of the TCR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TCR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu-.j, see TABLE 2) can be conjugated to the TCR/TRIOPEP sequences In one embodiment, imaging (visualization) of TCR
levels using PET and/or other imaging techniques can be used to diagnose TCR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
C. NKG2D-Related Trifunctional Peptides NKG2D is an activating receptor expressed by natural killer (NK) and T cells.
The NKG2D is a complex of an NKG2D chain, which is responsible for ligand recognition, and DAP10 homodimer, which is responsible for transmembrane signal transduction (see e.g.
Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589;
Sigalov. Self Nonself 2010, 1:4-39; and Sigalov. Self Nonself 2010, 1:192-224, all of which are herein incorporated by reference in their entirety). NKG2D ligands show a restricted expression in normal tissues, but they are frequently overexpressed in cancer and infected cells. The binding of NKG2D to its ligands activates NK and T cells and promotes cytotoxic lysis of the cells expressing these molecules. The mechanisms involved in the expression of the ligands of NKG2D play a role in the recognition of stressed cells by the immune system and represent a promising therapeutic target for improving the immune response against cancer or autoimmune disease (see e.g. Gonzalez, et al. Trends Immunol 2008, 29:397-403; Ito, et al. Am J Physiol Gastrointest Liver Physiol 2008, 294:G199-207; Vilarinho, et al. Proc Natl Acad Sci U S A
2007, 104:18187-18192; Van Belle, et al. J Autoimmun 2013, 40:66-73; Lopez-Soto, et al. Int J
Cancer 2015, 136:1741-1750; and Urso, et al. US 9,127,064, each of which is herein incorporated by reference in it's entirety).
The preferred NKG2D-related peptides and compositions of this class comprise the domain A comprising the NKG2D modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating NKG2D (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524;
Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, all of which are herein incorporated by reference in their entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative NKG2D-related trifunctional peptides: NKG2D/TRIOPEP IA36 (IAVAMGIRFIIMVAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 27) and NKG2D/TRIOPEP 1E36 (IAVAMGIRFIIMVAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 28). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with NKG2D in the cell membrane and selectively disrupt intramembrane interactions of NKG2D chain with the DNAX-activation protein 10 (DAP-10) signaling homodimer, resulting to specific ligand-independent inhibition of NKG2D upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
and Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein incorporated by reference in their entirety). In one embodiment, methionine residues of NKG2D/TRIOPEP peptides are modified.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit NKG2D can be used to treat and/or prevent NKG2D-related diseases and conditions including but not limiting to, celiac disease, type I diabetes, hepatitis, and rheumatoid arthritis, and any other disorder where NKG2D cells are involved/recruited. In one embodiment, the present invention provides methods and compositions for preventing NK cell-mediated graft rejection.
In certain embodiments, the capability of the NKG2D-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with NKG2D can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the NKG2D/TRIOPEP sequences In one embodiment, imaging (visualization) of NKG2D levels using PET and/or other imaging techniques can be used to diagnose NKG2D-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
D. GPVI-Related Trifunctional Peptides In recent years, the central activating platelet collagen receptor, glycoprotein (GP) VI, has emerged as a promising antithrombotic target because its blockade or antibody-mediated depletion in circulating platelets was shown to effectively inhibit experimental thrombosis and thromboinflammatory disease states, such as stroke, without affecting hemostatic plug formation.
GPVI is a complex of a GPVI chain, which is responsible for ligand recognition, and FcRg homodimer, which is responsible for transmembrane signal transduction (see e.g. Sigalov.
Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov.
Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. J
Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692;
Dutting, et al. Trends Pharmacol Sci 2012, 33:583-590; Ungerer, et al. PLoS One 2013, 8:e71193;
Sigalov, US
8,278,271; Sigalov, US 8,614,188, all of which are herein incorporated by reference in their entirety). The binding of GPVI to collagen or other antagonists ligands induces platelet adhesion, activation and aggregation. Platelet activation is a step in the pathogenesis of ischemic cardio-and cerebrovascular diseases, which represent the leading causes of death and severe disability worldwide. Although existing antiplatelet drugs have proved beneficial in the clinic, their use is limited by their inherent effect on primary hemostasis, making the identification of novel pharmacological targets for platelet inhibition a goal of cardiovascular research.
The preferred GPVI-related peptides and compositions of this class comprise the domain A comprising the GPVI modulatory peptide sequences designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating GPVI (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative GPVI-related trifunctional peptides: GPVI/TRIOPEP GA32 (GNLVRICLGAPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 29) and GPVI/TRIOPEP
GE32 (GNLVRICLGAPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 30). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with GPVI in the cell membrane and selectively disrupt intramembrane interactions of GPVI chain with the FcRg signaling homodimer, resulting to specific ligand-independent inhibition of GPVI upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99;
Sigalov. J Thromb Haemost 2007, 5:2310-2312; Sigalov. Expert Opin Ther Targets 2008, 12:677-692, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of GPVI/TRIOPEP peptides are modified.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit GPVI can be used to treat and/or prevent GPVI-related diseases and conditions including but not limiting to, ischemic and thromboinflammatory diseases, and any other disorder where platelets are involved/recruited.
In certain embodiments, the capability of the GPVI-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with GPVI can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g. [64cu], see TABLE 2) can be conjugated to the GPVI/TRIOPEP sequences In one embodiment, imaging (visualization) of GPVI levels using PET and/or other imaging techniques can be used to diagnose GPVI-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
E. DAP-10- and DAP-12-Related Trifunctional Peptides The DAP10 and DAP12 signaling subunits are highly conserved in evolution and associate with a large family of receptors in hematopoietic cells, including dendritic cells, plasmacytoid dendritic cells, neutrophils, basophils, eosinophils, mast cells, monocytes, macrophages, natural killer cells, and some B and T cells. Some receptors are able to associate with either DAP10 or DAP12, which contribute unique intracellular signaling functions. DAP-10- and DAP-12-associated receptors have been shown to recognize both host-encoded ligands and ligands encoded by microbial pathogens, indicating that they play a role in innate immune responses. See e.g. Lanier. Immunol Rev 2009, 227:150-160; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224, all of which are herein incorporated by reference in their entirety.
The preferred DAP-10 and DAP-12-related peptides and compositions of this class comprise the domain A comprising the DAP-10 or DAP-12 modulatory peptide sequences, respectively, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating DAP-10- and associated receptors (see e.g., Sigalov. Self Nonself 2010, 1:4-39; Sigalov.
Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, each of which is herein incorporated by reference in it's entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP
structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219;
Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-10-related trifunctional peptides: DAP-10/TRIOPEP LA32 (LVAADAVASLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 33) and DAP-10/TRIOPEP LE32 (LVAADAVASLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 34).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-10-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-10 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of DAP-10/TRIOPEP
peptides are modified.
In certain embodiments, TABLE 2 demonstrates the following structures of representative DAP-12-related trifunctional peptides: DAP-12/TRIOPEP VA32 (VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 31) and DAP-12/TRIOPEP VE32 (VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 32).
While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with DAP-12-associated cell receptors in the cell membrane and selectively disrupt intramembrane interactions of the receptor with the DAP-12 signaling homodimer, resulting to specific ligand-independent inhibition of the receptor upon ligand stimulation (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, each of which is herein incorporated by reference in it's entirety). In one embodiment, methionine residues of DAP-12/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the DAP-10- and DAP-12-associated receptors, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the DAP-10- and DAP-12-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the DAP-10- and DAP-12-associated receptors, respectively, can be used to .. visualize (image) these receptors and evaluate their expression in the areas of interest. In one 64cut embodiment, for this purpose, an imaging probe (e.g. [
see TABLE 2) can be conjugated to the DAP-10/TRIOPEP and DAP-12/TRIOPEP sequences. In one embodiment, imaging (visualization) of levels of the DAP-10- and DAP-12-associated receptors using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
6. EGFR-Related Trifunctional Peptides The epidermal growth factor (EGF) receptor (EGFR) family, or ErbB family, is the best studied example of oncogenic receptor tyrosine kinases (RTKs). HER2/ErbB2 is overexpressed on the surface of 25-30% of breast cancer cells, and it has been associated with a high risk of relapse and death. EGFR amplification and mutations have been associated with many carcinomas. In particular, the EGFR pathway appears to play a role in pancreatic carcinoma. See e.g. Overholser, et al. Cancer 2000, 89:74-82; Bennasroune, et al. Mol Biol Cell 2004, 15:3464-3474; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov. Trends Immunol 2004, 25:583-589; Sigalov. Self Nonself 2010, 1:4-39; Sigalov. Self Nonself 2010, 1:192-224, each of which is herein incorporated by reference in it's entiretyy. Short hydrophobic peptides corresponding to the transmembrane domains of EGFR, ErB2 and insulin receptors inhibit specifically the autophosphorylation and signaling pathway of their cognate receptor (see Bennasroune, et al.
Mol Biol Cell 2004, 15:3464-3474, all of which are herein incorporated by reference in their entirety).
The preferred EGFR-related peptides and compositions of this class comprise the domain A comprising the EGFR modulatory peptide sequences, designed using a well-known in the art novel model of cell receptor signaling, the Signaling Chain HOmoOLigomerization model, capable of modulating EGFR (see e.g., Sigalov. Self Nonself 2010, 1:4-39;
Sigalov. Self Nonself 2010, 1:192-224; Sigalov. Trends Pharmacol Sci 2006, 27:518-524; Sigalov.
Trends Immunol 2004, 25:583-589; Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-9, all of which are herein incorporated by reference in their entirety). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US 20130045161; Shen, et al.
PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety).
As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In one embodiment, TREM-1 inhibitory SCHOOL peptide GF9 described herein is incorporated into SLP that contain apo A-I peptide fragments comprising 22 amino acid residue-long peptide sequences of the apo A-I helix 4 and/or helix 6. In one embodiment, the inclusion of an amphipathic apo A-I sequences in the peptides and compositions of the invention further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form lipopeptide particles (LP) that mimic native lipoproteins. It further aids the ability to cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB).
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative EGFR-related trifunctional peptides: EGFR/TRIOPEP SA47 (SIATGMVGALLLLLVVALGIGLFMRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO.
35) and EGFR/TRIOPEP 5E47 (SIATGMVGALLLLLVVALGIGLFMRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO.
36). While not being bound to any particular theory, it is believed that in one embodiment, these peptides colocalize with EGFR in the cell membrane and selectively disrupts intramembrane interactions between the receptors, resulting to specific ligand-independent inhibition of the receptor (see e.g. Sigalov. Self Nonself 2010, 1:4-39.; Sigalov. Self Nonself 2010, 1:192-224;
Sigalov. Adv Protein Chem Struct Biol 2018, 111:61-99, all of which are herein incorporated by reference in their entirety). In one embodiment, methionine residues of EGFR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the EGFR and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit the receptors of the EGFR and/or ErB receptor families, respectively, can be used to treat and/or prevent any diseases and conditions where these receptors are involved.
In certain embodiments, the capability of the EGFR- and/or ErB-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with the receptors of the EGFR and/or ErB receptor families can be used to visualize (image) these receptors and evaluate their expression in the areas of interest. In one embodiment, 64co for this purpose, an imaging probe (e.g. [ can be conjugated to the EGFR/TRIOPEP
sequence. In one embodiment, imaging (visualization) of levels of the receptors of the EGFR
and/or ErB receptor families using PET and/or other imaging techniques can be used to diagnose any diseases and conditions where these receptors are involved as well as to monitor novel therapies for these diseases and conditions.
F. Additional Trifunctional Peptides Additional therapeutic peptide sequences (see e.g., Vlieghe, et al. Drug Discov Today 2010, 15:40-56; Tsung, et al. Shock 2007, 27:364-369; Chang, et al. PLoS One 2009, 4:e4171;
Tjin Tham Sjin, et al. Cancer Res 2005, 65:3656-3663; Ladetzki-Baehs, et al.
Endocrinology 2007, 148:332-336; Khan, et al. Hum Immunol 2002, 63:1-7; Banga. Therapeutic peptides and proteins: formulation, processing, and delivery systems. 2nd ed. Boca Raton, FL: Taylor &
Francis Group; 2006; Stevenson. Curr Pharm Biotechnol 2009, 10:122-137; Wu and Chi, US
9,387,257; Wu, et al., US 8,415,453; Faure, et al., US 8,013,116; Faure, et al., US 9,273,111;
Eggink and Hoober, US 7,811,995; Eggink and Hoober, US 8,496,942; Morgan and Pandha. US
2012/0177672 Al; Broersma, et al., US 5,681,925, each of which is herein incorporated by reference in it's entirety) and/or other therapeutic agents can comprise the domain A of the peptides and compositions of the present invention.
In one embodiment, this domain comprises the Toll Like Receptor (TLR) modulatory sequence (see e.g. Tsung, et al. Shock 2007, 27:364-369, herein incorpoated by referene in it's entirety). The preferred peptides and compositions of this class further comprise the domain B
comprising at least one modified or unmodified amphipathic apo A-I and/or A-II
peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP
structures that can be spherical or discoidal (described herein and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov. Int Immunopharmacol 2014, 21:208-219; Sigalov. US
20110256224;
Sigalov. US 20130045161; Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov.
Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, each of which is herein incorporated by reference in it's entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP
and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB
and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative TLR-related trifunctional peptides: TLR/TRIOPEP DA32 (DIVKLTVYDCIRRRRRRRRRPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 37) and TLR/TRIOPEP DE32 (DIVKLTVYDCIRRRRRRRRRPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 38). In one embodiment, methionine residues of TLR/TRIOPEP
peptides are modified.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit TLR can be used .. to treat and/or prevent TLR-related diseases and conditions including but not limiting to, sepsis and other infectious diseases, and any other disorder where TLR receptors are involved.
In certain embodiments, the capability of the TLR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with TLR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the TLR/TRIOPEP sequences In one embodiment, imaging (visualization) of TLR levels using PET
and/or other imaging techniques can be used to diagnose TLR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In one embodiment, the domain A of the peptides and compositions of the invention comprises the Atrial Natriuretic Peptide (ANP) receptor (ANPR)-modulatory sequence (see e.g.
Ladetzki-Baehs, et al. Endocrinology 2007, 148:332-336). The preferred peptides and compositions of this class further comprise the domain B comprising at least one modified or unmodified amphipathic apo A-I and/or A-II peptide fragment to form upon interaction with lipid and/or lipid mixtures, SLP structures that can be spherical or discoidal (described herein .. and in e.g., Sigalov. Contrast Media Mol Imaging 2014, 9:372-382; Sigalov.
Int Immunopharmacol 2014, 21:208-219; Sigalov. US 20110256224; Sigalov. US
20130045161;
Shen, et al. PLoS One 2015, 10:e0143453; Shen and Sigalov. Sci Rep 2016, 6:28672; Shen and Sigalov. J Cell Mol Med 2017, 21:2524-2534; Shen and Sigalov. Mol Pharm 2017, 14:4572-4582; Rojas, et al. Biochim Biophys Acta 2018, 1864:2761-2768, all of which are herein incorporated by reference in their entirety). As described above, the inclusion of an amphipathic apo A-I sequences aids the assistance in the self-assembly of SLP and the structural stability of the particle formed, particularly when the particle has a discoidal shape. It further aids the ability to provide targeted delivery to the cells of interest. It further aids the ability to interact with lipids and/or lipoproteins in a bloodstream in vivo and form LP that mimic native lipoproteins. It further aids the ability to cross the BBB, BRB and BTB.
In certain embodiments, exemplary trifunctional peptides comprise the domain B
comprises with the amino acid sequence selected from the amino acid sequences of the major HDL protein constituent, apo A-I. In certain embodiments, this sequence comprises 22 amino acid residue-long peptide sequence of the apo A-I helix 4. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide. In one embodiment, the domain B of the peptides and compositions of the invention comprises 22 amino acid residue-long peptide sequence of the apo A-I
helix 6. In one embodiment, this sequence contains a modified amino acid residue. In one embodiment, this modified amino acid residue is methionine sulfoxide.
In certain embodiments, TABLE 2 demonstrates the following structures of representative ANPR-related trifunctional peptides: ANPR/TRIOPEP SA50 (SLRRS SCFGGRMDRIGAQ SGLGCNSFRYPLGEEMRDRARAHVDALRTHLA) (SEQ ID
NO. 39) and ANPR/TRIOPEP SE50 (SLRRSSCFGGRMDRIGAQSGLGCNSFRYPYLDDFQKKWQEEMELYRQKVE) (SEQ ID
NO. 40). In one embodiment, methionine residues of ANPR/TRIOPEP peptides are modified.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to inhibit ANPRs can be used to treat and/or prevent ANPR-related diseases and conditions including but not limiting to, cardiovascular and inflammatory diseases, and any other disorder where ANP
receptors are involved.
In certain embodiments, the capability of the ANPR-related peptides and compounds of the present invention including but not limiting to those described above, to colocalize with ANPR can be used to visualize (image) this receptor and evaluate its expression in the areas of interest. In one embodiment, for this purpose, an imaging probe (e.g.
[64Cu]) can be conjugated to the ANPR/TRIOPEP sequences In one embodiment, imaging (visualization) of ANPR levels using PET and/or other imaging techniques can be used to diagnose ANPR-related diseases and conditions as well as to monitor novel therapies for these diseases and conditions.
In certain embodiments, other therapeutic agents including but not limiting to, to those described in Page and Takimoto. Principles of chemotherapy. In: Pazdur R, Wagman LD, Camphausen KA, editors. Cancer Management: A Multidisciplinary Approach. 11th ed.
Manhasset, NY: Cmp United Business Media; 2009. p. 21-37; Sipsas, et al., Therapy of Mucormycosis, J Fungi (Basel) 2018, 4; Lin, et al. Chem Commun (Camb) 2013, 49:4968-4970;
and in Turner, et al. Peptide Conjugates of Oligonucleotide Analogs and siRNA
for Gene Expression Modulation. In: Langel U, ed, editor. Handbook of Cell-Penetrating Peptides. 2nd edition ed. Boca Raton: CRC Press; 2007. p. 313-328 and disclosed in Schiffman and Altman, US 4,427,660; Castaigne, et al., US 9,161,988; Castaigne, et al., US
8,921,314; and in Castaigne, et al., US 9,173,891, all of which are herein incorporated by reference in their entirety, (see also TABLE 2) can comprise the domain A of the peptides and compositions of the present invention.
V. Diseases Contemplated For Treatment Using Peptides and Compositions Described Herein.
A. Overview.
The present invention encompasses the recognition that it is possible to produce compositions that possess the advantages typically associated with a fully synthetic material and yet also possess certain desirable features of materials derived from natural sources.
In some embodiments, peptides and compounds of the present invention, e.g.
trifunctional peptides, with rHDLs (including discoidal and/or spherical HDLs) or without rHDLs (such as in therapeitic compositions as free trifunctional peptides), are contemplated for use in preventative treatments for diseases associated with activated macrophages and/or T-cells, in particular for preventing one or more symptoms associated with the disease.
In some embodiments, peptides and compounds of the present invention are contemplated for use preventative treatments for diseases associated with activated macrophages and/or T-cells, in particular for reducing one or more symptoms associated with the disease. In some embodiments, peptides and compounds of the present invention are contemplated for use diagnostic applications for detecting/identifying; determining disease progression; determining results of disease treatment, for diseases associated with activated macrophages and/or T-cells.
Such diseases associated with activated macrophages and/or T-cells include but are not limited to including but not limited to lung cancer, such as non small-cell lung cancer (NSCLC); pancreatic cancer (PC); glioblastoma multiforme (GBM, or brain cancer), with or without radiation therapy;
breast cancer with or without radiation therapy; sepsis; retinopathy;
rheumatoid arthritis (RA);
sepsis; and alcoholic liver disease (ALD). Furthermore, diseases associated with activated macrophages and/or T-cells include but are not limited to 1) Alcohol-induced neuroinflammation .. and brain damage; 2) Radiation-induced multiple organ dysfunction syndrome;
3) Scleroderma;
4) Atopic dermatitis; 5) Atherosclerosis; 6) Alzheimer's, Parkinson's and/or Huntington's diseases. In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, liver diseases, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other rheumatic diseases, sepsis and other inflammatory diseases or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, including but not limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases.
Thus, in some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a disease symptom. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for delaying onset of a disease symptom. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for preventing a disease symptom. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject receiving therapy for a disease. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject receiving anti-cancer therapy.
In some embodiments, trifunctonal peptides as described herein, are contemplated for administration to a subject as anti-cancer therapy. In some embodiments, trifunctonal peptides as described herein, further comprising a drug compound are contemplated for administration to a subject as anti-cancer therapy. In some embodiments, trifunctonal peptides as described herein, further comprising a Paclitaxel compound are contemplated for administration to a subject as anti-cancer therapy.
As disease progression of a liver in a subject proceeds from epatosteatosis, steatohepatitis, and fibrosis to cirrhosis, it is contemplated that a trifunctonal peptide as described herein, is administered to said subject at any point along the disease progression for reducing disease progression, in part as described herein. Thus, in some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a liver disease symptom, including but not limited to reducing one or more of ALT, procollegen I-alpha and alpha-SMA.
In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a liver disease symptom, in combination with one or more of steroid drugs, ursodiol, etc., in order to delay or prevent further progression of liver .. degeneration to cirrhosis. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for reducing a liver disease symptom in combination with one or more of steroid drugs, ursodiol, etc., in order to improve function of a diseased liver.
In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for treating severe hemorrhagic shock. In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for treating colitis and colitis-associated tumorigenesis.
In some embodiments, trifunctonal peptides as described herein are contemplated for administration to a subject for decreasing neovascularization.
In some embodiments, trifunctonal peptides as described herein are selected from the group consisting of G-KV21, G-HV21, G-TE21, M-VE32 and M-TK32, and mixtures thereof. In some embodiments, a trifunctonal peptide as described herein is GE31. In some embodiments, a trifunctonal peptide as described herein is GA31. In some embodiments, a trifunctonal peptide as described herein is a mixture of GE31 and GA31.
FIG. 4B illustrates a hypothesized molecular mechanism of action of one embodiment of a trifunctional peptide (TRIOPEP) of the present invention comprising amino acid domains A
and B where domain A is a 9 amino acids-long TREM-1 inhibitory therapeutic peptide sequence and functions to treat and/or prevent a TREM-1-related disease or condition (shown for cancer), whereas domain B is a 22 amino acids-long apolipoprotein A-I helix 4 or 6 peptide sequence with a sulfoxidized methionine residue and functions to assist in the self-assembly of synthetic lipopeptide particles (SLP) and to target the particles to TREM-1-expressing macrophages as applied to the treatment and/or prevention of cancer. While not being bound to any particular theory, it is believed that chemical and/or enzymatic modification of protein sequence in domain B leads to the recognition of SLP of the present invention by the macrophage scavenger receptors and results in an irreversible binding to and consequent uptake by macrophages of such particles. It is further believed that accumulation of these particles in tumor-associated macrophages is accompanied by accumulation of TRIOPEP in these cells. In contrast, native HDL particles that contain only unmodified apolipoprotein molecules are not recognized by tumor-associated macrophages and return to the circulation.
FIG. 4C shows a symbol key used in FIGS. 4A-B.
B. Cancer.
Approximately 8.8 million people are dying each year of cancer, amounting to one out of six deaths globally, and cancer incidence is estimated to double by 2035 (Prager et al.
2018). Combination-therapy treatments for cancer have become more common, in part due to the perceived advantage of attacking the disease via multiple avenues. Although many effective combination-therapy treatments have been identified over the past few decades;
in view of the continuing high number of deaths each year resulting from cancer, a continuing need exists to identify effective therapeutic regimens for use in anticancer treatment.
The present invention encompasses the recognition that it is possible to prevent and treat different types of cancer including but not limited to, pancreatic cancer, multiple myeloma, leukemia, prostate cancer, breast cancer, liver cancer, bladder cancer, colorectal cancer, lung cancer, CNS cancer, melanoma, ovarian cancer, renal cancer, or osteosarcoma and other cancers, and cancer cachexia by blocking the TREM-1 signaling pathway using the peptide variants and compositions that possess the advantages typically associated with a fully synthetic material and yet possess certain desirable features of materials derived from natural sources. The invention further encompasses the recognition that it is possible to use imaging techniques and the peptide variants and compositions of the invention conjugated to an imaging probe for detecting the labeled probe in an individual with cancer, wherein the location of the labeled probe corresponds to at least one symptom of the myeloid cell-related condition. The invention further encompasses that it is possible to predict the efficacy of the peptides and compositions of the invention by determining the number of myeloid cells in the biological sample from the individual with cancer and determining the expression levels of TREM-1 in the cells contained within the biological sample.
Cancer continues to have a huge Social and economic impact. In 2011, 571,950 Americans died of cancer (-25% of all deaths), with US cancer-associated costs of S263.8 billion: S102.8 billion for direct medical costs (total health expenditures);
$20.9 billion for indirect morbidity costs (lost productivity); and S140.1 billion for indirect mortality costs (lost productivity from premature death).
Inflammatory responses play decisive roles at different stages of tumor development, including initiation, promotion, malignant conversion, invasion, and metastasis (Grivennikov et al. 2010). Inflammation also affects immune surveillance and responses to therapy (Grivennikov et al. 2010). Many solid tumors are characterized by a marked infiltration of macrophages, inflammatory cells, into the stromal compartment (Shih et al. 2006, Solinas et al. 2009), a process which is mediated by cancer-associated fibroblasts (CAFs) and plays a key role in disease progression and its response to therapy (see FIG. 49). These tumor-associated macrophages (TAMs) secrete a variety of growth factors, cytokines, chemokines, and enzymes .. that regulate tumor growth, angiogenesis, invasion, and metastasis (Shih et al. 2006). See FIG.
49. High macrophage infiltration correlates with the promotion of tumor growth and metastasis development (Solinas et al. 2009, Grivennikov et al. 2010). In patients with PC, macrophage infiltration begins during the preinvasive stage of the disease and increases progressively (Clark et al. 2007). The number of TAMs is significantly higher in patients with metastases (Gardian et al. 2012). TREM-1 is upregulated in cancer and its overexpression correlates with survival of cancer patients. In NSCLC, TREM-1 expression in TAMs is associated with cancer recurrence and poor survival of patients with NSCLC: patients with low TREM-1 expression have a 4-year survival rate of over 60%, compared with less than 20% in patients with high expression (Ho et al. 2008). Activation of the TREM-1/DAP-12 signaling pathway results in release of multiple cytokines, chemokines and growth factors most of which are increased in cancer patients and their upregulation correlates with poor prognosis (See FIG. 1).
The present invention encompasses the recognition that it is possible to prevent and treat different types of cancer in which myeloid cells are involved or recruited and cancer cachexia by combining cancer therapies with a therapeutically effective amount of at least one compound and/or composition ("modulator") which affects myeloid cells by action on the signaling pathway.
The infiltrate of most solid tumors contains tumor-associated macrophages (TAMs) that are attracted by chemokines including CCL2 and represent attractive treatment targets in oncology (Shih et al. 2006, Mantovani et al. 2017). The increased TAM content in NSCLC
(Yusen et al. 2018) is associated with poor prognosis in NSCLC (Welsh et al.
2005). TAM
recruitment, activation, growth and differentiation are regulated by CSF-1 (Elgert et al. 1998, Laoui et al. 2014). Many tumor cells or tumor stromal cells have been found to produce CSF-1, which activates monocyte/macrophage cells through CSF-1 receptor (CSF-1R). The level of CSF-1 in tumors has been shown to correlate with the level of TAMs in the tumor. Higher levels of TAMs have been found to correlate with poorer patient prognoses in the majority of cancers.
Increased pretreatment serum CSF-1 is a strong independent predictor of poor survival in NSCLC (Baghdadi et al. 2018). In addition, CSF-1 has been found to promote tumor growth and progression to metastasis in, for example, human breast cancer xenografts in mice (Paulus et al.
2006). Further, CSF-1R plays a role in osteolytic bone destruction in bone metastasis (Ohno et al. 2006). TAMs promote tumor growth, in part, by suppressing anti-tumor T
cell effector function through the release of immunosuppressive cytokines and the expression of T cell inhibitory surface proteins. Blockade of CSF-1 or CSF-1R not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota et al. 2009) but also improves response to T-cell checkpoint immunotherapies that target programmed cell death protein 1 (PD-1), programmed death-ligand 1 (PD-L1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu et al. 2014).
Importantly, continuous CSF-1 inhibition affects pathological angiogenesis but not healthy vascular and lymphatic systems outside tumors (Kubota et al. 2009). In contrast to blockade of vascular endothelial growth factor (VEGF), interruption of CSF-1 inhibition does not promote rapid vascular regrowth (Kubota et al. 2009).
The present invention provides a method of treating these and other types of cancers by using modulators of the TREM-1/DAP-12 signaling pathway that are capable of binding TREM-1 and modulating TREM-1/DAP-12 receptor complex activity in combination-therapy treatments together with other cancer therapies. The invention further provides the methods for predicting response of a cancer patient to the treatment by using these modulators in combination-therapy regimen. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
The invention further encompasses the recognition that it is possible to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regimen by: (a) obtaining a biological sample from the subject; (b) determining the expression of CSF-1, CSF-1R, IL-6, TREM-1 and/or number of CD68-positive .. TAMs or a combination thereof, wherein the higher is the expression of CSF-1, CSF-1R, IL-6, TREM-1 or the higher is number of CD68-positive TAMs or a combination thereof, the better the patient is predicted to respond to a therapy that involves the modulators.
The invention further encompasses the recognition that it is possible to use imaging techniques and the modulators conjugated to an imaging probe for detecting the labeled probe in an individual with cancer in which myeloid cells are involved or recruited, wherein the location and the measured intensity of the labeled probe can diagnose cancer and/or predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway, the higher the measured intensity of the labeled probe, the better the patient is predicted to respond to a therapy that involves the modulators.
1. Lung Cancer.
Lung cancer, including NSCLC, is the leading cause of cancer deaths worldwide (Wong et al. 2018) and has a poor prognosis. Despite advances made in chemotherapy, NSCLC is responsible for over 1.1 million deaths annually worldwide, and the 5-year survival rate for patients with NSCLC is reported to be only 15% or less than 18% (Zappa et al.
2016), showing .. an urgent need for new therapies.
FIG. 11A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer (NSCLC) H292 (FIG. 11A) and A549 (FIG. 11B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form. PTX, paclitaxel.
****, P < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 12A-B presents the exemplary data showing inhibition of tumor growth in the human non-small cell lung cancer H292 (FIG. 12A) and A549 (FIG. 12B) xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel. ****,p < 0.0001 as compared with vehicle-treated animals.
FIG. 13 presents the exemplary data showing average tumor weights in the A549 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. PTX, paclitaxel.
<0.0001 as compared with vehicle-treated animals.
2. Pancreatic cancer.
Pancreatic cancer (PC, 85% of which are pancreatic ductal adenocarcinomas, PDAC) is the fourth leading cause of cancer-related mortality across the world with very poor clinical outcome. (Ilic et al. 2016). Current treatments of PC marginally prolong survival or relieve symptoms in patients with PC (Ilic and Ilic 2016). There has been no significant progress in the field of targeted therapy for PC (Walker et al. 2014) and despite tremendous efforts, the 5-year survival rate remains less than 5% (Ilic and Ilic 2016).This highlights the urgent need for novel approaches to prevent and treat PC and other types of cancer. However, it should be noted that the techniques and compositions listed and described herein are applicable to a broad range of other types of cancer and cancer cachexia. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
Current treatments of PC marginally prolong survival or relieve symptoms in patients with PC (Schneider 2005). There has been no significant progress in the field of targeted therapy for PC (Walker and Ko 2014) and despite tremendous efforts, the 5-year survival rate remains less than 5% (2010).
3. Additional Neoplasms: Giant Cell Tumor and PVNS.
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the inflammatory response (Colonna et al. 2003) and is upregulated under inflammatory conditions including .. cancer (Wang et al. 2004). For downstream signal transduction, TREM-1 is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor, DNAX
activation protein of 12kDa (DAP12). TREM-1/DAP-12 receptor complex activation enhances release of multiple cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNFa), interleukin-la (IL-1a), IL-1I3, IL-6 and colony-stimulating factor 1 (referred to herein as CSF1; also referred to in the art as M-CSF) (Schenk et al. 2007, Lagler et al. 2009, Sigalov 2014).
Binding of CSF1 or the interleukin 34 ligand (referred to herein as IL-34) to receptor (referred to herein as CSF1R) leads to receptor dimerization, upregulation of CSF1R
protein tyrosine kinase activity, phosphorylation of CSF1R tyrosine residues, and downstream signaling events. CSF1R activation by CSF1 or IL-34 leads to the trafficking, survival, proliferation, and differentiation of monocytes and macrophages, as well as other monocytic cell lineages such as osteoclasts, dendritic cells, and microglia.
Many tumor cells or tumor stromal cells have been found to produce CSF1, which activates monocyte/macrophage cells through CSF1R. The level of CSF1 in tumors has been shown to correlate with the level of tumor-associated macrophages (TAMs) in the tumor. Higher levels of TAMs have been found to correlate with poorer patient prognoses in the majority of cancers. In addition, CSF1 has been found to promote tumor growth and progression to metastasis in, for example, human breast cancer xenografts in mice (Paulus et al. 2006). Further, CSF1R plays a role in osteolytic bone destruction in bone metastasis (Ohno et al. 2006). TAMs promote tumor growth, in part, by suppressing anti-tumor T cell effector function through the release of immunosuppressive cytokines and the expression of T cell inhibitory surface proteins.
Blockade of CSF1 or CSF1R not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota et al. 2009) but also improves response to T-cell checkpoint immunotherapies that target programmed cell death protein 1 (PD1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu et al. 2014). Importantly, continuous CSF1 inhibition affects pathological angiogenesis but not healthy vascular and lymphatic systems outside tumors (Kubota et al. 2009). In contrast to blockade of vascular endothelial growth factor (VEGF), interruption of CSF1 inhibition does not promote rapid vascular regrowth (Kubota et al. 2009).
Giant cell tumor of the tendon sheath (GCTTS), tenosynovial giant cell tumor (TGCT;
also referred to in the art as TSGCT), and pigmented villonodular synovitis (PVNS) are the common names for a group of rare proliferative disorders that involve synovial joints and tendon sheaths. PVNS is a solid tumor of the synovium with features of both reactive inflammation and clonal neoplastic proliferation in which CSF1 is over expressed. A common translocation of the CSF1 gene (1p13) to the COL6A3 promoter (2q35) is present in approximately 60%
of PVNS
patients. The translocation is accompanied by CSF1 overexpression in the synovium. In addition, approximately 40% of PVNS patients have CSF1 overexpression in the absence of an identified CSF1 translocation. The consistent presence of CSF1 overexpression in all cases of PVNS and reactive synovitis suggests both an important role for CSF1 in the spectrum of synovial pathologies and the utility of targeting the CSF1/CSF1R signaling pathway therapeutically (West et al. 2006). In PVNS, CSF1 overexpression is present in a minority of synovial cells, whereas the majority of the cellular infiltrate expresses CSF1R but not CSF1. This has been characterized as a tumor-landscaping effect with aberrant CSF1 expression in the tumor cells, leading to the abnormal accumulation of non-neoplastic cells that form a mass.
Surgery is the treatment of choice for patients with localized PVNS.
Recurrences occur in 8-20% of patients and are often managed by re-excision. Diffuse tenosynovial giant cell tumor (TGCT/PVNS or PVNS/dtTGCT) tends to recur more often (33-50%) and has a much more aggressive clinical course. Patients are often symptomatic and require multiple surgical procedures during their lifetime and even amputation. For patients with unresectable disease or multiple recurrences, systemic therapy using CSF1R inhibitors may help delay or avoid surgical procedures and improve functional outcomes (Radi et al. 2011).
Imatinib, a non-specific inhibitor of CSF1R, has undergone evaluation in PVNS
patients (Cassier et al. 2012). Twenty-nine patients from 12 institutions in Europe, Australia, and the United States were included. The median age was 41 years and the most common site of disease was the knee (n=17; 59%). Two patients had metastatic disease to the lung and/or bone. Five of 27 evaluable patients had complete (n=1) or partial (n=4) responses per RECIST
for an overall response rate of 19%. Twenty of 27 patients (74%) had stable disease.
Symptomatic improvement was noted in 16 of 22 patients (73%) who were assessable for symptoms. Despite a high rate of symptomatic improvement and an overall favorable safety profile, 10 patients discontinued treatment for toxicity or other reasons.
Pexidartinib (PLX3397), a potent, selective oral CSF1R inhibitor, that traps the kinase in the autoinhibited conformation, has undergone evaluation in TGCT patients (Tap et al. 2015). A
total of 41 patients were enrolled in the dose-escalation study, and an additional 23 patients were enrolled in the extension study. In the extension study, 12 patients with TGCTs had a partial response and 7 patients had stable disease. The most common adverse events included fatigue, change in hair color, nausea, dysgeusia, and periorbital edema; adverse events rarely led to discontinuation of treatment. Despite treatment of TGCTs with PLX3397 resulted in a prolonged regression in tumor volume in most patients of this Phase 2 study, later the Phase 3 study was suspended after two reported cases of nonfatal, serious liver toxicity.
Anti-CSF1R antibodies alone or in combination with antibodies against PD1 or against PDL1, one of the ligands for PD1, were proposed as less toxic alternative treatments for PVNS.
See, e.g., US Pat 10,040,858 B2 and US Pat 10,221,224. As with most combination therapies, the promise of increased clinical activity is accompanied by the risk of additive toxicity and therefore requires careful assessment.
Liver enzyme elevations can be considered a class effect of CSF1R-targeting compounds (Cannarile et al. 2017). In addition, the oversuppression of the CSF1/CSF1R
signaling pathway may result in potential serious long term adverse effects (AEs). In animals, CSF1 deficiency results in a range of developmental abnormalities, including skeletal, neurological, growth and fertility defects (Michaelson et al. 1996, Hume et al. 2012, Jones et al.
2013).
Thus, PVNS is a rare, locally aggressive neoplasm of the joint or tendon sheath with features of both reactive inflammation and clonal neoplastic proliferation in which CSF-1 is over expressed (Tap et al. 2015). Surgical resection is the primary treatment;
however, diffuse TGCT
is more difficult to resect and often involves total synovectomy, joint replacement, or amputation (Tap et al. 2015). There are no approved systemic therapies. Therefore, alternative, less toxic and more targeted treatments for PVNS are needed.
Inhibition of TREM-1 lowers levels of proinflammatory cytokines including CSF1 and is a promising approach in a variety of inflammation-associated disorders including cancer (Colonna and Facchetti 2003, Schenk et al. 2007, Pelham et al. 2014, Sigalov 2014, Shen et al.
2017, Shen et al. 2017, Rojas et al. 2018). In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Treml-/- mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines.
Treml-/- mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major (Weber et al. 2014). Furthermore, reduced morbidity was observed for influenza virus-infected Treml-/- mice (Weber et al. 2014).
Importantly, while immune-associated pathologies were significantly reduced, Treml-/- mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Treml+/+ controls (Weber et al. 2014). Humans lacking DAP-12 do not have problems resolving infections with viruses or bacteria (Lanier 2009). Collectively, these findings suggest that in contrast to single cytokine blockers including CSF1 and CSF1R
blockers, therapeutic blocking of TREM-1/DAP-12 signaling in distinct inflammatory disorders including CSF1-dependend TGCTs holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
The present invention provides a method of using the well-tolerable TREM-1/DAP-modulatory peptides and compositions for treatment of PVNS. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
Methods of treating tenosynovial giant cell tumor (TGCT) or pigmented villonodular synovitis (PVNS) with peptide variants and compositions that modulate activity of the receptor complex formed by triggering receptor expressed on myeloid cells 1 (TREM-1) and DNAX
activation protein of 12kDa (DAP12) are provided.
Inhibition of TREM-1 lowers levels of proinflammatory cytokines including CSF1 and is a promising approach in a variety of inflammation-associated disorders including cancer (Colonna and Facchetti 2003, Schenk et al. 2007, Pelham et al. 2014, Sigalov 2014, Shen et al.
2017, Shen et al. 2017, Rojas et al. 2018). In CD4+ T cell- and dextran sodium sulfate-induced models of colitis, Treml-/- mice displayed significantly attenuated disease that was associated with reduced inflammatory infiltrates and diminished expression of pro-inflammatory cytokines.
Treml-/- mice also exhibited reduced neutrophilic infiltration and decreased lesion size upon infection with Leishmania major (Weber et al. 2014). Furthermore, reduced morbidity was observed for influenza virus-infected Treml-/- mice (Weber et al. 2014).
Importantly, while immune-associated pathologies were significantly reduced, Treml-/- mice were equally capable of controlling infections with L. major, influenza virus, but also Legionella pneumophila as Treml+/+ controls (Weber et al. 2014). Humans lacking DAP-12 do not have problems resolving infections with viruses or bacteria (Lanier 2009). Collectively, these findings suggest that in contrast to single cytokine blockers including CSF1 and CSF1R
blockers, therapeutic blocking of TREM-1/DAP-12 signaling in distinct inflammatory disorders including CSF1-dependend TGCTs holds considerable promise by blunting excessive inflammation while preserving the capacity for microbial control.
The present invention provides a method of using the well-tolerable TREM-1/DAP-modulatory peptides and compositions for treatment of PVNS. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
4. Liver Cancer.
Globally, liver cancer is the fifth commonest cancer in 2012, accounting for 9.1% of all cancer deaths worldwide with the overall 5-year relative survival rate for patients with liver cancer of 17%. Owing to its extremely aggressive nature and poor survival rate, it remains an important public health issue worldwide (Wong et al. 2017) 5. Breast Cancer.
Breast cancer is the most common malignancy in women around the world (Ghoncheh et al. 2016). Ii is the most common cancer in women, accounting for 25.1% of all cancers. Breast cancer incidence in developed countries is higher, while relative mortality is greatest in less developed countries (Ghoncheh et al. 2016). Despite significant improvements in clinical outcomes within the field of breast cancer in the last 50 years, the triple-negative breast cancer (TNBC) subtype remains an area of huge unmet clinical need (Partridge et al.
2017).
6. Glioblastoma.
Glioblastoma Multiforme (GBM) is the most common and lethal type of brain cancer (Shergalis et al. 2018). For adults with GBM, treated with standard first-line therapy ¨
concurrent radiation and temozolomide (TMZ) therapy followed by TMZ
monotherapy, the median survival is about 14.6 months (Grossman et al. 2010, Shergalis et al.
2018). Little progress has been made over the past several decades in the treatment of GBM, highlighting an urgent need for new therapies.
7. Colorectal Cancer.
Colorectal cancer (CRC) has a considerable impact on patients and healthcare systems in developed countries and around 25% of patients present with metastatic disease that significantly impacts on prognosis (Van Cutsem et al. 2013). For those with localized CRC of stages I and II, the 5-year survival rate is as high as 93%, declining to 60%, 42% and 25% for patients with stages IIIA, IIIB and IIIC, respectively. However, most patients with metastatic CRC (stage IV) are not curable, with the 5-year survival rate falling to less than 10%. While early diagnosis of CRC in recent years combined with advances in treatment has considerably improved survival, management of the disease remains challenging and further progress is needed (Van Cutsem et al. 2013).
Scleroderma, related autoimmune conditions and fibrotic conditions.
It is estimated that scleroderma or systemic sclerosis (SSc) affects 100,000-300,000 Americans, predominantly young to middle aged women. Systemic sclerosis is a progressive and untreatable disease of unknown cause and high mortality. Fibrosis in SSc resembles uncontrolled .. wound healing, where healing occurs by intractable fibrosis rather than normal tissue regeneration.
It is believed that SSc is associated with the highest case-fatality rates among the rheumatic diseases or connective tissue diseases. Currently, there are no validated biomarkers for diagnosis. Furthermore, no effective disease-modifying therapies are currently available. In fact, .. while some treatment can alleviate the pain associated with SSc, to date no therapy has been shown to significantly alter survival. The pathogenesis of SSc is characterized by early vascular injury, with inflammation followed by progressive tissue damage and fibrosis.
Excessive production of collagen and ECM and accumulation of myofibroblasts in lesional tissues are believed to be responsible for progressive organ failure. Pathological fibrosis resembles a normal wound healing response that has become deregulated. It is estimated that fibrosis accounts for>25% of all deaths in the U.S. Thus, fibrosis represents one of the major unmet medical needs.
Accordingly, there is a need for an effective anti-fibrotic therapy.
Project Summary/Abstract Scleroderma that includes localized scleroderma (LS) and systemic sclerosis (SSc) is a rare but devastating autoimmune disorder. Current therapies all have side effects, are limited and associate with 10 year survival of 55%, showing the need for novel approaches.
The long-term goal of this project is to develop a new mechanism-based, efficient and well tolerable scleroderma therapy.
Triggering receptor expressed on myeloid cells 1 (TREM-1), an inflammation amplifier, contributes to the development of fibrosis in SSc. In patients, number of activated macrophages in the fibrotic areas is increased and associates with fibrosis severity.
Activation of TREM-1 leads to overproduction of MCP-1/CCL2 and M-CSF/CSF-1, resulting in macrophage recruitment to an injured area and the sclerotic lesion formation in rats with scleroderma. In animal models, TREM-1 blockade inhibits inflammation and ameliorates a variety of autoimmune diseases. The hypothesis of the "proof-of-concept" Phase I is that blockade can prevent and treat scleroderma.
Current TREM-1 inhibitors all attempt to block binding of TREM-1 to its still uncertain ligand(s). To minimize risk of failure in clinical development, we developed a first-in-class ligand-independent TREM-1 inhibitory peptide GF9 that is well-tolerated and can be formulated into SignaBlok's long half-life macrophage-specific lipopeptide complexes (LPC) to improve its half-life and targeting to the inflammation areas. The major goal of the Phase I study is to show that TREM-1 blockade by GF9-LPC alleviates the disease in a bleomycin (BLM)-induced mouse model of scleroderma.
Phase I specific aims are to: 1) optimize TREM-1 inhibitory compositions for their functionality in vitro and pharmacokinetics in vivo and select the lead, 2) test two doses of the lead selected in a BLM-induced mouse model of scleroderma. We will generate, optimize and select the lead based upon its functionality in vitro and its PK profile in vivo. We will test two doses of the lead for its ability to prevent and treat lung, heart, muscle and skin fibrosis in a mouse model of multiorgan fibrosis in vivo. Histology/IHC studies will be performed. Serum and tissue cytokines will be evlauated, nonlmiting examples including MCP-1, CSF-1, VEGF, TGF-beta, TNF-alpha, IL-6, and IL-1-beta, will be analyzed.
It is anticipated that the Phase I study will identify a novel, first-in-class, well tolerable agent as a powerful platform for development of an effective and well-tolerable systemic scleroderma therapy, thereby improving treatment and survival of patients. Its anticipated safety is supported by good tolerability of SignaBlok's GF9-based formulations by long term-treated mice. Prototypes of SignaBlok's LPC are well tolerated in humans. TREM-1 blockade by SignaBlok competitor's inhibitory peptide LR12 (Inotrem, France) was safe in healthy and septic subjects. If successful, Phase I will be followed in Phase II by toxicology, ADME, pharmacology and CMC studies, filing an IND and subsequent evaluation in humans.
Project Narrative.
Scleroderma (also known as systemic sclerosis) is a rare autoimmune disorder that affects about 20 to 24 people per million population in the US each year, with the majority being women of childbearing age. There is no approved drug for scleroderma. Current therapies all have side effects, are limited and associated with 10 year survival of 55%, highlighting the urgent need for novel approaches The proposed research is anticipated to result in the development of novel mechanism-based first-in-class therapeutics that could substantially improve treatment of scleroderma and patient survival.
SPECIFIC AIMS.
The Product. The final product will represent a new mechanism-based, efficient, stable, well tolerable systemic immunomodulatory therapy for scleroderma in order to significantly decrease long-term disability, morbidity and mortality of the patients with scleroderma and improve the quality of their life.
Scleroderma is a rare but devastating autoimmune disorder (Lawrence et al.
1998, Mayes et al. 2003, Helmick et al. 2008) with no approved drug available. Current main treatments all have side effects, are limited and associated with 10 year survival of 55%
(Badea et al. 2009, Kowal-Bielecka et al. 2009, Shah et al. 2013), highlighting an urgent need for new therapies.
Macrophages are associated with fibrosis (Ishikawa et al. 1992, Kraling et al.
1995, Lech et al.
2013, Chia et al. 2015) and are recruited to inflammation sites by MCP-1 which is significantly elevated in patients with systemic sclerosis (SSc) (Hasegawa et al. 1999).
Activated macrophages produce VEGF, IL-lbeta, TNFalpha, IL-6, TGFbeta and PDGF that play a role in scleroderma (Bonner et al. 1991, Clouthier et al. 1997, Yamamoto 2011, Yamamoto et al. 2011, Liu et al. 2013, Manetti 2015). In animals, blockers of IL-6 receptor (IL-6R) (Kitaba et al. 2012), VEGF (Koca et al. 2016), TNFalpha (Koca et al. 2008) and TGFalpha (Varga et al. 2009, Varga et al. 2009) alleviate scleroderma but all may have serious side effects including fatal infections and sepsis (Varga 2004). CSF-1/M-CSF plays a role in pulmonary fibrosis that occurs in 90% of scleroderma patients (Baran et al. 2007). TREM-1 mediates release of MCP-1/CCL2, TNFalpha, IL-lbeta, IL-6 and CSF-1 (Schenk et al. 2007, Dower et al. 2008, Lagler et al.
2009, Sigalov 2014, Shen et al. 2015). TREM-1 expression is increased in the lungs of mice with BLM-induced pulmonary fibrosis (Peng et al. 2016). Together, this implicates TREM-1 as a new target to develop a first-in-class therapy for scleroderma.
Innovation. At least two aspects: /. This is the first project to study TREM-1 blockade in an animal model of scleroderma. 2. To block TREM-1, we use a proprietary peptide formulated into macrophage-specific LipoPeptide Complexes (LPC) to extend its half-life and increase targeting (Sigalov 2014, Shen et al. 2017, Shen et al. 2017). Other TREM-1 blockers (e.g., LR12 peptide by Inotrem, France (Cuvier et al. 2018)) all attempt to block binding of currently uncertain ligands of TREM-1 and have a risk of failure in clinics, while GF9 is an advantageously ligand-independent.
Previously ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai et al.
2019), Preliminary Data), we found that TREM-1 blockade using GF9: ameliorates disease in mice with collagen-induced arthritis (CIA); reduces serum CSF-1, TNFalpha, IL-lalpha, IL-6 in mice with CIA, cancer, and liver disease; and inhibits expression of MCP-1/CCL2, TNFalpha, Pro-Colll-alpha and alpha -SMA in mice with liver disease.
The goal of this project is to develop TREM-1-targeting drug for the treatment of scleroderma.
Aim 1: Optimize TREM-1 inhibitory compositions for their functionality in vitro and pharmacokinetics in vivo and select the lead. GF9-LPC will be generated using GF9, lipids and two modified peptides that mediate macrophage uptake of GF9-LPC and affect their half-life in vivo. We will vary lipid/peptide composition and peptide ratios to prepare long half-life GF9-LPC with fast and high uptake by J774 cells and high inhibitory effect on cytokine release by LPS-stimulated J774 cells. Three most promising GF9-LPC injectables selected based on their functionality in vitro will be tested in rats for their pharmacokinetic (PK) profiles. To analyze GF9 in animal serum, we will develop and validate an LC-MS assay with ZATA
Pharmaceuticals. Milestone 1 includes development of the long half-life lead, which is efficient in inhibiting cytokine release in vitro. Completion of the Aim 1 will answer the question on the possibility of generating of the lead optimized to provide fast, efficient and long-lasting therapeutic effect.
Aim 2: Test two doses of the GF9-LPC lead in a bleomycin-induced mouse model of scleroderma. We have shown that chronic subcutaneous injection of BLM in mice results in the development of progressive multiple organ fibrosis with histological changes in the skin, muscle and lungs that resemble those seen in patients with SSc (Bhattacharyya et al.
2018, Bhattacharyya et al. 2018). Two doses of the GF9-LPC lead generated in the Aim 1 will be tested for its effect on lung, heart, muscle and skin fibrosis in this mouse model. Studies will be performed at Northwestern Scleroderma by lab of Dr. John Varga, a world-renowned expert in autoimmune diseases with special emphasis on scleroderma. Histology/IHC
studies will be performed. Serum and tissue CCL2, CSF-1, VEGF, TGFbeta, TNFalpha, IL-6, and IL-lbeta will be analyzed. Milestone 2 includes in vivo testing of suitability of TREM-1 blockade to prevent and treat scleroderma. Completion of the Aim 2 will answer a question about feasibility of using GF9-LPC as a first-in-class therapy for scleroderma.
The project is anticipated to identify the lead that will set the stage for development of first-in-class, safe and effective scleroderma therapies. If successful, Phase I will be followed in Phase II by toxicology, pharmacology ADME, PK/PD, and CMC studies, filing an IND and subsequent evaluation in humans.
Anticipated low toxicity of GF9 therapy is supported by the safety and well tolerability of 300 mg/kg GF9 in healthy mice (Sigalov 2014) (while its therapeutic dose varies from 2.5 mg/kg for GF9-LPC to 25 mg/kg for free GF9 (Sigalov 2014, Shen and Sigalov 2015, Rojas et al. 2017, .. Shen and Sigalov 2017, Tornai et al. 2019)) and lack of body weight changes in cancer and arthritic mice long-term treated with GF9-LPC (Sigalov 2014, Shen and Sigalov 2017).
Prototypes of SignaBlok's LPC are safe in humans (Newton et al. 2002, Kingwell et al. 2013).
TREM-1 blockade using peptide LR12 developed by SignaBlok's top competitor (Inotrem, France) is safe in humans (Cuvier et al. 2018, Francois et al. 2018).
Successful completion of Phase I will provide the animal proof of concept that might be applicable not only to scleroderma but also to other rare musculoskeletal, rheumatic or skin diseases.
Research Strategy Scleroderma: An unmet need for an effective and low toxic treatment options Scleroderma is a rare but devastating autoimmune disorder (Lawrence et al.
1998, Mayes et al.
2003, Helmick et al. 2008) with no approved drug available. Current main treatments all have side effects, are limited and associated with 10 year survival of 55% (Badea et al. 2009, Kowal-Bielecka et al. 2009, Shah and Wigley 2013), highlighting an urgent need for new therapies. The long-term goal of the proposed project is to develop a novel, first-in-class, efficient and well tolerable systemic therapy for scleroderma.
Macrophages and scleroderma.
Macrophages are the predominant infiltrating cells in skin lesions of patients with scleroderma and are associated with fibrosis (Ishikawa and Ishikawa 1992, Kraling et al. 1995, Lech and Anders 2013, Chia and Lu 2015). MCP-1 recruits macrophages to inflammation sites and is significantly elevated in patients with systemic sclerosis (S Sc) (Hasegawa et al. 1999).
Activated macrophages produce VEGF, IL-1-beta, TNFalpha, IL-6, TGF-beta and PDGF, which are of crucial importance in the profibrogenic role of fibroblasts in scleroderma (Bonner et al. 1991, Clouthier et al. 1997, Yamamoto 2011, Yamamoto and Katayama 2011, Liu et al. 2013, Manetti 2015). In animals, blockers of IL-6 receptor (IL-6R) (Kitaba et al.
2012), VEGF (Koca et al. 2016), TNF-alpha (Koca et al. 2008) and TGF-beta (Varga and Pasche 2009, Varga and Whitfield 2009) alleviate scleroderma but all may have serious side effects including fatal infections and sepsis (Varga 2004). M-CSF plays a role in pulmonary fibrosis that occurs in 90%
of scleroderma patients (Baran et al. 2007). In rats, elevated MCP-1 and M-CSF
lead to macrophage recruitment in an injured area and to the lesion formation (Juniantito et al. 2013).
Inhibition of TRE11-1 signaling: A new approach to disorders associated with systemic inflammation Triggering Receptor Expressed on Myeloid cells-1 (TREM-1), an inflammation amplifier, plays a role in immune response (Bouchon et al. 2000, Bouchon et al. 2001, Bleharski et al. 2003, Colonna et al. 2003, Klesney-Tait et al. 2006, Tessarz et al.
2008) and is upregulated upon inflammation (Wang et al. 2004, Gonzalez-Roldan et al. 2005, Koussoulas et al. 2006, Schenk et al. 2007). TREM-1 mediates release of multiple cytokines including MCP-1, TNF 0, IL-1 0, IL-6 and M-CSF (Schenk et al. 2007, Dower et al. 2008, Lagler et al.
2009, Sigalov 2014, Shen and Sigalov 2015). TREM-1 blockade is a new approach to inflammatory disorders (Bouchon et al. 2001, Colonna and Facchetti 2003, Schenk et al. 2007, Gibot et al. 2008, Ho et al. 2008, Ford et al. 2009, Gibot et al. 2009, Murakami et al. 2009, Luo et al. 2010, Pelham et al.
2014, Pelham et al. 2014, Bosco et al. 2016), In mice, TREM-1 blockade inhibits M-CSF, TNFalpha, IL-lbeta and IL-6, suppresses tumor growth and ameliorates autoimmune arthritis (Sigalov 2014, Shen and Sigalov 2017).
TREM-1 blockade blunts excessive inflammation but in contrast to single cytokine blockers, preserves the capacity for microbial control (Weber et al. 2014).
TREM-1 blockade was suggested as a treatment of neonatal infection (Qian et al. 2014).
Endotoxic and septic mice lacking DAP12, a signaling adapter of TREM-1, have improved survival (Turnbull et al. 2005).
Humans lacking DAP12 do not have problems resolving infections (Lanier 2009).
Inhibition of TRE11-1 signaling: A new approach to preventing and treating scleroderma TREM-1 is overexpressed in the lungs of mice with BLM-induced pulmonary fibrosis (Peng et al. 2016). In experimental autoimmune arthritis, cancer and retinopathy, TREM-1 blockade reduces inflammation and inhibits the macrophage infiltration / activation (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) (Section 3.3.3.1). In mice with alcohol-induced liver disease (ALD), TREM-1 blockade inhibits expression of TREM-1, MCP-1/CCL2, TNF
0, Pro-Colll 0 and 0 -SMA (Tornai et al. 2019). Collectively, these findings implicate TREM-1 as a target for development of new therapy for scleroderma.
The main concepts of the proposed project: Silencing the scleroderma-related specific inflammatory response can be superior to anti-single cytokine strategies in the treatment of scleroderma in terms of safety and efficacy; Delivery of systemically administered TREM-1 blockers to macrophages may have several advantages: (a) striking the target cell population, (b) sparing other cells that have no (or marginal) effects on scleroderma, (c) minimizing off-target effects, and (d) reducing the therapeutic dose; and Rate and efficiency of intracellular delivery of TREM-1 blockers to macrophages may be important to provide a prompt and effective therapeutic response during scleroderma progression.
INNOVATION
TREM-1 Blockade Major challenge. Current approaches (eg Inotrem's LR12) that all attempt to block TREM-1 binding to its ligand(s) (Fig. 97A) have a risk of failure since exact nature of TREM-1 ligand(s) is still uncertain (Tammaro et al. 2017).
SignaBlok's solution. Using our new model of signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model (Sigalov 2006, Sigalov 2010), we developed a first-in-class ligand-independent TREM-1 inhibitory peptide GF9 (US 8,513,185) that disrupts recognition and signaling functions of TREM-1 in the membrane (Fig. 97B) (Sigalov 2010, Sigalov 2013, Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017).
As other peptides (Graff et al. 2003, Lien et al. 2003, Gotthardt et al. 2004, Ladner et al.
2004, Prive et al. 2006, Sato et al. 2006, Antosova et al. 2009, Koskimaki et al. 2010), GF9 is advantageous compared to large protein molecules. Mechanistically, GF9 self-penetrates into the cell membrane and can reach its site of action from both inside and outside the cell (Fig. 97B
and Fig. 97C). GF9 is well-tolerated by healthy mice (up to 300 mg/kg; Fig.
98A). GF9 at 25 mg/kg in a free form or at 2.5 mg/kg when formulated into LipoPeptide Complexes (LPC, below), reduces tissue TREM-1 and M-CSF upon inflammation (shown on the example of the retina of mice with oxygen-induced retinopathy, OIR) (Fig. 98B), and ameliorates diseases in mouse models of cancer (Sigalov 2014, Shen and Sigalov 2017), retinopathy (Rojas et al. 2018), ALD (Tornai et al. 2019), sepsis and autoimmune arthritis (Sigalov 2014, Shen and Sigalov 2017).
LPC mimic human High Density Lipoproteins (HDL) and consist of lipids and peptides of human apolipoprotein (apo) A-I, the major protein of HDL. In contrast to native HDL, these peptides contain naturally occurring modifications that target LPC to macrophages. SignaBlok's LPC can deliver GF9 to macrophages in vitro and in vivo (Fig. 99A-C) and increase its therapeutic efficacy (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017). NOTE:
GF9-LPC describes GF9 formulated into either discoidal (GF9-dLPC, short 612:
hrs) or spherical (GF9-sLPC; long 612: days) LPC.
Epitope-based rational design of long half-life GF9-LPC fast and effective in delivery of GF9.
GF9-LPC tested to date, all contained a fixed amount of GF9 and an equimolar mixture of oxidized (MetS0) 22-mer peptides with sequences from either helix 4 (PE22) or 6 (PA22) of human apo A-I. Although these modifications increase macrophage uptake of LPC
in vitro and in vivo (Sigalov 2014, Sigalov 2014, Shen et al. 2015) (Fig. 99A), the uptake can be optimized to make it faster and more efficient. Oxidized PE22 and PA22 contain different MetS0 epitopes for binding to Scavenger Receptor (SR) SR-A (Apo A-I peptides contain putative epitopes for binding with SR-A (italics-M(0)) and SR-BI (bold). PE22:
PYLDDFQKKWQEEM(0)ELYRQKVE. PA22: PLGEEM(0)RDRARAHVDALRTHLA) (Neyen et al. 2009). In addition, PA22 contains an epitope for binding to macrophage and hepatocyte SR-BI (Liadaki et al. 2000, Cai et al. 2012). Its exposure affects binding to SR-BI (de .. Beer et al. 2001) and can determine the LPC half life.
APPROACH
Overall strategy, methodology, and analyses to be used to accomplish the specific aims Towards the overall goal of the proposed Phase I research, we will:
Aim 1. Optimize TREM-1 inhibitory compositions for their functionality in vitro and pharmacokinetics in vivo and select the lead.
1) generate and characterize GF9-LPC of different GF9/1pid/PE22/PA22 compositions; 2) use J774 cells and the relevant antibodies to explore mechanisms of SR-mediated uptake of GF9-LPC; 3) use the mechanistic data to optimize GF9/1pid/PE22/PA22 compositions and generate .. long half-life GF9-LPC with high GF9 load and high rate and efficiency of macrophage delivery of GF9; 4) functionally test the generated GF9-LPC for inhibition of cytokine release in LPS-stimulated J774 cells; 5) develop an LC-MS assay for analysis of GF9 in rat serum;
6) test three most promising formulations in PK studies in Sprague-Dawley (SD) rats;
7) analyze the data obtained and select the lead GF9-LPC formulation for further animal testing.
Aim 2: Test two doses of the GF9-LPC lead in a bleomycin-induced mouse model of scleroderma 1) test two doses of the GF9-LPC lead generated in the Aim 1 in preventative and established BLM-induced mouse models of scleroderma for its efficacy in preventing and treating the disease;
2) perform comprehensive histology / immunohistochemistry studies;
3) analyze serum and tissue GF9 and cytokines (LC-MS; ELISA).
Preliminary data Preliminary data, rationale, methodology, and analyses to be used to accomplish the Aim 1. Previously (Sigalov 2014), we showed that oxidation of PE22 and PA22 results in increased in vitro J774 cell uptake of GF9-LPC (Fig. 99A-C) and that GF9 (but not a control peptide) either in a free form (not shown) or formulated into LPC of discoidal (GF9-dLPC) or spherical (GF9-sLPC) shape inhibits cytokine release both in vitro and in vivo and protects mice from LPS-induced sepsis-related death (ig. 100A-D). GF9-dLPC and GF9-sLPC
both contained the same amount of GF9 and an equimolar mixture of oxidized PE22 and PA22.
Rationale GF9-dLPC and GF9-sLPC both inhibit LPS-stimulated cytokine release in vitro and in vivo to about the same degree (Fig. 100A, Fig. 100B) but their protective effect at the dose of 5 mg/kg in LPS-induced septic mice differs: GF9-sLPC provide less effective but longer-lasting protection as compared with GF9-dLPC (Fig. 100C). Further, despite the same GF9 load and 1:1 PE22:PA22 molar ratio, these GF9-LPC differ in rate and efficiency of the macrophage uptake in vitro (Fig. 101) (Sigalov 2014). Stronger protection by GF9-dLPC may result from higher efficiency and rate of their uptake (Fig. 101), while longer protection by GF9-sLPC ¨ from their longer half-life. Thus, uptake of GF9-LPC may depend on exposure of SR-binding apo A-I
epitopes (Liu et al. 2002, Horiuchi et al. 2003) (Apo A-I peptides contain putative epitopes for binding with SR-A (italics-M(0)) and SR-BI (bold). PE22:
PYLDDFQKKWQEEM(0)ELYRQKVE.
PA22: PLGEEM(0)RDRARAHVDALRTHLA) that affect both rate and efficiency of the uptake.
In Phase I Aim 1, we will optimize exposure of SR-A- and SR-BI-binding epitopes and GF9 content of long half-life GF9-LPC by varying of GF9/lipid/PE22/PA22 ratios to increase GF9 load and rate and efficiency of its delivery in vivo and thus to provide prompt, effective and long-.. term therapeutic response.
Methodology and analyses Peptides. GF9 and two oxidized 22-mer peptides PE22 and PA22 will be ordered from Bachem, Inc. and characterized as described previously (Sigalov et al. 1998, Sigalov et al. 2001, Sigalov et al. 2002, Sigalov 2014, Shen et al. 2015, Shen and Sigalov 2017, Shen and Sigalov 2017).
Long half-life GF9-LPC (spherical). Previously used non-optimized GF9-LPC be synthesized as described (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) and used as a reference in all in vitro studies. In some studies, GF9 and/or PE22 will be Dylight (Dy) 488-labeled. In some studies, GF9-LPC will be Rhodamine B (Rho B)-labeled.
Optimization. The following parameters will be varied: a) phospholipid chain length; 2) lipid composition; 3) lipid/PE22/PA22 composition/ ratio; and 4) GF9 content. The obtained GF9-LPC will be purified and their integrity, stability, and GF9 content will be analyzed as reported (Sigalov 2014, Shen and Sigalov 2017). As analyzed by Dynamic Light Scattering (DLS), GF9-LPC are stable at 4 C for at least, up to 6 months (Fig. 102).
In vitro macrophage uptake assay. GF9-LPC will be characterized by in vitro macrophage uptake assay as reported (Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai et al. 2019). To explore the mechanisms of GF9-LPC uptake, cells will be incubated with either anti-SR-BI, anti-SR-A, or isotype controls for 15 min on ice before adding Rho B-labeled GF9-LPC with Dy 488-labeled GF9 and/or PE22. After incubation, cells will be washed, lysed and fluorescence and protein concentrations in the lysates will be measured.
In vitro cytokine release. The assay will be performed in LPS-stimulated J774 macrophages (Fig. 100B) as previously reported (Sigalov 2014).
Confocal analysis. J774A.1 cells will be grown at 37 C in 6 well tissue culture plates containing glass coverslips. After reaching target confluency of ¨ 50%, cells will be incubated for 6 h at 37 C with Rho B-GF9-LPC. In subsets of experiments, Rho B- GF9-LPC
that contain Dylight 488-PE22 or Dylight 488-GF9 will be used. TREM-1 staining will be performed as described (Shen and Sigalov 2017). The slides will be imaged as reported (Shen and Sigalov 2017).
Integrity and stability studies. RP-HPLC, SEC, and DLS will be used as described (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017) to study structural integrity and stability of GF9-LPC.
LC-MS for GF9 analysis in animal serum. LC-MS assay for analysis of GF9 in rat serum in PK studies in rats will be developed and validated (with ZATA). The assay will include ultracentrifugation step followed by LC-MS. The snap-frozen samples of rat serum will be ordered from BioreclamationIVT (Westbury, NY) and processed as reported (Walther et al.
2011, Yanachkov et al. 2011, Yanachkova et al. 2015, Yanachkov et al. 2016).
GF9-LPC will be added to serum and GF9 will be assayed by LC-MS. The assay will be validated using the FDA
guidelines (https://www.fda.gov/downloads/drugs/guidances/ucm368107.pdf).
PK studies in SD rats. Animal studies will be provided by WBI. SignaBlok will perform LC-MS/histology/IHC. Sex as a biological variable. To exclude differences in PK
in male and female rats (Shelnutt et al. 1999), we propose to use both sexes. 3 most promising GF9-LPC
selected based on their TREM-1 inhibitory activity in vitro will be tested in 8 wk-old SD rats (200-250 g) (3 groups; 3 males+3 females / group, 18 SD rats). Briefly, SD
rats will be IV
administered with 2.5 mg/kg GF9-sLPC. Serum samples will be collected at 8 post-injection timepoints within 24 hrs, frozen and shipped to SignaBlok for LC-MS analysis of GF9.
Ultracentrifugation of serum to float lipoproteins and GF9-LPC will be performed as reported (Sigalov et al. 1991, Sigalov 1993, Sigalov et al. 1997, Sigalov and Stern 1998, Sigalov and Stern 2001). Briefly, 50 OL serum, 50 OL saline, 0.5 mM EDTA, and 130 OL KBr (d = 1.37 g/mL) will be mixed (final d=1.21 g/mL) and centrifuged in a 42.2 TI rotor at 42,000 rpm for 12 h at 10 C. 50 OL will be taken from top, dialyzed for 4 h at 4 C and analyzed for GF9 by LC-MS.
Statistical analysis. GraphPad Prism will be used for statistical testing. In in vitro uptake assay and cytokine assay data, statistical significances will be determined by two-tailed Student's t test as described (Sigalov 2014). Results will be considered significant atp<0.05.
PK data will be analyzed using PKSolver, a menu-driven add-in Microsoft Excel software (Zhang et al. 2010).
Outcome measures Stability of GF9-LPC will be tested by DLS (Fig. 102). GF9 in GF9-LPC will be analyzed as reported (Sigalov 2014, Shen and Sigalov 2017) and by LC-MS. In vitro J774 cell uptake will be measured by Rho B fluorescence in cell lysates (Sigalov 2014). Activity of GF9-LPC in reduction of cytokine release by LPS-stimulated cells will be tested as reported (Sigalov 2014).
In PK studies, half-life, Cmax, Tmax and the area under the AUC will be analyzed.
Anticipated results and interpretations Native dHDL and sHDL have half-lives of 12-20 hrs and 3-5 days, respectively (Scanu et al.
1962, Furman et al. 1964). We expect that formulation of GF9 into spherical LPC will extend its half-life closer to that for sHDL. Based on our preliminary data and (Sigalov 2014), we predict that: 1) GF9-sLPC of different compositions will have different exposure of SR-A and SR-BI
epitopes, and 2) use of SR inhibitors will allow to find the preferential receptor involved in cell uptake. We predict that PK profiles of GF9-LPC formulations with different exposure of SR-A
and SR-BI epitopes will differ. Thus, we anticipate to optimize SR-A/SR-BI
epitope exposure and prepare GF9-LPC with high in vitro efficacy and favorable PK in vivo.
Completion of Aim 1 will show the feasibility of using of epitope-based design to optimize GF9-LPC
for effective and long-term inhibition of TREM-1 in vitro and in vivo. Milestone 1 includes selection of the lead based on its stability, in vitro activity and PK profile.
Anticipated problems, alternative strategies and future directions.
We do not expect technical problems as we at SignaBlok, Drs. Tabatadze and Yanachkov at ZATA, and the WBI's staff have expertise in all methods (Yanachkov et al.
2011, Sigalov 2014, Sigalov 2014, Shen et al. 2015, Yanachkova et al. 2015, Shen et al. 2016, Yanachkov et al. 2016, Shen and Sigalov 2017, Shen and Sigalov 2017, Yanachkov et al. 2017).
Preliminary data Previously, using non-optimized GF9-LPC, we demonstrated that:
1) in mice with ALD, systemic 2.5 mg/kg GF9-LPC reduces TREM-1, MCP-1/CCL2, early fibrosis markers (alpha-smooth muscle actin [alpha-SMA] and procollagenl-alpha [Pro-Colll-alpha]) at the mRNA level (Tornai et al. 2019) Fig. 103A-D);
2) in cancer mice, systemic 25 mg/kg GF9 and 2.5 mg/kg GF9-LPC are well-tolerated (Fig.
Fig. 104A), reduce macrophage infiltration into the tumor (Fig. 103B, Fig.
103C) and inhibit CSF-1/M-CSF (Fig. 104D) (Shen and Sigalov 2017);
3) in mice with CIA, systemic 25 mg/kg GF9 and 2.5 mg/kg GF9-LPC are well-tolerated (Fig. 105AA), ameliorate arthritis (Fig. 104B) and inhibit IL-1-beta, IL-6, TNF-alpha and CSF-1/M-CSF (Fig. 105AC) (Shen and Sigalov 2017).
Rationale TREM-1 blockade by GF9-LPC suppress macrophage infiltration and activation, reduce cytokine, CSF-1/ M-CSF and early fibrosis markers and ameliorate disease in ALD, cancer, septic and arthritic mice ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Tornai et al. 2019), Fig. 103A-D-Fig. 105A- C). This suggests that GF9-LPC will be effective in the treatment of scleroderma (Ishikawa and Ishikawa 1992, Kraling et al. 1995, Lech and Anders 2013, Chia and Lu 2015). BLM mouse model is a valuable tool for drug development for scleroderma (Yamamoto et al. 1999, Yamamoto et al. 1999, Huber et al. 2007, Beyer et al. 2010, Kitaba et al. 2012, Avci et al. 2013, Artlett 2014, Toyama et al. 2016). We have shown that chronic subcutaneous (s.c.) injection of BLM in mice results in development of progressive multiple organ fibrosis (Bhattacharyya et al. 2018, Bhattacharyya et al.
2018). In Aim 2, we will use this model to test GF9- LPC ability to prevent and treat organ fibrosis.
Serum and tissue CCL2, CSF-1/M-CSF, VEGF, TGF 0, TNF 0, IL-6, and IL-10 will be analyzed.
Methodology and analyses We will design perform and analyze animal studies with Dr. John Varga, M.D.
(Director, Northwestern Scleroderma, Northwestern University Feinberg School of Medicine (Chicago, IL).
Sex (gender) as a biological variable. In a BLM-induced scleroderma mouse model, while a more pronounced fibrosis phenotype was reported for male compared with female mice (Ruzehaji et al. 2015), other data show no histologic differences between male and female mice (Yamamoto et al. 1999, Yamamoto et al. 1999) We suggest to use both sexes of mice in this project.
Mouse model of scleroderma. S.c. BLM delivery leads to slowly-progressive fibrosis in multiple organs with no mortality, and histological changes in the skin, muscle and lungs that resemble those seen in patients with SSc (Bhattacharyya et al. 2018, Bhattacharyya et al. 2018).
8-12 wk-old C57BL6 mice (288 in total) will be randomized and divided into 3 arms by 12 groups of 8 mice per group (6 male and 6 female groups). In preventative model (arms 1 and 2), mice will receive s.c. injections of 10 mg/kg BLM or PBS daily for 10 days (5 days/week), along with 2.5 or 5 mg/kg GF9-LPC by daily i.p. injections starting concurrently with BLM, and will be sacrificed on day 7 (arm 1) or 22 (arm 2). In established model (arm 3), mice will receive 2.5 or 5 mg/kg GF9-LPC daily starting at day 15, and continue until sacrifice at day 28. In all arms, control groups of mice will receive BLM or PBS alone daily for 10 (7, arm 1) days or 2.5 or 5 mg/kg GF9-LPC alone daily until sacrifice at days 7, 22 or 28.
Statistical analysis. Statistical significance of differences in parameters of fibrosis and inflammation between control and treated mice will be determined by F-test.
Comparison among three or more groups will be executed with one-way ANOVA followed by a post hoc Tukey's test. Based on our previous studies using this model (Bhattacharyya et al.
2018, Bhattacharyya et al. 2018), a sample size of 8 mice in each group is chosen to give a power of 80% to detect 10%
difference in mean values between experimental and control groups, with a significance level of 0.05.
Outcome measures Experiments will test efficacy of treatment given as prevention, as well as treatment, to determine if TREM-1 inhibition can promote regression of established skin, lung and heart fibrosis and resolution of tissue damage. Clinical observations (daily) and body weights (weekly) will be made until termination. DRAIZE scoring will be recorded once weekly for all groups.
Effect of TREM-1 blockade will be tested on early (day 3-7) inflammatory changes and monocyte/macrophage influx in the lungs and skin by IHC; subsequent development of fibrotic parenchymal changes (at day 10-20) by histology/IHC, biochemical and functional assays.
Tissues will be collected, prepared, stained with H&E and Trichrome and evaluated by board-certified pathologist. Part of collected tissues will be homogenized and along with blood and FFPE tissue samples shipped to SignaBlok for GF9, cytokine and IHC analysis.
Tissue collagen content will be determined by hydroxyproline assays (Bhattacharyya et al.
2016). Lung fibrosis will be quantitated in histological lung sections using the modified Ashcroft score determined from 5 h.p.f. per mice (Hubner score) (Hubner et al. 2008). Skin hardness will be measured using a Vesmeter three times at the injection area. Dermal thickness will be determined at three randomly selected sites in each animal. a-SMA-positive cells will be counted.
Macrophage infiltration will be evaluated by IHC. Serum and tissue CCL2, VEGF, CSF-1, TGF
0, TNF 0, IL-6 and IL-1 0 will be analyzed by ELISA. Tissue TREM-1 expression will be tested by Western Blot.
Anticipated results and interpretations These studies are expected to demonstrate if TREM-1 blockade using GF9-LPC
can, by attenuating TLR4 activity in target organs, prevent, slow the progression, and promote the recovery from, fibrotic injury in the skin, lungs, muscle and heart. Further, the results are expected to indicate whether observed beneficial effects are primarily due to attenuated early inflammation, reduced fibrosis due to attenuated activation of (myo)fibroblasts, or a combination of both of these mechanisms.
Based on our previous data ((Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017); Fig. 100A-D, Fig. 103A-D -Fig. 105A-C), we predict that treatment with GF9-LPC will be well-tolerated and associated with reductions in levels of CCL2, CSF-1, TNF-beta, TGF-alpha, IL-6 and IL-lbeta. We expect that GF9-LPC will be effective in a dose-dependent manner and that LPC (no GF9) will be without effect. Completion of Aim 2 will answer a question about the feasibility of using GF9-LPC as a first-in-class therapy for scleroderma.
Anticipated problems, alternative strategies and future directions.
We do not expect technical problems as we at SignaBlok and the Varga laboratory's and animal facility' staff have extensive expertise in all methods (Varga and Whitfield 2009, Sigalov 2014, Shen et al.
2015, Shen and Sigalov 2016, Shen and Sigalov 2017, Shen and Sigalov 2017, Bhattacharyya et al.
2018, Bhattacharyya et al. 2018, Yamashita et al. 2018, Lakota et al. 2019, Tornai et al. 2019).
Final product. SignaBlok's GF9-LPC consist of phospholipids widely used in pharmacology and synthetic peptides, all derived from human sequences, suggesting the lack of potential immunogenicity. Lipoprotein- and peptide-based drug formulations are currently on the market (Chang et al. 2012, Adler-Moore et al. 2016) or in clinical trials (Tricoci et al.
2015), which makes SignaBlok's efficient and well tolerable systemic therapy for scleroderma commercially viable.
Future directions. If successful, Phase I will be followed in Phase II where to evaluate the efficacy of TREM-1 blockade for mitigating organ fibrosis, we will use complementary mouse models of SSc, including the Tsk1/+ mouse, which (spontaneously) develop skin fibrosis in the absence of inflammation. Other administration schedules and regimen will be tested. The lead and its manufacturing technology will be further optimized and the more detailed safety, TOX, ADME, CMC and other IND-enabling studies will be performed. Upon completion, an IND will be filed for subsequent testing in humans.
Anticipated low toxicity of GF9-LPC is supported by the safety of 300 mg/kg GF9 in healthy mice (Sigalov 2014) (therapeutic doses are 25 mg/kg for GF9 or 2.5 mg/kg for GF9-LPC), lack of body weight changes in mice long-term treated with GF9-LPC (Sigalov 2014, Shen et al.
2017, Tornai et al. 2019), and by the fact that prototypes of SignaBlok's LPC
were well tolerated in humans (Newton and Krause 2002, Kingwell and Chapman 2013). TREM-1 blockade using inhibitory peptide LR12 which is in development by SignaBlok's top competitor (Inotrem, France) was well tolerated in healthy and septic subjects (Cuvier et al. 2018, Francois et al.
2018).
The decision to go to Phase II will be made if the significant (more than 50%) decrease in fibrosis is shown in treated mice as compared with those shown in control mice.
Bibliography and References Cited.
1. Helmick CG, Felson DT, Lawrence RC, Gabriel S, Hirsch R, Kwoh CK, et al. Estimates of the prevalence of arthritis and other rheumatic conditions in the United States. Part I. Arthritis Rheum 2008; 58:15-25. PMID: 18163481.
2. Lawrence RC, Helmick CG, Arnett FC, Deyo RA, Felson DT, Giannini EH, et al.
Estimates of the prevalence of arthritis and selected musculoskeletal disorders in the United States. Arthritis Rheum 1998; 41:778-99. PMID: 9588729.
3. Mayes MD, Lacey JV, Jr., Beebe-Dimmer J, Gillespie BW, Cooper B, Laing TJ, et al.
Prevalence, incidence, survival, and disease characteristics of systemic sclerosis in a large US
population. Arthritis Rheum 2003; 48:2246-55. PMID: 12905479.
4. Kowal-Bielecka 0, Landewe R, Avouac J, Chwiesko S, Miniati I, Czirjak L, et al.
EULAR recommendations for the treatment of systemic sclerosis: a report from the EULAR
Scleroderma Trials and Research group (EUSTAR). Ann Rheum Dis 2009; 68:620-8.
PMID:
19147617.
5. Badea I, Taylor M, Rosenberg A, Foldvari M. Pathogenesis and therapeutic approaches for improved topical treatment in localized scleroderma and systemic sclerosis. Rheumatology (Oxford) 2009; 48:213-21. PMID: 19022832.
6. Shah AA, Wigley FM. My approach to the treatment of scleroderma.
Mayo Clin Proc 2013; 88:377-93. PMID: 23541012.
7. Ishikawa 0, Ishikawa H. Macrophage infiltration in the skin of patients with systemic sclerosis. J Rheumatol 1992; 19:1202-6. PMID: 1404154.
8. Chia JJ, Lu TT. Update on macrophages and innate immunity in scleroderma. Curr Opin Rheumatol 2015; 27:530-6. PMID: 26352734.
9. Lech M, Anders HJ. Macrophages and fibrosis: How resident and infiltrating mononuclear phagocytes orchestrate all phases of tissue injury and repair.
Biochim Biophys Acta 2013; 1832:989-97. PMID: 23246690.
10. Kraling BM, Maul GG, Jimenez SA. Mononuclear cellular infiltrates in clinically involved skin from patients with systemic sclerosis of recent onset predominantly consist of monocytes/macrophages. Pathobiology 1995; 63:48-56. PMID: 7546275.
11. Hasegawa M, Sato S, Takehara K. Augmented production of chemokines (monocyte chemotactic protein-1 (MCP-1), macrophage inflammatory protein-lalpha (MIP-lalpha) and MIP-lbeta) in patients with systemic sclerosis: MCP-1 and MIP-lalpha may be involved in the development of pulmonary fibrosis. Clin Exp Immunol 1999; 117:159-65. PMID:
10403930.
10403930.
12. Liu J, Copland DA, Hone S, Wu WK, Chen M, Xu Y, et al. Myeloid cells expressing VEGF and arginase-1 following uptake of damaged retinal pigment epithelium suggests potential mechanism that drives the onset of choroidal angiogenesis in mice. PLoS One 2013; 8:e72935.
PMID: 23977372.
PMID: 23977372.
13. Yamamoto T, Katayama I. Vascular changes in bleomycin-induced scleroderma. Int J
Rheumatol 2011; 2011:270938. PMID: 22028717.
Rheumatol 2011; 2011:270938. PMID: 22028717.
14. Yamamoto T. Autoimmune mechanisms of scleroderma and a role of oxidative stress.
Self Nonself 2011; 2:4-10. PMID: 21776329.
Self Nonself 2011; 2:4-10. PMID: 21776329.
15. Clouthier DE, Comerford SA, Hammer RE. Hepatic fibrosis, glomerulosclerosis, and a lipodystrophy-like syndrome in PEPCK-TGF-betal transgenic mice. J Clin Invest 1997;
100:2697-713. PMID: 9389733.
100:2697-713. PMID: 9389733.
16. Bonner JC, Osornio-Vargas AR, Badgett A, Brody AR. Differential proliferation of rat lung fibroblasts induced by the platelet-derived growth factor-AA, -AB, and -BB isoforms secreted by rat alveolar macrophages. Am J Respir Cell Mol Biol 1991; 5:539-47. PMID:
1958381.
1958381.
17. Manetti M. Deciphering the alternatively activated (M2) phenotype of macrophages in scleroderma. Exp Dermatol 2015; 24:576-8. PMID: 25869115.
18. Kitaba S, Murota H, Terao M, Azukizawa H, Terabe F, Shima Y, et al.
Blockade of interleukin-6 receptor alleviates disease in mouse model of scleroderma. Am J
Pathol 2012;
180:165-76. PMID: 22062222.
Blockade of interleukin-6 receptor alleviates disease in mouse model of scleroderma. Am J
Pathol 2012;
180:165-76. PMID: 22062222.
19. Koca SS, Ozgen M, Dagli AF, Gozel N, Ozercan IH, Isik A. The Protective Effects of Bevacizumab in Bleomycin-Induced Experimental Scleroderma. Adv Clin Exp Med 2016;
25:249-53. PMID: 27627557.
25:249-53. PMID: 27627557.
20. Koca SS, Isik A, Ozercan IH, Ustundag B, Evren B, Metin K.
Effectiveness of etanercept in bleomycin-induced experimental scleroderma. Rheumatology (Oxford) 2008;
47:172-5.
PMID: 18174229.
Effectiveness of etanercept in bleomycin-induced experimental scleroderma. Rheumatology (Oxford) 2008;
47:172-5.
PMID: 18174229.
21. Varga J, Whitfield ML. Transforming growth factor-beta in systemic sclerosis .. (scleroderma). Front Biosci (Schol Ed) 2009; 1:226-35. PMID: 19482698.
22. Varga J, Pasche B. Transforming growth factor beta as a therapeutic target in systemic sclerosis. Nat Rev Rheumatol 2009; 5:200-6. PMID: 19337284.
23. Varga J. Antifibrotic therapy in scleroderma: extracellular or intracellular targeting of activated fibroblasts? Curr Rheumatol Rep 2004; 6:164-70. PMID: 15016348.
24. Baran CP, Opalek JM, McMaken S, Newland CA, O'Brien JIM, Jr., Hunter MG, et al.
Important roles for macrophage colony-stimulating factor, CC chemokine ligand 2, and mononuclear phagocytes in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med 2007; 176:78-89. PMID: 17431224.
Important roles for macrophage colony-stimulating factor, CC chemokine ligand 2, and mononuclear phagocytes in the pathogenesis of pulmonary fibrosis. Am J Respir Crit Care Med 2007; 176:78-89. PMID: 17431224.
25. Lagler H, Sharif 0, Haslinger I, Matt U, Stich K, Furtner T, et al.
TREM-1 activation .. alters the dynamics of pulmonary IRAK-M expression in vivo and improves host defense during pneumococcal pneumonia. J Immunol 2009; 183:2027-36. PMID: 19596984.
TREM-1 activation .. alters the dynamics of pulmonary IRAK-M expression in vivo and improves host defense during pneumococcal pneumonia. J Immunol 2009; 183:2027-36. PMID: 19596984.
26. Dower K, Ellis DK, Saraf K, Jelinsky SA, Lin LL. Innate immune responses to TREM-1 activation: overlap, divergence, and positive and negative cross-talk with bacterial lipopolysaccharide. J Immunol 2008; 180:3520-34. PMID: 18292579.
27. Schenk M, Bouchon A, Seibold F, Mueller C. TREM-1--expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases. J Clin Invest 2007; 117:3097-106. PMID: 17853946.
28. Shen ZT, Sigalov AB. Novel Ligand-Independent Peptide Inhibitors of Triggering Receptor Expressed on Myeloid Cells 1 (TREM-1) and T Cell Receptor (TCR):
Efficacy in a Collagen-Induced Arthritis Model Suggests New Targeted Treatment for Rheumatoid Arthritis [abstract]. Arthritis Rheumatol 2015; 67:1347-8. PMID.
Efficacy in a Collagen-Induced Arthritis Model Suggests New Targeted Treatment for Rheumatoid Arthritis [abstract]. Arthritis Rheumatol 2015; 67:1347-8. PMID.
29. Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-induced septic shock. Int Immunopharmacol 2014; 21:208-19. PMID: 24836682.
30. Peng L, Zhou Y, Dong L, Chen RQ, Sun GY, Liu T, et al. TGF-betal Upregulates the Expression of Triggering Receptor Expressed on Myeloid Cells 1 in Murine Lungs. Sci Rep 2016; 6:18946. PMID: 26738569.
31. Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide inhibitors of TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017; 21:2524-34.
PMID:
28382703.
PMID:
28382703.
32. Shen ZT, Sigalov AB. Novel TREM-1 Inhibitors Attenuate Tumor Growth and Prolong Survival in Experimental Pancreatic Cancer. Mol Pharm 2017; 14:4572-82. PMID:
29095622.
29095622.
33. Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-induced septic shock. Int Immunopharmacol 2014; 21:208-19. PMID: 24836682.
34. Cuvier V, Lorch U, Witte S, Olivier A, Gibot S, Delor I, et al. A first-in-man safety and pharmacokinetics study of nangibotide, a new modulator of innate immune response through TREM-1 receptor inhibition. Br J Clin Pharmacol 2018; 84:2270-9. PMID:
29885068.
29885068.
35. Tornai D, Fun i I, Shen ZT, Sigalov AB, Coban S, Szabo G. Inhibition of Triggering Receptor Expressed on Myeloid Cells 1 Ameliorates Inflammation and Macrophage and Neutrophil Activation in Alcoholic Liver Disease in Mice. Hepatol Commun 2019;
3:99-115.
PMID: 30619998.
3:99-115.
PMID: 30619998.
36. Bhattacharyya S, Wang W, Tamaki Z, Shi B, Yeldandi A, Tsukimi Y, et al.
Pharmacological Inhibition of Toll-Like Receptor-4 Signaling by TAK242 Prevents and Induces Regression of Experimental Organ Fibrosis. Front Immunol 2018; 9:2434. PMID:
30405628.
Pharmacological Inhibition of Toll-Like Receptor-4 Signaling by TAK242 Prevents and Induces Regression of Experimental Organ Fibrosis. Front Immunol 2018; 9:2434. PMID:
30405628.
37. Bhattacharyya S, Wang W, Qin W, Cheng K, Coulup S, Chavez S, et al.
dependent fibroblast activation drives persistent organ fibrosis in skin and lung. JCI Insight 2018; 3. PMID: 29997297.
dependent fibroblast activation drives persistent organ fibrosis in skin and lung. JCI Insight 2018; 3. PMID: 29997297.
38. Rojas MA, Caldwell RB, Shen Z, Sigalov A. TREM-1 blockade prevents vitreoretinal neovascularization in a mouse model of retinopathy of prematurity [abstract].
Invest Ophthalmol Vis Sci 2017; 58:3452. PMID.
Invest Ophthalmol Vis Sci 2017; 58:3452. PMID.
39. Kingwell BA, Chapman MJ. Future of high-density lipoprotein infusion therapies:
potential for clinical management of vascular disease. Circulation 2013;
128:1112-21. PMID:
24002713.
potential for clinical management of vascular disease. Circulation 2013;
128:1112-21. PMID:
24002713.
40. Newton RS, Krause BR. HDL therapy for the acute treatment of atherosclerosis.
Atheroscler Suppl 2002; 3:31-8. PMID: 12573361.
Atheroscler Suppl 2002; 3:31-8. PMID: 12573361.
41. Francois B, Wittebole X, Mira JP, Dugernier T, Gibot S, Derive M, et al. P1 Safety and pharmacodynamic activity of a novel TREM-1 pathway inhibitory peptide in septic shock patients: phase Ha clinical trial results. Intensive Care Med Exp 2018;
6(Suppl 1). PMID.
6(Suppl 1). PMID.
42. Juniantito V, Izawa T, Yuasa T, Ichikawa C, Yano R, Kuwamura M, et al.
Immunophenotypical characterization of macrophages in rat bleomycin-induced scleroderma.
Vet Pathol 2013; 50:76-85. PMID: 22700848.
Immunophenotypical characterization of macrophages in rat bleomycin-induced scleroderma.
Vet Pathol 2013; 50:76-85. PMID: 22700848.
43. Bouchon A, Dietrich J, Colonna M. Cutting edge: inflammatory responses can be triggered by TREM-1, a novel receptor expressed on neutrophils and monocytes.
J Immunol 2000; 164:4991-5. PMID: 10799849.
J Immunol 2000; 164:4991-5. PMID: 10799849.
44. Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature 2001; 410:1103-7. PMID:
11323674.
11323674.
45. Bleharski JR, Kiessler V, Buonsanti C, Sieling PA, Stenger S, Colonna M, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol 2003; 170:3812-8. PMID:
12646648.
12646648.
46. Tessarz AS, Cerwenka A. The TREM-1/DAP12 pathway. Immunol Lett 2008;
116:111-6. PMID: 18192027.
116:111-6. PMID: 18192027.
47. Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and signal integration. Nat Immunol 2006; 7:1266-73. PMID: 17110943.
48. Colonna M, Facchetti F. TREM-1 (triggering receptor expressed on myeloid cells): a new player in acute inflammatory responses. J Infect Dis 2003; 187 Suppl 2:S397-401. PMID:
12792857.
12792857.
49. Gonzalez-Roldan N, Ferat-Osorio E, Aduna-Vicente R, Wong-Baeza I, Esquivel-Callejas N, Astudillo-de la Vega H, et al. Expression of triggering receptor on myeloid cell 1 and histocompatibility complex molecules in sepsis and major abdominal surgery.
World J
Gastroenterol 2005; 11:7473-9. PMID: 16437719.
World J
Gastroenterol 2005; 11:7473-9. PMID: 16437719.
50. Koussoulas V, Vassiliou S, Demonakou M, Tassias G, Giamarellos-Bourboulis EJ, Mouktaroudi M, et al. Soluble triggering receptor expressed on myeloid cells (5TREM-1): a new mediator involved in the pathogenesis of peptic ulcer disease. Eur J
Gastroenterol Hepatol 2006;
18:375-9. PMID: 16538108.
Gastroenterol Hepatol 2006;
18:375-9. PMID: 16538108.
51. Wang DY, Qin RY, Liu ZR, Gupta MK, Chang Q. Expression of TREM-1 mRNA
in acute pancreatitis. World J Gastroenterol 2004; 10:2744-6. PMID: 15309732.
in acute pancreatitis. World J Gastroenterol 2004; 10:2744-6. PMID: 15309732.
52. Gibot S, Massin F, Alauzet C, Montemont C, Lozniewski A, Bollaert PE, et al. Effects of the TREM-1 pathway modulation during mesenteric ischemia-reperfusion in rats.
Crit Care Med 2008; 36:504-10. PMID: 18091551.
Crit Care Med 2008; 36:504-10. PMID: 18091551.
53. Luo L, Zhou Q, Chen XJ, Qin SM, Ma WL, Shi HZ. Effects of the TREM-1 pathway modulation during empyema in rats. Chin Med J (Engl) 2010; 123:1561-5. PMID:
20819512.
20819512.
54. Murakami Y, Akahoshi T, Aoki N, Toyomoto M, Miyasaka N, Kohsaka H.
Intervention of an inflammation amplifier, triggering receptor expressed on myeloid cells 1, for treatment of autoimmune arthritis. Arthritis Rheum 2009; 60:1615-23. PMID: 19479878.
Intervention of an inflammation amplifier, triggering receptor expressed on myeloid cells 1, for treatment of autoimmune arthritis. Arthritis Rheum 2009; 60:1615-23. PMID: 19479878.
55. Gibot S, Massin F, Alauzet C, Derive M, Montemont C, Collin S, et al.
Effects of the TREM 1 pathway modulation during hemorrhagic shock in rats. Shock 2009; 32:633-7. PMID:
19333144.
Effects of the TREM 1 pathway modulation during hemorrhagic shock in rats. Shock 2009; 32:633-7. PMID:
19333144.
56. Ho CC, Liao WY, Wang CY, Lu YH, Huang HY, Chen HY, et al. TREM-1 expression in tumor-associated macrophages and clinical outcome in lung cancer. Am J Respir Crit Care Med 2008; 177:763-70. PMID: 18096709.
57. Bosco MC, Raggi F, Varesio L. Therapeutic Potential of Targeting TREM-1 in Inflammatory Diseases and Cancer. Curr Pharm Des 2016; 22:6209-33. PMID:
27568730.
27568730.
58. Ford JW, McVicar DW. TREM and TREM-like receptors in inflammation and disease.
Curr Opin Immunol 2009; 21:38-46. PMID: 19230638.
Curr Opin Immunol 2009; 21:38-46. PMID: 19230638.
59. Pelham CJ, Agrawal DK. Emerging roles for triggering receptor expressed on myeloid cells receptor family signaling in inflammatory diseases. Expert Rev Clin Immunol 2014;
10:243-56. PMID: 24325404.
10:243-56. PMID: 24325404.
60. Pelham CJ, Pandya AN, Agrawal DK. Triggering receptor expressed on myeloid cells receptor family modulators: a patent review. Expert Opin Ther Pat 2014;
24:1383-95. PMID:
25363248.
24:1383-95. PMID:
25363248.
61. Weber B, Schuster S, Zysset D, Rihs S, Dickgreber N, Schurch C, et al.
Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance. PLoS Pathog 2014; 10:e1003900. PMID: 24453980.
Deficiency Can Attenuate Disease Severity without Affecting Pathogen Clearance. PLoS Pathog 2014; 10:e1003900. PMID: 24453980.
62. Qian L, Weng W, Chen W, Sun CH, Wu J. TREM-1 as a potential therapeutic target in neonatal sepsis. Int J Clin Exp Med 2014; 7:1650-8. PMID: 25126161.
63. Turnbull IR, McDunn JE, Takai T, Townsend RR, Cobb JP, Colonna M. DAP12 (KARAP) amplifies inflammation and increases mortality from endotoxemia and septic peritonitis. J Exp Med 2005; 202:363-9. PMID: 16061725.
64. Lanier LL. DAP10- and DAP12-associated receptors in innate immunity.
Immunol Rev .. 2009; 227:150-60. PMID: 19120482.
Immunol Rev .. 2009; 227:150-60. PMID: 19120482.
65. Derive M, Boufenzer A, Bouazza Y, Groubatch F, Alauzet C, Barraud D, et al. Effects of a TREM-like transcript 1-derived peptide during hypodynamic septic shock in pigs. Shock 2013;
39:176-82. PMID: 23324887.
39:176-82. PMID: 23324887.
66. Derive M, Boufenzer A, Gibot S. Attenuation of responses to endotoxin by the triggering receptor expressed on myeloid cells-1 inhibitor LR12 in nonhuman primate.
Anesthesiology 2014; 120:935-42. PMID: 24270127.
Anesthesiology 2014; 120:935-42. PMID: 24270127.
67. Tammaro A, Derive M, Gibot S, Leemans JC, Florquin S, Dessing MC. TREM-1 and its potential ligands in non-infectious diseases: from biology to clinical perspectives. Pharmacol Ther 2017; 177:81-95. PMID: 28245991.
68. Sigalov AB. Immune cell signaling: a novel mechanistic model reveals new therapeutic targets. Trends Pharmacol Sci 2006; 27:518-24. PMID: 16908074.
69. Sigalov AB. The SCHOOL of nature. III. From mechanistic understanding to novel therapies. Self/Nonself - Immune Recognition and Signaling 2010; 1:192-224.
PMID.
PMID.
70. Sigalov AB. New therapeutic strategies targeting transmembrane signal transduction in the immune system. Cell Adh Migr 2010; 4:255-67. PMID: 20519929.
71. Sigalov AB. Inhibition of TREM receptor signaling with peptide variants. Pat. US
8,513,185. In: USPTO, ed. USA: SignaBlok, Inc., Shrewsbury, MA (US), 2013.
8,513,185. In: USPTO, ed. USA: SignaBlok, Inc., Shrewsbury, MA (US), 2013.
72. Ladner RC, Sato AK, Gorzelany J, de Souza M. Phage display-derived peptides as therapeutic alternatives to antibodies. Drug Discov Today 2004; 9:525-9. PMID:
15183160.
15183160.
73. Antosova Z, Mackova M, Kral V, Macek T. Therapeutic application of peptides and proteins: parenteral forever? Trends Biotechnol 2009; 27:628-35. PMID:
19766335.
19766335.
74. Gotthardt M, Boermann OC, Behr TM, Behe MP, Oyen WJ. Development and clinical application of peptide-based radiopharmaceuticals. Curr Pharm Des 2004;
10:2951-63. PMID:
15379661.
10:2951-63. PMID:
15379661.
75. Koskimaki JE, Karagiannis ED, Tang BC, Hammers H, Watkins DN, Pili R, et al.
Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29. PMID: 20122172.
Pentastatin-1, a collagen IV derived 20-mer peptide, suppresses tumor growth in a small cell lung cancer xenograft model. BMC Cancer 2010; 10:29. PMID: 20122172.
76. Lien S, Lowman HB. Therapeutic peptides. Trends Biotechnol 2003; 21:556-62. PMID:
14624865.
14624865.
77. Prive GG, Melnick A. Specific peptides for the therapeutic targeting of oncogenes. Curr Opin Genet Dev 2006; 16:71-7. PMID: 16377176.
78. Sato AK, Viswanathan M, Kent RB, Wood CR. Therapeutic peptides:
technological advances driving peptides into development. Curr Opin Biotechnol 2006; 17:638-42. PMID:
17049837.
technological advances driving peptides into development. Curr Opin Biotechnol 2006; 17:638-42. PMID:
17049837.
79. Graff CP, Wittrup KD. Theoretical analysis of antibody targeting of tumor spheroids:
importance of dosage for penetration, and affinity for retention. Cancer Res 2003; 63:1288-96.
PMID: 12649189.
importance of dosage for penetration, and affinity for retention. Cancer Res 2003; 63:1288-96.
PMID: 12649189.
80. Rojas MA, Shen ZT, Caldwell RB, Sigalov AB. Blockade of TREM-1 prevents vitreoretinal neovascularization in mice with oxygen-induced retinopathy.
Biochim Biophys Acta 2018; 1864:2761-8. PMID: 29730341.
Biochim Biophys Acta 2018; 1864:2761-8. PMID: 29730341.
81. Sigalov AB. Nature-inspired nanoformulations for contrast-enhanced in vivo MR
imaging of macrophages. Contrast Media Mol Imaging 2014; 9:372-82. PMID:
24729189.
imaging of macrophages. Contrast Media Mol Imaging 2014; 9:372-82. PMID:
24729189.
82. Sigalov AB. Methods and Compositions for Targeted Imaging. US
20130045161.
Publication Date: Feb 21, 2013.
20130045161.
Publication Date: Feb 21, 2013.
83. Sigalov AB. Methods and Compositions for Targeted Delivery. US
20110256224.
Publication Date: Oct. 20, 2011. PMID.
20110256224.
Publication Date: Oct. 20, 2011. PMID.
84. Shen ZT, Zheng S, Gounis MJ, Sigalov AB. Diagnostic Magnetic Resonance Imaging of Atherosclerosis in Apolipoprotein E Knockout Mouse Model Using Macrophage-Targeted Gadolinium-Containing Synthetic Lipopeptide Nanoparticles. PLoS One 2015;
10:e0143453.
PMID: 26569115.
10:e0143453.
PMID: 26569115.
85. Neyen C, Pluddemann A, Roversi P, Thomas B, Cai L, van der Westhuyzen DR, et al.
Macrophage scavenger receptor A mediates adhesion to apolipoproteins A-I and E. Biochemistry 2009; 48:11858-71. PMID: 19911804.
Macrophage scavenger receptor A mediates adhesion to apolipoproteins A-I and E. Biochemistry 2009; 48:11858-71. PMID: 19911804.
86. Liadaki KN, Liu T, Xu S, Ishida BY, Duchateaux PN, Krieger JP, et al.
Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger
Binding of high density lipoprotein (HDL) and discoidal reconstituted HDL to the HDL receptor scavenger
87 receptor class B type I. Effect of lipid association and APOA-I mutations on receptor binding. J
Biol Chem 2000; 275:21262-71. PMID: 10801839.
87. Cai L, Wang Z, Meyer JM, Ji A, van der Westhuyzen DR. Macrophage SR-BI
regulates LPS-induced pro-inflammatory signaling in mice and isolated macrophages. J
Lipid Res 2012;
53:1472-81. PMID: 22589557.
Biol Chem 2000; 275:21262-71. PMID: 10801839.
87. Cai L, Wang Z, Meyer JM, Ji A, van der Westhuyzen DR. Macrophage SR-BI
regulates LPS-induced pro-inflammatory signaling in mice and isolated macrophages. J
Lipid Res 2012;
53:1472-81. PMID: 22589557.
88. de Beer MC, Durbin DM, Cai L, Jonas A, de Beer FC, van der Westhuyzen DR.
Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI. J Lipid Res 2001; 42:309-13. PMID: 11181762.
Apolipoprotein A-I conformation markedly influences HDL interaction with scavenger receptor BI. J Lipid Res 2001; 42:309-13. PMID: 11181762.
89. Horiuchi S, Sakamoto Y, Sakai M. Scavenger receptors for oxidized and glycated proteins. Amino Acids 2003; 25:283-92. PMID: 14661091.
90. Liu T, Krieger M, Kan HY, Zannis VI. The effects of mutations in helices 4 and 6 of ApoA-I on scavenger receptor class B type I (SR-BI)-mediated cholesterol efflux suggest that formation of a productive complex between reconstituted high density lipoprotein and SR-BI is required for efficient lipid transport. J Biol Chem 2002; 277:21576-84. PMID:
11882653.
11882653.
91. Sigalov AB, Stern U. Enzymatic repair of oxidative damage to human apolipoprotein A-I. FEBS Lett 1998; 433:196-200. PMID: 9744793.
92. Sigalov AB, Stern U. Oxidation of methionine residues affects the structure and stability of apolipoprotein A-I in reconstituted high density lipoprotein particles.
Chem Phys Lipids 2001;
113:133-46. PMID: 11687233.
Chem Phys Lipids 2001;
113:133-46. PMID: 11687233.
93. Sigalov AB, Stern U. Dihydrolipoic acid as an effective cofactor for peptide methionine sulfoxide reductase in enzymatic repair of oxidative damage to both lipid-free and lipid-bound apolipoprotein a-I. Antioxid Redox Signal 2002; 4:553-7. PMID: 12215223.
94. Yanachkov IB, Chang H, Yanachkova MI, Dix EJ, Berny-Lang MA, Gremmel T, et al.
New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors. Eur J Med Chem 2016; 107:204-18. PMID:
26588064.
New highly active antiplatelet agents with dual specificity for platelet P2Y1 and P2Y12 adenosine diphosphate receptors. Eur J Med Chem 2016; 107:204-18. PMID:
26588064.
95. Yanachkov IB, Dix EJ, Yanachkova MI, Wright GE. P1,P2-diimidazoly1 derivatives of pyrophosphate and bis-phosphonates--synthesis, properties, and use in preparation of dinucleoside tetraphosphates and analogs. Org Biomol Chem 2011; 9:730-8. PMID:
21082127.
21082127.
96. Yanachkova M, Xu WC, Dvoskin S, Dix EJ, Yanachkov IB, Focher F, et al.
Prodrugs of herpes simplex thymidine kinase inhibitors. Antivir Chem Chemother 2015; 24:47-55. PMID:
26463822.
Prodrugs of herpes simplex thymidine kinase inhibitors. Antivir Chem Chemother 2015; 24:47-55. PMID:
26463822.
97. Walther DM, Mann M. Accurate quantification of more than 4000 mouse tissue proteins reveals minimal proteome changes during aging. Mol Cell Proteomics 2011;
10:M110 004523.
PMID: 21048193.
10:M110 004523.
PMID: 21048193.
98. Shelnutt SR, Gunnell M, Owens SM. Sexual dimorphism in phencyclidine in vitro metabolism and pharmacokinetics in rats. J Pharmacol Exp Ther 1999; 290:1292-8. PMID:
10454506.
10454506.
99. Sigalov A, Alexandrovich 0, Strizevskaya E. Large-scale isolation and purification of human apolipoproteins A-I and A-II. J Chromatogr 1991; 537:464-8. PMID:
1904881.
1904881.
100. Sigalov AB. Comparison of apolipoprotein A-I values assayed in lyophilized and frozen pooled human sera by a non-immunochemical electrophoretic method and by immunoassay. Eur J Clin Chem Clin Biochem 1993; 31:579-83. PMID: 8260529.
101. Sigalov AB, Petrichenko IE, Kolpakova GV. The ratio of non-oxidized/oxidized forms of apolipoprotein A-I can affect cholesterol efflux from human skin fibroblasts mediated by high density lipoprotein. Eur J Clin Chem Clin Biochem 1997; 35:395-6. PMID:
9189746.
.. 102. Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed 2010;
99:306-14. PMID: 20176408.
103. Scanu A, Hughes WL. Further characterization of the human serum D 1.063-1.21, alpha-lipoprotein. J Clin Invest 1962; 41:1681-9. PMID: 14497795.
104. Furman RH, Sanbar SS, Alaupovic P, Bradford RH, Howard RP. Studies of the Metabolism of Radioiodinated Human Serum Alpha Lipoprotein in Normal and Hyperlipidemic Subjects. J Lab Clin Med 1964; 63:193-204. PMID: 14125106.
105. Shen ZT, Sigalov AB. SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice. Sci Rep 2016; 6:28672. PMID:
27349522.
.. 106. Yanachkov I, Zavizion B, Metelev V, Stevens LJ, Tabatadze Y, Yanachkova M, et al.
Self-neutralizing oligonucleotides with enhanced cellular uptake. Org Biomol Chem 2017;
15:1363-80. PMID: 28074950.
107. Huber LC, Distler JH, Moritz F, Hemmatazad H, Hauser T, Michel BA, et al.
Trichostatin A prevents the accumulation of extracellular matrix in a mouse model of bleomycin-induced skin fibrosis. Arthritis Rheum 2007; 56:2755-64. PMID: 17665426.
108. Toyama T, Asano Y, Akamata K, Noda S, Taniguchi T, Takahashi T, et al.
Tamibarotene Ameliorates Bleomycin-Induced Dermal Fibrosis by Modulating Phenotypes of Fibroblasts, Endothelial Cells, and Immune Cells. J Invest Dermatol 2016; 136:387-98. PMID:
26967475.
109. Yamamoto T, Takagawa S, Katayama I, Yamazaki K, Hamazaki Y, Shinkai H, et al.
Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma. J Invest Dermatol 1999; 112:456-62. PMID: 10201529.
110. Yamamoto T, Takahashi Y, Takagawa S, Katayama I, Nishioka K. Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice. J Rheumatol 1999; 26:2628-34. PMID: 10606374.
111. Avci P, Sadasivam M, Gupta A, De Melo WC, Huang YY, Yin R, et al. Animal models of skin disease for drug discovery. Expert Opin Drug Discov 2013; 8:331-55.
PMID: 23293893.
112. Artlett CM. Animal models of systemic sclerosis: their utility and limitations. Open Access Rheumatol 2014; 6:65-81. PMID: 27790036.
113. Beyer C, Schett G, Distler 0, Distler JH. Animal models of systemic sclerosis: prospects and limitations. Arthritis Rheum 2010; 62:2831-44. PMID: 20617524.
114. Ruzehaji N, Avouac J, Elhai M, Frechet M, Frantz C, Ruiz B, et al.
Combined effect of genetic background and gender in a mouse model of bleomycin-induced skin fibrosis. Arthritis Res Ther 2015; 17:145. PMID: 26025306.
115. Bhattacharyya S, Wang W, Morales-Nebreda L, Feng G, Wu M, Zhou X, et al.
Tenascin-C drives persistence of organ fibrosis. Nat Commun 2016; 7:11703. PMID:
27256716.
116. Hubner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, et al.
Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques 2008; 44:507-11, 14-7. PMID: 18476815.
117. Lakota K, Hanumanthu VS, Agrawal R, Cams M, Armanios M, Varga J. Short lymphocyte, but not granulocyte, telomere length in a subset of patients with systemic sclerosis.
Ann Rheum Dis 2019. PMID: 30679155.
118. Yamashita T, Lakota K, Taniguchi T, Yoshizaki A, Sato S, Hong W, et al.
An orally-active adiponectin receptor agonist mitigates cutaneous fibrosis, inflammation and microvascular pathology in a murine model of systemic sclerosis. Sci Rep 2018; 8:11843.
PMID: 30087356.
.. 119. Chang HI, Yeh MK. Clinical development of liposome-based drugs:
formulation, characterization, and therapeutic efficacy. Int J Nanomedicine 2012; 7:49-60.
PMID: 22275822.
120. Adler-Moore JP, Gangneux JP, Pappas PG. Comparison between liposomal formulations of amphotericin B. Med Mycol 2016; 54:223-31. PMID: 26768369.
121. Tricoci P, D'Andrea DM, Gurbel PA, Yao Z, Cuchel M, Winston B, et al.
Infusion of Reconstituted High-Density Lipoprotein, CSL112, in Patients With Atherosclerosis: Safety and Pharmacokinetic Results From a Phase 2a Randomized Clinical Trial. J Am Heart Assoc 2015;
4:e002171. PMID: 26307570.
122. Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide inhibitors of TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017. PMID:
28382703.
123. Deshmukh SV, Durston J, Shomer NH. Validation of the use of nonnaive surgically catheterized rats for pharmacokinetics studies. J Am Assoc Lab Anim Sci 2008;
47:41-5. PMID:
19049252.
124. Wang X, Song L, Li N, Qiu Z, Zhou S, Li C, et al. Pharmacokinetics and biodistribution study of paclitaxel liposome in Sprague-Dawley rats and Beagle dogs by liquid chromatography-tandem mass spectrometry. Drug Res (Stuttg) 2013; 63:603-6. PMID: 23842945.
125. Kuwahara Y, Shima Y, Shirayama D, Kawai M, Hagihara K, Hirano T, et al.
Quantification of hardness, elasticity and viscosity of the skin of patients with systemic sclerosis using a novel sensing device (Vesmeter): a proposal for a new outcome measurement procedure.
Rheumatology (Oxford) 2008; 47:1018-24. PMID: 18440998.
Additional Advantages of Using Peptides and Compositions as described herein.
As well-known in the art and described in Irby, et al. Mol Pharm 2017, 14:1325-1338, most anticancer chemotherapeutic agents as well as many other therapeutic agents (TA) are toxic and hydrophobic and cannot be administered by themselves as pure chemicals but have to be included in biocompatible formulations to enhance solubility, increase circulatory residence time of the therapeutic agents, minimize the undesirable side effects and alleviate drug resistance.
Numerous formulation approaches have been developed, including solid lipid particles, emulsions, liposomes, etc., however, the delivery of the poorly water soluble (hydrophobic, or lipophilic) pharmaceuticals remains especially problematic as most of the body compartments, including the blood circulation and intracellular fluids, represent an aqueous environment. As a result, the direct injection of hydrophobic TAs often results in harmful side effects due to hypersensitivity, hemolysis, cardiac and neurological symptoms.
As described in Vlieghe, et al. Drug Discov Today 2010, 15:40-56, the main limitations generally attributed to therapeutic peptides are: a short half-life because of their rapid degradation by proteolytic enzymes of the digestive system and blood plasma;
rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance); poor ability to cross physiological barriers because of their general hydrophilicity; high conformational flexibility, resulting sometimes in a lack of selectivity involving interactions with different receptors/targets (poor specific biodistribution), causing activation of several targets and leading to side effects; eventual risk of immunogenic effects; and high synthetic and production costs (the production cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500 Da molecular mass small molecule by more than 10-fold but clearly not 100-fold).
Consequently, there is need for more effective and low toxic therapies for PC
and other types of cancer as well as new formulations of hydrophobic drugs and therapeutic peptides to improve their targeted delivery, prolonged half-life, biocompatibility and therapeutic efficiency.
As described herein, it is surprisingly found that the peptides and compositions of the present invention capable of modulating the TREM-1 signaling pathway can be synthesized and used for targeted treatment of cancer and imaging. The advantageous trifunctional peptides and compositions are demonstrated by the present invention to solve numerous problems which otherwise are associated with high dosages of TAs and imaging probes required and the lack of control and reproducibility of formulations, especially in large-scale production.
As many other solid tumors, PC is characterized by a marked infiltration of macrophages into the stromal compartment (Shih 2006, Solinas 2009), a process, which is mediated by cancer-associated fibroblasts (CAFs) (FIG. 49) and plays a role in disease progression and its response to therapy. These tumor-associated macrophages (TAMs) secrete a variety of growth factors, cytokines, chemokines, and enzymes that regulate tumor growth, angiogenesis, invasion, and metastasis (Feurino 2006, Lewis and Pollard 2006, Shih 2006). High macrophage infiltration correlates with the promotion of tumor growth and metastasis development (Lin 2006, Lin 2001, Solinas 2009). In patients with PC, macrophage infiltration begins during the pre-invasive stage of the disease and increases progressively (Clark 2007). The number of TAMs is significantly higher in patients with metastases (Gardian 2012). Presence of TAMs in the PC
stroma correlates with increased angiogenesis (Esposito 2004), a known predictor of poor prognosis (Kuwahara 2003). TAM recruitment, activation, growth and differentiation are regulated by macrophage colony-stimulating factor (M-CSF, also known as colony-stimulating factor 1, CSF-1) (Elgert 1998, Varney 2005). High pretreatment serum M-CSF is a strong independent predictor of poor survival in PC patients (Groblewska 2007). In PC mouse models, blockade of M-CSF or its receptor not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota 2009) but also improves response to T-cell checkpoint immunotherapies that target programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu 2014).
Importantly, continuous M-CSF inhibition affects pathological angiogenesis but not healthy vascular and lymphatic systems outside tumors (Kubota 2009). In contrast to blockade of vascular endothelial growth factor (VEGF), interruption of M-CSF inhibition does not promote rapid vascular regrowth (Kubota 2009). Collectively, these findings further suggest that targeting TAMs is a promising strategy for treating cancer (Bowman and Joyce 2014, Jinushi and Komohara 2015, Komohara 2016).
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the inflammatory response (Colonna and Facchetti 2003) and is upregulated under inflammatory conditions including including cancer (Ho et al. 2008, Yuan et al. 2014, Nguyen et al.
2015), brain and spinal cord injuries (Li et al 2019) and acute pancreatitis (D. Y. Wang 2004).
For downstream signal transduction, TREM-1 is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor, DNAX activation protein of 12 kDa (DAP-12).
Activation of TREM-1/DAP-12 receptor complex enhances release of multiple cytokines including monocyte chemoattractant protein-1 (MCP-1; also referred to in the art as CCL2), tumor necrosis factor-a (TNFa), interleukin-la (IL-1a), IL-1I3, IL-6 and macrophage colony-stimulating factor 1 (CSF-1; also referred to in the art as M-CSF) (Schenk et al. 2007, Dower et al.
2008, Sigalov 2014, Shen et al. 2017, Shen et al. 2017, Rojas et al. 2018, Tornai et al. 2019).
Most of these cytokines are increased in cancer patients (Tjomsland et al. 2011, Rossi et al. 2015, Yako et al. 2016, Tsukamoto et al. 2018, Yoshimura 2018)and play a vital role in creating and sustaining inflammation in the tumor favorable microenvironment, thus affecting patient survival.
TREM-1 activation enhances release of multiple cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNFa), interleukin-la (IL-1a), IL-113, IL-6 and M-CSF (Lagler 2009, Schenk 2007, Sigalov 2014). Most of these cytokines are increased in patients with PC (Tjomsland 2011, Yako 2016) and play a vital role in creating and sustaining inflammation in the tumor favorable microenvironment, thus affecting patient survival. Inhibition of TREM-1 lowers levels of proinflammatory cytokines and is a promising approach in a variety of inflammation-associated disorders (Colonna and Facchetti 2003, Pelham and Agrawal 2014, Schenk 2007, Shen and Sigalov 2017, Sigalov 2014).
Importantly, in contrast to cytokine blockers, blockade of TREM-1 can blunt excessive inflammation while preserving the capacity for microbial control (Weber 2014). In vitro silencing of TREM-1 suppresses cancer cell invasion (Ho 2008). In patients with non-small cell lung cancer (NSCLC), expression on TAMs is associated with cancer recurrence and poor survival:
patients with low TREM-1 expression have a 4-year survival rate of over 60%, compared with less than 20% in patients with high TREM-1 expression (Ho 2008).
Inhibition of TREM-1 lowers levels of proinflammatory cytokines and chemokines including CSF-1 (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Rojas et al.
2018, Tornai et al. 2019) and as recently demonstrated in experimental cancer including NSCLC, pancreatic cancer and liver cancer, TREM-1 blockade inhibits tumor growth and improves survival (Wu et al. 2012, Sigalov 2014, Shen and Sigalov 2017, Wu et al.
2019). In vitro silencing of TREM-1 suppresses cancer cell invasion (Ho et al. 2008). In patients with NSCLC, TREM-1 expression on TAMs is associated with cancer recurrence and poor survival: patients with low TREM-1 expression have a 4-year survival rate of over 60%, compared with less than 20% in patients with high TREM-1 expression (Ho et al. 2008). Importantly, in contrast to cytokine blockers, blockade of TREM-1 can blunt excessive inflammation while preserving the capacity for microbial control (Weber et al. 2014). Septic mice lacking DAP-12, a signaling adapter of TREM-1, have improved survival (Turnbull et al. 2005). Humans lacking DAP12 do not have problems resolving infections (Lanier 2009). TREM-1 blockade is safe in healthy and septic subjects (Cuvier et al. 2018, Francois et al. 2018). Taken together, these finding make TREM-1 a promising therapeutic target in oncology.
The present invention provides the low toxic peptides and compositions for targeted treatment of cancer, e.g. PC, and other myeloid cell-related diseases and conditions and the methods for predicting the efficacy of these compositions. The invention further provides a method of using these peptides and compositions. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *,p < 0.05; **,p < 0.01, ****,p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP-bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar= 200 pm.
C. Sepsis; Severe Sepsis and Septic Shock.
Sepsis is another disorder with a high mortality rate. Currently, no approved sepsis drugs are available and over 30 drug candidates have failed late-stage clinical trials. Sepsis refers to a potentially life-threatening complication of an infection. Sepsis occurs when endogenous chemicals released into the bloodstream to fight the infection trigger inflammatory responses throughout the body. This inflammation can trigger a cascade of changes that can damage multiple organ systems, causing them to fail. If sepsis progresses to septic shock, blood pressure drops dramatically, which may lead to death.
Anyone can develop sepsis, but it's most common and most dangerous in older adults or those with weakened immune systems. Risk factors include but are not limited to: young or elderly; Have a compromised immune system; Are already very sick, often in a hospital's intensive care unit; Have wounds or injuries, such as burns; Have invasive devices, such as intravenous catheters or breathing tubes; etc.
Early treatment of sepsis, usually with antibiotics and large amounts of intravenous fluids, improves chances for survival. While any type of infection: ncluding bacterial, viral or fungal, can lead to sepsis, the most likely varieties include: Pneumonia;
Abdominal infection;
Kidney infection; Bloodstream infection (bacteremia); etc.
The incidence of sepsis appears to be increasing in the United States. The causes of this increase may include: Aging population. Americans are living longer, which is swelling the ranks of the highest risk age group ¨ people older than 65; Drug-resistant bacteria. Many types of bacteria can resist the effects of antibiotics that once killed them.
These antibiotic-resistant bacteria are often the root cause of the infections that trigger sepsis.; Weakened immune systems. More Americans are living with weakened immune systems, caused by HIV, cancer treatments or transplant drugs.; etc.
Sepsis ranges from less to more severe. As sepsis worsens, blood flow to vital organs, such as brain, heart and kidneys, becomes impaired. Sepsis can also cause blood clots to form in organs and in arms, legs, fingers and toes, leading to varying degrees of organ failure and tissue death (gangrene). Most people recover from mild sepsis, but the mortality rate for septic shock is nearly 50 percent. Also, an episode of severe sepsis may place you at higher risk of future infections.
Sepsis may present as a three-stage syndrome, starting with sepsis and progressing through severe sepsis to septic shock. The goal is to treat sepsis during its early stage, before it becomes more dangerous.the following symptoms, plus a probable or confirmed infection: Body temperature above 101 F (38.3 C) or below 96.8 F (36 C); Heart rate higher than 90 beats a minute; Respiratory rate higher than 20 breaths a minute, etc.
Severe sepsis refers to having at least one of the following signs and symptoms, which indicate an organ may be failing: Significantly decreased urine output; Abrupt change in mental status; Decrease in platelet count; Difficulty breathing; Abnormal heart pumping function;
Abdominal pain; etc.
Septic shock refers to having at least one of the following signs and symptoms of severe sepsis, plus extremely low blood pressure that doesn't adequately respond to simple fluid replacement.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
D. Rheumatoid arthritis (RA).
Rheumatoid arthritis (RA) refers to a chronic inflammatory disorder that can affect more than just your joints. In some people, the condition also can damage a wide variety of body systems, including the skin, eyes, lungs, heart and blood vessels.
RA affects as much as 1% of the worldwide population. There is no cure for RA
yet and up to 80% or more of RA patients are disabled after 20 years of symptoms.
Unlike the wear-and-tear damage of osteoarthritis, rheumatoid arthritis affects the lining of your joints, causing a painful swelling that can eventually result in bone erosion and joint deformity.
The inflammation associated with rheumatoid arthritis is what can damage other parts of the body as well. While new types of medications have improved treatment options dramatically, severe rheumatoid arthritis can still cause physical disabilities.
Signs and symptoms of rheumatoid arthritis may include: Tender, warm, swollen joints;
Joint stiffness that is usually worse in the mornings and after inactivity;
Fatigue, fever and weight loss; etc.
Early rheumatoid arthritis tends to affect your smaller joints first, particularly the joints that attach your fingers to your hands and your toes to your feet.
As the disease progresses, symptoms often spread to the wrists, knees, ankles, elbows, hips and shoulders. In most cases, symptoms occur in the same joints on both sides of your body.
About 40 percent of the people who have rheumatoid arthritis also experience signs and symptoms that don't involve the joints. Rheumatoid arthritis can affect many nonjoint structures, including: Skin; Eyes; Lungs; Heart; Kidneys; Salivary glands; Nerve tissue;
Bone marrow;
Blood vessels; etc.
Rheumatoid arthritis signs and symptoms may vary in severity and may even come and go. Periods of increased disease activity, called flares, alternate with periods of relative remission ¨ when the swelling and pain fade or disappear. Over time, rheumatoid arthritis can cause joints to deform and shift out of place.
Rheumatoid arthritis increases your risk of developing: Osteoporosis.
Rheumatoid arthritis itself, along with some medications used for treating rheumatoid arthritis, can increase your risk of osteoporosis ¨ a condition that weakens your bones and makes them more prone to fracture. Rheumatoid nodules. These firm bumps of tissue most commonly form around pressure points, such as the elbows. However, these nodules can form anywhere in the body, including the lungs. Dry eyes and mouth. People who have rheumatoid arthritis are much more .. likely to experience Sjogren's syndrome, a disorder that decreases the amount of moisture in your eyes and mouth. Infections. The disease itself and many of the medications used to combat rheumatoid arthritis can impair the immune system, leading to increased infections. Abnormal body composition. The proportion of fat compared to lean mass is often higher in people who have rheumatoid arthritis, even in people who have a normal body mass index (BMI). Carpal .. tunnel syndrome. If rheumatoid arthritis affects your wrists, the inflammation can compress the nerve that serves most of your hand and fingers. Heart problems. Rheumatoid arthritis can increase your risk of hardened and blocked arteries, as well as inflammation of the sac that encloses your heart. Lung disease. People with rheumatoid arthritis have an increased risk of inflammation and scarring of the lung tissues, which can lead to progressive shortness of breath.
Lymphoma. Rheumatoid arthritis increases the risk of lymphoma, a group of blood cancers that develop in the lymph system.
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *,p <0.05, **, p <0.01;
***,p <0.001 as compared with vehicle-treated or naive animals.
FIG. 42A-B presents exemplary data showing average clinical arthritis score (Collagen-induced arthritis: Score 42A) and Collagen-induced arthritis: Body weight change mean BW changes (42B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the CIA mice treated with PBS (vehicle), DEX, TREM-1-related control peptide G-TE21, TCR-related control peptide M-TK32, TCR-related trifunctional peptide M-VE32 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to the relevant control peptides, G-HV21, G-KV21 and M-VE32 all ameliorate the disease (A) and are well-tolerated by arthritic mice (B). *, p <0.05, **,p <0.01; ***,p <0.001 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; CIA, collagen-induced arthritis; PBS, phosphate-buffer saline; DEX, dexamethasone;
TCR, T cell receptor; BW, body weight.
E. Retinopathy.
Pathological retinal neovascularization (RNV) causes angiogenesis-related vision impairment in retinopathy of prematurity (ROP), diabetic retinopathy (DR), and retinal vein occlusion (RVO), which are the most common causes of vision loss and blindness in each age group. Conventional therapeutic options include laser ablation and the anti-vascular endothelial growth factor (VEGF) therapy, which both have their limitations and complications. Laser therapy is often accompanied by corneal edema, anterior chamber reaction, intraocular hemorrhage, cataract formation, and intraocular pressure changes, while the VEGF-targeted therapy can be complicated by damage of healthy vessels, potential side effects on neurons, rapid vascular regrowth upon interrupting the VEGF blockade, and limited effectiveness in some patients.
F. Cirrhosis Of The Liver And Alcoholic Liver Disease.
The human liver is located in the upper right side of the abdomen below the ribs. It has many essential body functions. These include: producing bile, which helps your body absorb dietary fats, cholesterol, and vitamins A, D, E, and K; storing sugar and vitamins for later use by the body; removing toxins such as alcohol and bacteria from your system:
creating blood clotting proteins; etc.
Several of the most common causes of cirrhosis of the liver in the United States are long-term viral hepatitis C infection and chronic alcohol abuse. Obesity is also a cause of cirrhosis, although it is not as prevalent as alcoholism or hepatitis C. Obesity can be a risk factor by itself, or in combination with alcoholism and hepatitis C.
According to the The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and other components of the National Institutes of Health (NIH), cirrhosis can develop in women who drink more than two alcoholic drinks per day (including beer and wine) for many years. For men, drinking more than three drinks a day for years can put them at risk for cirrhosis.
However, the amount is different for every person, and this doesn't mean that everyone who has ever drunk more than a few drinks will develop cirrhosis. Cirrhosis caused by alcohol is usually the result of regularly drinking more than these amounts over the course of 10 or 12 years.
Cirrhosis causes the liver to shrink and harden. This makes it difficult for nutrient-rich blood to flow into the liver from the portal vein. The portal vein carries blood from the digestive organs to the liver. The pressure in the portal vein rises when blood can't pass into the liver. The end result is a serious condition called portal hypertension, in which the vein develops high blood pressure.
The unfortunate consequence of portal hypertension is that this high-pressure system causes a backup, which leads to esophageal varices (like varicose veins), which can then burst and bleed.
Cirrhosis of the liver refers to severe scarring of the liver and poor liver function seen at the terminal stages of chronic liver disease. The scarring is most often caused by long-term exposure to toxins such as alcohol or viral infections.
Alcoholic liver cirrhosis is directly related to alcohol intake and is the final phase of alcoholic liver disease. Symptoms including but not limited to: anemia (low blood levels due to too little iron); high blood ammonia level); high blood sugar levels;
leukocytosis (large amount of white blood cells) ; unhealthy liver tissue when a sample is removed from a biopsy and studied in a laboratory; liver enzyme blood tests that show the level of aspartate aminotransferase (AST) is two times that of alanine aminotransferase (ALT); low blood magnesium levels; low blood potassium levels; low blood sodium levels; portal hypertension; etc.
Alcoholic liver cirrhosis can cause serious complications. This is known as decompensated cirrhosis. Examples of these complications include: ascites, or a buildup of fluid in the stomach; encephalopathy, or mental confusion; internal bleeding, known as bleeding varices; jaundice, which makes the skin and eyes have a yellow tint; etc.
Those with this the more severe form of cirrhosis often require a liver transplant to survive.; etc.
According to the Cleveland Clinic, patients with decompensated alcoholic liver cirrhosis who receive a liver transplant have a five-year survival rate of 70 percent.
Alcoholic liver disease (ALD) occurs after years of heavy drinking. The chances of getting liver disease go up the longer you have been drinking and more alcohol you consume.
Typically, a person has drank heavily for at least eight years. The National Institute on Alcohol Abuse and Alcoholism defines heavy drinking as drinking five or more drinks in one day on at least five of the past 30 days.
Symptoms of alcoholic liver cirrhosis typically develop when a person is between the ages of 30 and 40. A human body will be able to compensate for it's liver's limited function in the early stages of the disease. As the disease progresses, symptoms will become more noticeable. The disease is common in people between 40 and 50 years of age.
Men are more likely to have this problem. However, Women are also more at-risk for alcoholic liver disease.
Women don't have as many enzymes in their stomachs to break down alcohol particles. Because of this, more alcohol is able to reach the liver and make scar tissue.
Alcoholic liver disease can also have some genetic factors. For example, some people are born with a deficiency in enzymes that help to eliminate alcohol. Obesity, a high-fat diet, and having hepatitis C can also increase a person's likelihood they will have alcoholic liver disease.
women may develop the disease after less exposure to alcohol than men. Some people may have an inherited risk for the disease. The disease is part of a progression. It may start with fatty liver disease, then progress to alcoholic hepatitis, and then to alcoholic cirrhosis. However, it's possible a person can develop alcoholic liver cirrhosis without ever having alcoholic hepatitis.
When a person drinks alcohol heavily over the course of decades, the body starts to replace the liver's healthy tissue with scar tissue. Doctors call this condition alcoholic liver cirrhosis.
Alcoholic liver disease affects millions of people globally and often leads to fibrosis and cirrhosis. Liver cirrhosis is the 12th leading cause of death in the United States and costs society more than $15 billions annually. Despite this profound economic and health impact, there are currently no approved drugs to treat ALD. Current treatments including corticosteroids, immunosuppressants, and antioxidants have multiple shortcomings including a high level of serious side effects and insufficient efficacy.
slow the disease's progress and reduce your symptoms.
In some emboidments, either or both of the TREM-1 rHDLS and TREM-1 trifunctional peptides may be used in combination with treatments including but not limited to: Medications:
including but not limited to corticosteroids, calcium channel blockers, insulin, antioxidant supplements, and S-adenosyl-L-methionine (SAMe).; Nutritional Counseling:
Alcohol abuse can lead to malnutrition.; Extra protein: Patients often require extra protein in certain forms to help reduce the likelihood for developing brain disease (encephalopathy).; Liver Transplant.; etc.
investigated the role of TREM-1 in ALD and the potential therapeutic effect of the TREM-1 inhibitory GF9-HDL and GA/ E31-HDL formulations in the Lieber-DeCarli ALD
mouse model.
1. TREM-1 BLOCKADE AMELIORATES EXPRESSION OF EARLY
FIBROSIS MARKER GENES INDUCED BY CHRONIC ALCOHOL CONSUMPTION.
The clinical progression of ALD is associated with liver fibrosis.27 Our mouse model of ALD mimics the early phase of the human disease, yet mRNA levels of early fibrosis markers Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of these makers was remarkably attenuated in the vehicle-treated group and further decreased by the TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B a-Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
2. TREM-1 INHIBITORY FORMULATIONS AND HDL
AMELIORATE CHRONIC ALCOHOL-INDUCED LIVER INJURY AND
STEATOSIS.
We evaluated the impact of the TREM-1 inhibitors on hepatocyte damage and steatosis in liver. Serum ALT levels obtained during week 5 of the alcohol feeding showed significant increases in alcohol-fed mice compared to PF controls. This ALT
increase was attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig. 21A). Interestingly, vehicle treatment (HDL) also showed a similar protective effect (Fig. 21A).
Consistent with steatosis, we found a significant increase in Oil Red 0 staining in livers of alcohol-fed mice compared to PF controls (Fig. 21C). Oil Red 0 (Fig.
21B-D) and H& (Fig. 21D) staining revealed attenuation of steatosis in the alcohol-fed inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig. 21B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
WooP < 0.01;*"/###P <0.001; ****P < 0.0001. ***, 0.001 > P> 0.0001; ##, 0.01 >
P> 0.001.
3. BLOCKADE OF TREM-1 SIGNALING REDUCES THE EXPRESSION
OF INFLAMMATION-ASSOCIATED GENES IN ALD IN MICE.
Previous reports showed that TREM-1 activation leads to the expression and release of proinflammatory cytokines and chemokines through nuclear factor kB activation, which also regulates the expression of TREM-1, providing a positive feedback loop on the expression of the receptor.4 Proinflammatory cytokine expression is increased in ALDA1-3123124, therefore, we hypothesized that TREM-1 signaling contributes to the amplification of proinflammatory pathways in ALD.
To evaluate this hypothesis, first we tested whole-liver mRNAs of Et0H-fed and PF mice with or without treatment with two different TREM-1 inhibitory formulations and a vehicle control in a 5-week alcohol administration model of ALD in mice.(25) We found that mRNA
levels of TREM-1 and MCP-1 were significantly increased in livers of alcohol-fed mice compared to PF controls (Fig. 1A,B).
In contrast, in mice treated with the TREM-1 inhibitors, both GF9-HDL and HDL inhibited alcohol-related changes in TREM-1; in addition, MCP-1 mRNA
levels corresponded to those of the PF controls (Fig. 1A,B). Although induction of TNF-a and IL-11s in alcohol-fed mice did not reach statistical significance compared to PF
controls, TREM-1 block-ade by GF9-HDL resulted in a significant inhibition of TNF-a mRNA in the alcohol-fed mice compared to vehicle treatment (Fig. 1C), while IL-lfi mRNA expression was also significantly attenuated by both the GF9-HDL and GA/E31-HDL formulations in the alcohol-fed as well as in the PF groups (Fig. ID). MIP-la mRNA levels were increased in alcohol-fed mice, but TREM-1 blockade with GF9-HDL or GA/E31-HDL significantly attenuated this increase compared to the vehicle control (Fig. IE). Regulated on activation, normal T cell expressed, and secreted (RANTES) mRNA levels did not change regardless of alcohol feeding or TREM-1 treatment (Fig. IF).
FIG. 45A-E TREM-1 pathway inhibition. TREM-1 pathway inhibition suppresses the expression of (FIG. 45A) TREM-1 and inflammatory cytokines (FIG. 45B) MCP-1, (FIG. 45C) TNF-a, (FIG. 45D) IL-113, and (FIG. 45E) MIP-la but not (F) RANTES at the mRNA level as measured in whole-liver lysates by real-time quantitative PCR. * indicates significance level compared to nontreated PF group; # indicates significance level compared to nontreated alcohol-fed group; o indicates significance level compared to vehicle-treated alcohol-fed group.
Significance levels are as follows: * /#/o P <0.05; ** /##/oo P <0.01; *** /000 P <0.001; ****P
<0.0001.
Abbreviation: CCL, chemokine (C-C motif) ligand.
Next, we used specific ELISA kits to assess the protein levels of cytokines in the serum and in whole-liver lysates (Fig. 2). We found a significant increase in MCP-1 level in the serum and liver and TNF-a in the liver of alcohol-fed mice compared to PF controls (Fig. 2A-D). All these alcohol-induced increases were prevented both in the serum and liver by administration of either TREM-1 inhibitor. Interestingly, we found attenuation of alcohol-induced liver MCP-1 and TNF-a induction even in the vehicle-treated (HDL only) groups (Fig. 2A-C).
The increase in total IL-lfs levels after alcohol feeding and its attenuation by TREM-1 inhibition did not reach statistical significance (Fig. 2D).
Because TREM-1 is a membrane-associated molecule that triggers SYK activation as one of its proximal signaling molecules and we previously found increased SYK
phosphorylation in liver in ALD/24" we EBtested the levels of total and activated phospho-SYK
(p_syKY525/526) in the livers. We found significantly increased total and p-SYKY525/s26 levels after alcohol feeding .. (Fig. 2E-G). Treatment with GA/E31-HDL significantly decreased the p-levels in alcohol-fed mice compared to the untreated and vehicle-treated alcohol-fed groups, while GF9-HDL decreased p-SYKYs25/526 levels compared to the vehicle-treated group.
(Fig. 2E,F).
FIG. 46AE-G TREM-1 blockade and inflammatory cytokine levels. TREM-1 blockade reduces inflammatory cytokine levels in (FIG. 46A) serum and (FIG. 46B-D) whole-liver lysates as .. measured with specific ELISA kits. (FIG. 46E-G) Total liver protein was analyzed for total SYK
and activated p-SYK Y525/526 expression by western blotting using 13-actin as a loading control.
Statistical analysis was performed by evaluating two blots (n = 4/group).*
indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##
P < 0.01; ***P <
0.001; **** / P < 0.0001.
5. BLOCKADE OF TREM-1 ACTIVATION REDUCES EXPRESSION OF
MACROPHAGE AND NEUTROPHIL MARKERS IN LIVER
In agreement with previous studies indicating that chronic alcohol use causes hepatic macrophage infiltration and activation/1'3'26^ we found increased expression of the Kupffer cell/macrophage markers F4/80 and CD68 at the mRNA level. Treatment with the inhibitors significantly attenuated alcohol-induced expression of both F4/80 and CD68 in the liver, indicating anti-significant decrease in F4/80 expression on paraffin-embedded liver sections by IHC in alcohol-fed mice treated with either GF9-HDL or GA/E31-HDL
compared to the Et0H-fed vehicle-treated group (Fig. 3C,D).
Neutrophil infiltration of the liver is a characteristic of alcoholic hepatitis; therefore, we investigated markers associated with this cell population. Expression of the neutrophil markers Ly6G and MPO were significantly increased in livers of alcohol-fed mice compared to PF
controls. This was fully prevented by TREM-1 blockade (Fig. 3E,F).
Interestingly, the HDL
vehicle alone also resulted in a decreasing trend of Ly6G and MPO expression in alcohol-fed mice; however, the GF9-HDL and GA/E31-HDL TREM-1 inhibitors significantly attenuated Ly6G and MPO levels even when compared to the vehicle-treated alcohol-fed mice (Fig. 3E,F).
MPO staining on IHC confirmed that both TREM-1 inhibitors significantly reduced WO-positive cell numbers compared to the untreated alcohol-fed control group (Fig. 3G,H).
FIG. 47A-H. Effects of TREM-1 inhibition. (FIG. 47A, FIG. 47B) TREM-1 inhibition suppresses the mRNA expression of macrophage cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47C, FIG. 47D) Both TREM-1 inhibitors attenuated F4/80 as shown by IHC. (FIG. 47E, FIG. 47F) TREM-1 inhibition suppresses the mRNA
expression of neutrophil cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47G, H) Both TREM-1 inhibitors attenuated MPO-positive cell infiltration as shown by IHC. * indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P <
0.05; ** /## P < 0.01;
### P < 0.001; **** / P < 0.0001.
6. TREM-1 INHIBITORY FORMULATIONS AND HDL AMELIORATE
CHRONIC ALCOHOL-INDUCED LIVER INJURY AND STEATOSIS
To further assess the effects of the TREM-1 inhibitors on mechanisms of lipid metabolism, we tested genes involved in lipid synthesis (sterol regulatory element binding transcription factor 1 [SREBF1] and acetyl-coenzyme A carboxylase 1 [ACC1]) along with the lipid accumulation marker perilipin-2 (ADRP) (FIG. 48A-C). Both TREM-1 inhibitors but not vehicle treatment prevented alcohol-induced up-regulation of SREBF1, ACC1, and ADRP at the mRNA level (FIG. 48A-C). To assess lipid oxidation, we tested peroxisome proliferator-activated receptor a (PPARa), carnitine palmitoyl transferase 1A (CPT1A), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) mRNA levels in whole-liver samples (FIG.
48D-F).
Alcohol feeding significantly reduced mRNA expression of PPARa and CPT1A, while MCAD
had a decreasing trend. Both TREM-1 inhibitors as well as the vehicle treatment significantly increased PPARa and MCAD levels compared to the untreated alcohol-fed controls (FIG. 48D-F).
FIG. 48A-F Measurement of mRNA expression. mRNA expression of genes involved in (FIG.
48A, FIG. 48B) lipid synthesis (SERBF1, ACC1), (FIG. 48C) the lipid accumulation marker (ADRP), and (FIG. 48D-F) lipid oxidation (PPARa, CPT1a, MCAD) were measured in whole liver.
* indicates significance level compared to the nontreated PF group; #
indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P
<0.01; ### P <0.001; ****P <0.0001.
7. GF9HDL AND GA/E31HDL IS MAINLY MEDIATED BY S R A
We studied the uptake of GF9-HDL and GA/ E31-HDL in vitro in order to evaluate potential mechanisms of targeted delivery of GF9 (GA/E31). Kupffer cells and recruited hepatic macrophages express high levels of SRs, including SR-A, that are involved in phagocytosis and removal of oxidatively damaged lipoproteins and cells from the blood circulation.28'29 We previously demonstrated intracellu-SR lar macrophage delivery of GF9, GA31, and GE31 by macrophage-targeted GF9-HDL and GA/E31-HDL, respectively, and hypothesized that the observed macrophage endocytosis of these complexes is SR mediated.16'17 See, FIG 9A1 and 9A2. To further investigate the molecular mechanisms involved in this process, we used J774 macrophages as a model for Kupffer cells and incubated them with rho B-labeled GF9-HDL or GA/ E31-HDL in the presence or absence of cytochalasin D, fucoidan, or BLT-1, which are known to inhibit all SRs,(30) SR-A,(31) or SR-BI,(32) respectively.
In the presence of cytochalasin D, which inhibits both SR-A and SR-BI, the macrophage uptake of both TREM-1 inhibitor complexes was significantly inhibited, suggesting that this uptake is SR mediated. Fucoidan, an SR-A inhibitor, substantially suppressed endocytosis of TREM-1 inhibitor complexes at 22 hours but not at 4 hours, indicating time-dependent mechanisms of SR-A-mediated endocytosis (Fig. 9B). In contrast, BLT-1, which inhibits SR-BI, similarly inhibited the uptake of the complexes at both time points but to a lesser extent compared with that of fucoidan (Fig. 9C), presumably because of lower expression of SR-BI on J774 macrophages(33'1) These findings suggest that SR-A is the main contributor in SR-mediated endocytosis of both GF9-HDL and GA/E31-HDL.
Interestingly, quantitatively determined macrophage uptake levels in the presence or absence of fucoidan or BLT-1 were similar for GF9-HDL and GA/E31-HDL (Fig.
7B). This suggests that the combination of GF9 and apo AT peptide sequences in GA31 and sequences does not change the level and mechanisms of macrophage endocytosis of GA/E31-HDL compared with those of GF9-HDL.
8. Summary of TREM-1 in ALD.
Using a mouse model, significant up-regulation of TREM-1 was measured in livers of mice following chronic alcohol feeding. Treatment with novel ligand-independent TREM-1 inhibitors reduced the expression of the TREM-1 molecule itself, attenuated or fully prevented alcohol-induced increases in proinflammatory cytokines at the mRNA level, and inhibited SYK
activation. TREM-1 blockade provided by trifunctional peptides described herein, results in reduced macrophage and neutrophil infiltration and activation indicated by reduced F4/80, .. CD68, Ly6G, and MPO expression in the liver. These findings complement data demonstrating that TREM-1 blockade using GF9-HDL and GA/ E31-HDL suppresses macrophage infiltration of the tumor in cancer mice.(Reference 17) The TREM-1 inhibitors attenuated alcohol-induced liver steatosis. HDL and the TREM-1 inhibitors also attenuated liver injury and markers of early fibrosis in alcohol-fed mice. Interestingly, the HDL vehicle control showed similar efficiency as .. the inhibitory formulations at the protein level of the proinflammatory cytokines. Thereforeitwas also discovered that rHDL itself has some protective effects on ALD at the level of ALT and lipid oxidation.
While the ligand of TREM-1 is still unknown, it has been shown that TREM-1 activation amplifies inflammation and synergizes with TLR signaling pathways.(34) It was also observed that bacterial infection and challenge with LPS or lipoteichoic acid increase expression,(7) indicating a positive feedback loop among PAMP exposure, TREM-1 expression, and inflammatory cytokine induction. Different DAMPs, such as 3-hydroxy-3-methyl-glutaryl Bl and heat shock protein 70, have been suggested to stimulate TREM-1,(35)' while other studies found cell (granulocyte and platelet)-surface-associated activators as well.(35,36) Both PAMPs and DAMPs are present in ALD, providing potential mechanisms for TREM-1 up-regulation in this disease. Alcohol induces changes in the gut microbiome and disrupts the gut barrier function, resulting in increased levels of endotoxin and microbial PAMPs in circulation.(1,37) Alcohol also causes hepatocyte damage that leads to the release of DAMP5,(23) and these processes contribute to TREM-1 activation.
TREM-1 signaling leads to phosphorylation and activation of SYK, which has been indicated as a major regulator in inflammatory processes in ALD.(38) TREM-1 also amplifies TLR4 signaling that involves activation of SYK, which has been indicated as a downstream SYK
activation and phosphorylation.(38) Indeed, we found increased total and phosphorylated SYK
levels in the livers of alcohol-fed mice that was attenuated by TREM-1 inhibitor administration.
A previous study showed that inhibition of SYK activation attenuates alcohol-induced liver inflammation, cell death, and steatosis, suggesting that the SYK pathway could be a feasible therapeutic target in ALD.(24) SYK is expressed in a wild spectrum of cells, while TREM-1 inhibition may specifically modulate macrophages, neutrophils, and stellate cells that each play a role in ALD. Another advantage of TREM-1 inhibition is that it likely attenuates signaling from a broader spectrum ofTLRs, in addition toTLR4.
TREM-1 activation alone has been shown to increase the production of proinflammatory chemo-kines and cytokines.(39) Furthermore, simultaneous stimulation of TREM-1 and TLRs by an agonistic anti-TREM-1 antibody and different TLR ligands synergized in the induction of these proinflammatory molecules. TREM-1 and TLR4 costimulated monocytes showed increased production of MCP-1, IL-113, and IL-8. In contrast, the level of the anti-inflammatory cytokine IL-10 decreased when anti-TREM-1 antibody and the TLR3 ligand poly(LC) or the TLR4 ligand LPS simultaneously attached to their receptors.(40) Because self-perpetuating proinflammatory pathways are present in alcoholic hepatitis, interruption of these pathways using TREM-1 inhibition seems attractive.
By inducing TNF-a, IL-6, MCP-1, IL-8, and granulocyte-macrophage colony-stimulating factor and inhibiting IL-10 production, TREM-1 is involved in activation and recruitment of monocytes and modulation of inflammatory responses.(40) Furthermore, TREM-1 expression was highly up-regulated on the surface of infiltrating monocytes and neutrophils in human tissues infected by bacteria, highlighting the importance of this receptor in these processes.(7) In alcoholic hepatitis, neutrophils infiltrate the liver, inducing oxidative stress and cytotoxicity that contributes to the high mortality of the disease.(2) We showed that these processes can be attenuated by TREM-1 inhibitors. Mechanistically, the GF9-HDL and GA/E31-HDL
formulations target the liver more efficiently than peptides alone and release the TREM-1 inhibitory sequences inside the target cells where these peptides likely inhibit TREM-1 signaling by disrupting the intramembrane interactions of the TREM-1 receptor and its signaling adaptor molecule death-associated protein 12 (Fig. 27).(15-17) It was contemplated that observed preferential endocytosis of GF9-HDL and HDL by macrophages and hepatic clearance of these complexes is mediated by SR
recognition of putative epitopes in the modified apo A-I peptide constituents of GF9-HDL
and GA/E31-HDL.(16,17,19) Findings described herein indicate that GF9-HDL and GA/ E31-HDL
are largely recognized by SR-A on macrophages (Fig. 9A-B). We also observed SR-BI-mediated uptake, which likely explains the previously observed hepatic clearance for these complexes in another animal model.(19) While these data confirm our hypothesis, future studies are needed to determine the clearance properties for GF9-HDL and GA/E31-HDL in ALD.
Further, our present study demonstrates that GF9-HDL and GA/E31-HDL exhibit not only similar macrophage uptake in vitro largely driven by SR-A (Fig. 9A1) but also similar therapeutic effect in a mouse model of ALD (Figs. 20-21). This is in line with our previous studies where GF9-HDL and GA/E31-HDL exhibited similar therapeutic activities in cancer and arthritic mice. (16,17) We suggest that SR-A epitopes are similarly exposed on GA31 and GE31 in GA/ E31-HDL and on PA22 and PE22 in GF9-HDL, providing similar uptake of these complexes and as a result delivery of TREM-1 inhibitory GF9 peptide sequences in vivo. The use of GA/E31-HDL in the further development of effective and low-toxicity therapy for ALD is advantageous because it makes the entire manufacturing process easier and less expensive. We also suggest that the in vitro macrophage uptake assay can be potentially used to predict the outcomes for macrophage-targeted TREM-1 therapy in vivo.
In addition to attenuating inflammatory processes, the TREM-1 inhibitory formulations also ameliorated hepatocyte damage and steatosis. Serum ALT and liver triglyceride levels were both decreased in the GF9-HDL, GA/E31-HDL, and HDL-vehicle treated groups. The vehicle also had an inhibitory effect on TNF-a and MCP-1 protein levels as well as on mRNA expression of neutrophil and fibrosis markers, indicating that the HDL vehicle formulation can attenuate inflammation to a moderate extent. A previous study found evidence that HDL
can protect hepatocytes from endoplasmic reticulum stress, (41) while other publications reported a scavenger function of HDL for LPS and lipoteichoic acid(42,43) that could prevent immune cells from being activated by those molecules.(42,43) Further, the observed moderate beneficial effect of HDL treatment alone on fatty acid oxidation markers in alcohol-exposed mice (Fig. 47A-C) is in line with data that demonstrate infusion of reconstituted HDL reduces fatty acid oxidation in patients with type 2 diabetes mellitus. (44) In human and rat plasma, apo A-I, the major protein of HDL, has been shown to inhibit lipid peroxidation. (45) These data might provide an explanation for our findings of the hepatoprotective effects of HDL.
Our study shows that TREM-1 inhibitors with HDL formulation exerted significant inhibition on early signaling events of proinflammatory processes at ^the level of cytokine mRNA and the activated p-SYK protein levels compared to the HDL vehicle alone in a mouse model of ALD. This effect presumably would be even more obvious at the protein level of cytokines in a more severe liver injury. However, in mice, the most commonly used 5-week alcohol feeding that weused resulted in moderate liver damage and minimal(25) inflammation, which is a limitation of our study. As shown on the stained liver sections, the GF9-HDL and GA/E31-HDL formulations significantly inhibited immune cell infiltration and steatosis compared to the HDL vehicle only in mice with ALD. Thus, in some emboidments, inhibitors, such as the trifuncitonal peptides described herein, are contemplate for administration to patients showing at least one sympton, or at risk of developing a sympton, for ALD for decreasing inflammation in liver tissue for reducing said symptom or delaying/preventing said sympton.
Materials and Methods, for example, in relation to experiments assocated with treating ALD.
REAGENTS AND CELLS
The murine macrophage J774A.1 cell line was purchased from ATCC (Manassas, VA).
Cytochalasin D was purchased from MP Biomedicals (Solon, OH). Blocker of lipid transport 1 (BLT-1) was purchased from Calbiochem (Torrey Pines, CA). Sodium cho-late, cholesteryl oleate, fucoidan, and other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO). 1-Palmitoy1-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoy1-0T-glycero-3-phosphoeth-anolamine-N-(lissamine rhodamine B sulfonyl) (rho EEB-PE), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL).
PEPTIDE SYNTHESIS
The following synthetic peptides were ordered from Bachem (Torrance, CA): one 9-mer peptide, GFLSKSLVF (human TREM-1213- 221, GF9); two 22-mer methionine sulfoxidized peptides, PYLDDFQKKWQEEM(0)ELYRQKVE (H4) and PLG
EEM(0)RDRARAHVDALRTHLA (H6), which correspond to human apo A-I helices 4 (apo A-1123.144) and 6 (apo A-I167-188), respectively; and two 31-mer methionine sulfoxidized peptides, GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE (GE31) and GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (GA31).
LIPOPEPTIDE COMPLEXES
HDL-mimicking lipopeptide complexes of spherical morphology that contained either GF9 and an equimolar mixture of PE22 and PA22 (GF9-HDL) or an equimolar mixture of GA31 and GE31 (GA/ E31-HDL) were synthesized using the sodium cholate dialysis procedure, purified, and characterized as described.(16-18, 22) For GF9-HDL, the initial molar ratio was 125:6:2:3:1:210, corresponding to POPC:cholesterol:cholesteryl oleate:GF9:apo A-I:sodium cholate, respectively, where apo A-I was an equimolar mixture of PE22 and PA22. For GA/E31-HDL, the initial molar ratio was 125:6:2:1:210, corresponding to POPC:cholesterol:cholesteryl oleate:GA/ E31: sodium cholate, respectively, where GA/E31 was an equimolar mixture of GA31 and GE31.
A quantitative in vitro macrophage assay of endo-cytosis of rho B-labeled HDL-mimicking lipopeptide complexes by J774 macrophage was performed as described.(18-20) Briefly, BALB/c murine macrophage J774A.1 cells (ATCC) were cultured at 37 C
with 5% CO2 in Dulbecco's modified Eagle's medium (Cellgro Mediatech, Manassas, VA) with 2 mM
glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (Cellgro Mediatech) and grown to approximately 90% confluency in 12-well tissue culture plates (Corning Costar, Corning, NY). After reaching target confluency, cells were incubated for 1 hour in medium with or without fucoidan (400 ug/mL), BLT-1 ((1011M), or cytochalasin D (4011M). Cells were subsequently incubated for 4 hours and 22 hours at 37 C in medium containing 2 [EM of rho B-labeled GF9-HDL or GA/E31-HDL (as calculated for rho B).
Cells were washed twice using phosphate-buffered saline and lysed using Passive Lysis Buffer (Promega, Madison, WI). Rho B fluorescence was measured in the lysates with 544-nm excitation and 590-nm emission filters, using a Fluoroscan Ascent CF
fluorescence microplate reader (Thermo Labsystems, Vantaa, Finland). Protein concentrations in the lysates were measured using Bradford reagent (Sigma-Aldrich) and an MRX microplate reader (Dynex Technologies, Chantilly, VA) according to the manufacturer's recommended protocol.
ANIMALS
C57BL/6 female mice (10- to 12-week-old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed at the University of Massachusetts Medical School (UMMS) animal facility. Animals received humane care in accordance with protocols approved by the UMMS Institutional Animal Use and Care Committee. Mice (n = 6-9/group) were acclimated to a Lieber-DeCarli liquid diet of 5% eth-anol (Et0H) (volume [vol]/vol) over a period of 1 week, then maintained on the 5% diet for 4 weeks. Pair-fed (PF) control mice were fed a calorie-matched dextran-maltose diet. Animals had unrestricted access to water throughout the entire experimental period. In treated groups, mice were intraperitone-ally treated 5 days/week with vehicle (empty HDL) or the TREM-1 inhibitory formulations GF9-HDL (2.5 mg of GF9/kg) or GA/E31-HDL (4 mg equivalent of GF9/kg) (SignaBlok, Shrewsbury, MA) from the first day on a 5% Et0H diet. At the end of all animal experiments, cheek blood samples were collected in serum collection tubes (BD Biosciences, San Jose, CA) and processed within an hour. After blood collections, mice were euthanized and liver samples were harvested and stored at -80 C
until further analysis.
TOTAL PROTEIN ISOLATION FROM LIVER
Total protein was extracted from liver samples using radio immunoprecipitation assay buffer (BP-115; Boston BioProducts) supplemented with protease inhibitor cocktail tablets (11836153001; Roche) and Phospho Stop phosphatase inhibitor (04906837001;
Roche). Cell debris was removed from cell lysates by 10 minutes centrifugation at 2,000 rpm.
BIOCHEMICAL ASSAYS AND CYTOKINES
Serum ALT levels were determined by the kinetic method using commercially available reagents from Teco Diagnostics (Anaheim, CA). Cytokine levels were measured in serum samples, and whole-liver lysates were diluted in assay diluent following the manufacturer's instructions. Specific anti-mouse enzyme-linked immunosorbent assay (ELISA) kits were used for the quantification of MCP-1, TNF-a (BioLegend Inc., San Diego, CA), and IL-ip (R&D
Systems, Minneapolis, MN) levels. For normalization, the total protein concentration of the whole-liver lysate was determined using the Pierce bicinchoninic acid protein assay.
WESTERN BLOT ANALYSIS
Whole-liver proteins were boiled in Laemmli's buffer. Samples were resolved in 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel under reducing conditions, using a Tris-glycine buffer system; resolved proteins were transferred onto a nitrocellulose membrane.
SYK proteins were detected by specific primary antibodies (SYK, 2712 [Cell Signaling];
phospho-SYKY525/526, ab58575 [Abeam]) followed by an appropriate secondary horseradish peroxidase-conjugated immunoglobulin G antibody from Santa Cruz Biotechnology, p-actin, detected by an ab49900 antibody (Abeam), was used as a loading control. The specific immunoreactive bands of interest were visualized by chemiluminescence (Bio-Rad Laboratories) using the Fujifilm LAS-4000 luminescent image analyzer.
RNA EXTRACTION AND QUANTITATIVE REALTIME POLYMERASE
CHAIN REACTION ANALYSIS
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen) according to the manufacturer's instructions with on-column deoxyribonuclease treatment. RNA
was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and complementary DNA
synthesis was performed using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories) and 1 ug total RNA. Real-time quantitative polymerase chain reaction (PCR) was performed using Bio-Rad iTaq Universal SYBR Green Supermix and a CFX96 real-time detection system (Bio-Rad Laboratories). Relative gene expression was calculated by the comparative AAACt method. The expression level of target genes was normalized to the housekeeping gene 18S ribosomal RNA in each sample, and the fold change in the target gene expression among experimental groups was expressed as a ratio. Primers were synthesized by IDT, Inc.; exemplary sequences are listed in Table 1.
LIVER HISTOPATHOLOGY
Sections of formalin-fixed paraffin-embedded liver specimens from mice were stained with hematoxylin and eosin (H&E) or F4/80 (MF48000; Thermo Fisher Scientific) and MPO
(ab9535; Abeam) antibodies for immunohistochemistry (IHC). The fresh-frozen samples were stained with Oil Red 0 at the UMMS Diabetes and Endocrinology Research Center histology core facility.
STATISTICAL ANALYSIS
Statistical analyses were performed using GraphPad Prism 7.02 (GraphPad Software Inc.). Significance levels were determined using one-way analysis of variance followed by a post-hoc test for multiple comparisons. Data are shown as mean SEM, and differences were considered statistically significant when P < 0.05.
REFERENCES related to sections on ALD.
1) Szabo G, Bala S, Petrasek J, Gattu A. Gut-liver axis and sensing microbes.
Dig Dis 2010;28:737-744.
2) Bautista AP. Neutrophilic infiltration in alcoholic hepatitis. Alcohol 2002;27:17-21.
3) SzaboG, PetrasekJ, BalaS. Innate immunity and alcoholic liver disease. Dig Dis 2012;30(Suppl 1.):55-60.
4) Tessarz AS, Cerwenka A. The TREM-1/DAP12 pathway. Immunol Lett 2008;116:111-116.
5) Arts RJ, Joosten LA, Dinarello CA, Kullberg BJ, van der Meer JW, Netea MG.
interaction with the LPS/TLR4 receptor complex. Eur Cytokine Netw 2011;22:11-14.
6) Campanholle G, Mittelsteadt K, Nakagawa S, Kobayashi A, Lin SL, Gharib SA, et al. TLR-2/TLR-4 TREM-1 signaling pathway is dispensable in inflammatory myeloid cells during sterile kidney injury. PLoS One 2013;8:e68640.
7) Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature 2001;410:1103-1107.
8) Zysset D, Weber B, Rihs S, Brasseit J, Freigang S, Riether C, et al. TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis. Nat Commun 2016;7:13151.
9) Schenk M, Bouchon A, Seibold F, Mueller C. TREM-1¨expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases. J Clin Invest 2007;117:3097-3106.
10) Liao R, Sun TW, Yi Y, Wu H, Li YW, Wang FX, et al. Expression of TREM-1 in hepatic stellate cells and prognostic value in hepatitis B-related hepatocellular carcinoma. Cancer Sci 2012;103:984-992.
11) Wu J, Li J, Salcedo R, Mivechi NF, Trinchieri G, Horuzsko A. The proinflammatory myeloid cell receptor TREM-1 controls Kupffer cell activation and development of hepatocellular carcinoma. Cancer Res 2012;72:3977-3986.
12) ReadCB, KuijperJL,Hjorth SA, HeipelMD,TangX,Fleetwood AJ, et al. Cutting edge:
identification of neutrophil PGLYRP1 as a ligand for TREM-1.J Immunol 2015;194:1417-1421.
13) Sigalov AB. Multichain immune recognition receptor signaling: different players, same game? Trends Immunol 2004;25:583-589.
14) Sigalov AB. Immune cell signaling: a novel mechanistic model reveals new therapeutic targets. Trends Pharmacol Sci 2006;27:518-524.
15) Rojas MA, Shen ZT, Caldwell RB, Sigalov AB. Blockade of TREM-1 prevents vitreoretinal neovascularization in mice with oxygen-induced retinopathy. Biochim Biophys Acta Mol Basis Dis 2018;1864:2761-2768.
16) Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide inhibitors of TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017;21:2524-2534.
17) Shen ZT, Sigalov AB. Novel TREM-1 inhibitors attenuate tumor growth and prolong survival in experimental pancreatic cancer. Mol Pharm 2017;14:4572-4582.
18) Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-in-duced septic shock. Int Immunopharmacol 2014;21:208-219.
19) Shen ZT, Zheng S, Gounis MJ, Sigalov AB. Diagnostic magnetic resonance imaging of atherosclerosis in apolipoprotein E knockout mouse model using macrophage-targeted gadolinium-containing synthetic lipopeptide nanoparticles. PLoS One 2015;10:e0143453.
20) Sigalov AB. Nature-inspired nanoformulations for contrast-enhanced in vivo MR imaging of macrophages. Contrast Media Mol Imaging 2014;9:372-382.
21) Lieber CS, DeCarli LM. The feeding of alcohol in liquid diets: two decades of applications and 1982 update. Alcohol Clin Exp Res 1982;6:523-531.
22) Shen ZT, Sigalov AB. SARS coronavirus fusion peptide-derived sequence suppresses collagen-induced arthritis in DBA/If mice. Sci Rep 2016;6:28672.
23) Iracheta-Vellve A, Petrasek J, Satishchandran A, Gyongyosi B, Saha B, Kodys K, et al.
Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice. J Hepatol 2015;63:1147-1155.
24) Bukong TN, Iracheta-Vellve A, Saha B, Ambade A, Satishchandran A, Gyongyosi B, et al.
Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease. Hepatology 2016;64:1057-1071.
25) Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc 2013;8:627-637.
26) Ju C, Mandrekar P. Macrophages and alcohol-related liver inflammation.
Alcohol Res 2015;37:251-262.
27) Teli MR, Day CP, Burt AD, Bennett MK, James OF. Determinants of progression to cirrhosis or fibrosis in pure alcoholic fatty liver. Lancet 1995;346:987-990.
28) Terpstra V, van Berkel TJ. Scavenger receptors on liver Kupffer cells mediate the in vivo uptake of oxidatively damaged red blood cells in mice. Blood 2000;95:2157-2163.
29) Zingg JIM, Ricciarelli R, Azzi A. Scavenger receptors and modified lipoproteins: fatal attractions? ITJBMB Life 2000;49:397-403.
30) Gu BJ, Saunders BM, Jursik C, Wiley JS. The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonop-sonized particles and bacteria by a pathway attenuated by extracellular ATP. Blood 2010;115:1621-1631.
31) Sigola LB, Fuentes AL, Millis LM, Vapenik J, Murira A. Effects of Toll-like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis. Tissue Cell 2016;48:389-396.
32) Yu M, Romer KA, Nieland TJ, Xu S, Saenz-Vash V, Penman M, et al.
Exoplasmic cysteine Cys384 of the HDL receptor SR-BI is critical for its sensitivity to a small-molecule inhibitor and normal lipid transport activity. Proc Natl Acad Sci USA 2011;108:12243-12248.
33) Dong P, Xie T, Zhou X, Hu W, Chen Y, Duan Y, et al. Induction of macrophage scavenger receptor type BI expression by tamoxifen and 4-hydroxytamoxifen.
Atherosclerosis 2011;218:435-442.
34) Tammaro A, Derive M, Gibot S, Leemans JC, Florquin S, Dessing MC. TREM-1 and its potential ligands in non-infectious diseases: from biology to clinical perspectives. Pharmacol Ther 2017;177:81-95.
35) El Mezayen R, El Gazzar M, Seeds MC, McCall CE, Dreskin SC, Nicolls MR.
Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin.
Immunol Lett 2007;111:36-44.
36) Gibot S, Buonsanti C, Massin F, Romano M, Kolopp-Sarda MN, Benigni F, et al.
Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock. Infect Immun 2006;74:2823-2830.
37) Bala S, Marcos M, Gattu A, Catalano D, Szabo G. Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals. PLoS One 2014;9:e96864.
38) Arts RJ, Joosten LA, van der Meer JW, Netea MG. TREM-1: intracellular signaling pathways and interaction with pattern recognition receptors. J Leukoc Biol 2013;93:209-215.
39) Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and signal integration.
Nat Immunol 2006;7:1266-1273.
40) Bleharski JR, Kiessler V, Buonsanti C, Sieling PA, Stenger S, Colonna M, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol 2003;170:3812-3818.
41) Hong D, Li LF, Gao HC, Wang X, Li CC, Luo Y, et al. High-density lipoprotein prevents endoplasmic reticulum stress-induced downregulation of liver LOX-1 expression.
PLoS One 2015;10:e0124285.
42) Feingold KR, Grunfeld C. The role of HDL in innate immunity. J Lipid Res 2011;52:1-3.
43) Tobias PS, Ulevitch RJ. Control of lipopolysaccharide-high density lipoprotein binding by acute phase protein(s). J Immunol 1983;131:1913-1916.
44) Drew BG, Carey AL, Natoli AK, Formosa MF, Vizi D, Reddy-Luthmoodoo M, et al.
Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus. J Lipid Res 2011;52:572-581.
45) Mashima R, Yamamoto Y, Yoshimura S. Reduction of phosphatidylcholine hydroperoxide by apolipoprotein A-I: purification of the hydroperoxide-reducing proteins from human blood plasma. J Lipid Res 1998;39:1133-1140.
Taken together, this highlights the urgent need for novel approaches to prevent, treat and/or diagnose these diseases. However, it should be noted that the techniques and compositions listed and described herein are applicable to a broad range of disease states including, but not limiting to, cardiovascular disease, bacterial infectious diseases, diabetes, and autoimmune diseases. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
VI. Imaging probes.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to an imaging probe. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, liver diseases, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other rheumatic diseases, sepsis and other inflammatory diseases or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, including but not limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases.
VII. Exempalry Methods Of Providing Synthetic (recombinant) Lipopeptide Particles (SLPs or rHDLs) And Synthetic Peptides.
In one embodiment, the invention provides methods for making SLPs. The method comprises co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids.
The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof. The co-dissolving is conducted for a time period sufficient to allow the mixture to self-assemble into structures whereby particles are formed. The method further comprises isolating particles that have a size of between about 5 to about 200 nm diameter.
The lipid of the method may include PC, PE, PS, PI, PG, CL, SM, DOTAP or PA.
In certain embodiments, the invention provides a method for making SLP comprising co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with a predetermined amount of cholesterol, a predetermined amount of triglycerides and/or cholesteryl ester. The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of sodium cholate and a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof The co-dissolving is conducted for a time period sufficient to allow the components to coalesce into particles. The method still further comprises removing sodium cholate from the mixture, and isolating particles that have a size of between about 5 to about 200 nm diameter. The lipid of the method may include PC, PE, PS, PI, PG, CL, SM, DOTAP, or PA.
In one embodiment, in the methods of the present disclosure, the peptides and compounds of the invention are pre-formulated into synthetic lipopeptide particles (SLP). In one embodiment, SLPs are discoidal in shape. In one embodiment, SLPs are spherical in shape.
While the size of the particles is preferably between 5 nm and 50 nm, the diameter may be up to 200 nm. In one embodiment, the lipid of the particles may include cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, or a triacylglycerol. And further, the phospholipid may include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), or phosphatidic acid (PA), and any combinations thereof.
And even further, the cationic lipid can be 1,2-dioleoy1-3-trimethylammonium-propane (DOTAP). The lipid of the synthetic nanoparticle may be polyethylene glycol(PEG)ylated. In one embodiment, lipid is conjugated to at least one imaging probe.
In certain embodiments, an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, TC99111, Cr51, Ga67, Ga68, Cu64, Rb82,m099, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, Cll, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol, or a combination thereof.
In one embodiment, the imaging agent is a GBCA for MM. In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as PET imaging systems (and combined PET/CT and PET/MRI systems). In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In certain embodiments, the modulators and compositions described herein are incorporated into long half-life SLP. In certain embodiments, the modulators and compositions described herein may incorporate into lipopeptide particles (LP) in vivo upon administration to the individual. In certain embodiments, the peptides and compositions of the invention can cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention provides for a method for suppressing tumor growth in an individual in need thereof by administering to the individual an amount of a TREM-1 inhibitor that is effective for suppressing tumor growth.
A. Discoidal SLP (dSLP).
In one embodiment, the invention provides a method for making discoidal SLP
(dSLP).
The method comprises co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids. The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof The co-dissolving is conducted for a time period sufficient to allow the mixture to self-assemble into structures whereby particles are formed. The method further comprises isolating particles that have a size of between about 5 to about 200 nm diameter.
B. Spherical SLP (sSLP).
In one embodiment, the invention provides a method for making spherical SLP
(sSLP) comprising co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with a predetermined amount of cholesterol, a predetermined amount of triglycerides and/or cholesteryl ester. The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of sodium cholate and a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof The co-dissolving is conducted for a time period sufficient to allow the components to coalesce into particles. The method still further comprises removing sodium cholate from the mixture, and isolating particles that have a size of between about 5 to about 200 nm diameter.
From second prov C. Peptides.
Synthetic peptides, including trifunctional peptides of the present invention may include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH2(CH2)õCOOH
wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;
citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
Naturally occurring residues are divided into groups based on common side chain properties:
(1) hydrophobic: norleucine, methioninc (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe);
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr);
(3) acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu);
(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), Arginine (Arg);
(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro);
(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His);
(7) polar: Ser, Thr, Asn, Gln;
(8) basic positively charged: Arg, Lys, His; and;
(9) charged: Asp, Glu, Arg, Lys, His Analogues may be generated by substitutional mutagenesis and retain the biological activity of the original trifunctional peptides. Examples of substitutions identified as "conservative substitutions" are shown in TABLE 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened for their capability of executing three functions.
TABLE 1. Amino acid substitutions.
Amino acid substitution Original residue Exemplary substitution Conservative substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gin, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gin, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu TABLE 2A. Exemplary Trifunctional Peptides and Compositions.
## Exemplary Trifunctional Peptides and Compositions 2 GFL SKSLVFGEEM(0)RDRARAHV
4 GFL SKSLVFWQEEM(0)ELYRQKV
GFL SR SLVF GEEMRDRARAHV
6 GFL SR SLVF GEEM(0)RDRARAHV
8 GFL SRSLVFWQEEM(0)ELYRQKV
GLLSKSLVFGEEM(0)RDRARAHV
12 GLL SKSLVFWQEEM(0)ELYRQKV
GFL SKSLVFPLGEEMRDRARAHVDALRTHLA
17 GFL SKSLVFPLGEEM(0)RDRARAHVDALRTHLA
18 GFL SK SLVFPYLDDFQKKWQEEM(0)ELYRQKVE
GFL SKSLVFPYLDDFQKKWQEEMELYRQKVERGD
21 [64Cu]GFL SKSLVFGEEM(0)RDRARAHV
22 [64Cu]GFLSKSLVFWQEEM(0)ELYRQKV
23 [64Cu]GFL SK SLVFPLGEEM(0)RDRARAHVDALRTHLA
24 [64Cu]GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE
LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
26 LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
27 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
28 [64Cu]LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
29 LQEEDAGEYGCMGEEM(0)RDRARAHV
LQEEDAGEYGCMWQEEM(0)ELYRQKV
31 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
32 LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
33 [64Cu]L QVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
34 [64Cu]LQVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
35 LQVTD SGLYRCVIYHPPGEEM(0)RDRARAHV
36 LQVTD SGLYRCVIYHPPWQEEM(0)ELYRQKV
41 [64Cu]MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
42 [64Cu]MWKTPTLKYFPYLDDF QKKWQEEMELYRQKVE
45 [64Cu] GAR SMTL TVQARQLPLGEEMRDRARAHVDALRTHLA
46 [64Cu]GARSMTLTVQARQLPYLDDF QKKWQEEMELYRQKVE
49 [64Cu]GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
50 [64Cu]GVLRLLLFKLPYLDDF QKKWQEEMELYRQKVE
53 [64Cu]LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
54 [64Cu]LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
57 [64Cu]YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
58 [64C u] YLLD GILF IYP YLDDF QKKWQEEMELYRQKVE
61 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
62 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
65 [64Cu]FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
66 [64Cu]FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
69 [64Cu]IVIVDIC IT GPL GEEMRDRARAHVDALRTHLA
70 [64Cu]IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
75 [64C u] GNL VRICL GAPL GEEMRDRARAHVD ALRTHL A
76 [64C u] GNL VRICL GAP YLDDF QKKWQEEMELYRQKVE
79 [64Cu]VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA
80 [64C u] VIVIGDL VL T VLP YLDDF QKKWQEEMELYRQKVE
83 [64Cu]LVAADAVASLPLGEEMRDRARAHVDALRTHLA
84 [64Cu]LVAADAVASLPYLDDF QKKWQEEMELYRQKVE
91 PtxGFL SK SLVFPLGEEMRDRARAHVDALRTHLA
92 PtxGFL SK SLVFPYLDDF QKKWQEEMELYRQKVE
93 PtxGFL SK SLVFPLGEEM(0)RDRARAHVDALRTHLA
94 PtxGFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
TABLE 2B. Exemplary Trifunctional Peptides and Compositions.
## Exemplary Trifunctional Peptides and Compositions 3 GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
4 GFLSKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD
7 [64C u] GF L SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
8 [64Cu]GFL SKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
9 LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
11 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
12 [64Cu]LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
13 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
14 LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
[64Cu]LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
16 [64Cu]LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
19 [64Cu]MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
[64Cu]MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE
23 [64Cu] GAR SMTL TVQARQLPLGEEMRDRARAHVDALRTHLA
24 [64Cu]GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE
GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
27 [64Cu]GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
28 [64Cu]GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE
LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE
31 [64Cu]LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
32 [64Cu]LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
35 [64Cu]YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
36 [64Cu]YLLDGILFIYPYLDDF QKKWQEEMELYRQKVE
39 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
40 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
43 [64Cu]FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
44 [64Cu]FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
47 [64Cu]IVIVDIC IT GPL GEEMRDRARAHVDALRTHLA
48 [64Cu]IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
53 [64Cu]GNLVRICLGAPLGEEMRDRARAHVDALRTHLA
54 [64Cu]GNLVRICLGAPYLDDF QKKWQEEMELYRQKVE
57 [64C1.1]VMGDLVLTVLPLGEEIVIRDRARAHVDALRTHLA
58 [64CU]VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE
61 [64Cu]LVAADAVASLPLGEEMRDRARAHVDALRTHLA
62 [64Cu]LVAADAVASLPYLDDFQKKWQEEMELYRQKVE
69 Ptx-GFLSKSLVFPLGEEMRDRARAHVDALRTHLA
70 Ptx-GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
71 Ptx-GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
72 Ptx-GFLSKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
TREM-1 inhibitory trifunctional SCHOOL peptides In certain embodiments, the present invention relates to amphipathic TREM-1 inhibitory trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating cancer in combination with other cancer therapies. In one embodiment, these peptides may possess the antitumor activity. In one embodiment, these peptides may not possess the antitumor activity.
In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on myeloid cells including but not limited to, macrophages.
In one embodiment, the TREM-1 inhibitory trifunctional SCHOOL peptides (TRIOPEPs) of the present invention form self-assembling SLP in vitro. In one embodiment, TRIOPEPs are incorporated into self-assembled nanosized SLP of discoidal or spherical morphology (dSLP and sSLP, respectively) that contain apo A-I peptide fragments comprising 22 amino acid residue-long peptide sequences of the apo A-I helix 4 and/or helix 6. In one embodiment, the TREM-1 inhibitory trifunctional SCHOOL peptides described herein form naturally long half life lipopeptide particles in vivo. In certain embodiments, the present invention relates to peptides consisting of two amino acid domains, wherein upon binding to lipid or lipid mixtures, one amino acid domain assists in the self-assembly of naturally long half-life lipopeptide particles and targets these particles to macrophages, whereas another amino acid domain inhibits TREM-1/DAP-12 receptor complex expressed on macrophages.
In some embodiments of the present inventions, TABLE 3 presents a list of the peptides and therapeutic compositions that includes, but is not limited to the trifunctional SCHOOL
peptide-based TREM-1 inhibitors and therapeutic compositions that can be used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
Exemplary TREM-1 inhibitory trifunctional SCHOOL peptides include but are not limited to, 31 amino acid-long peptide TREM-1 inhibitory peptides GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID
NO. 26) and GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID NO. 27). In one embodiment, methionine residues of the peptides GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 25) and GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 24) are unmodified. See TABLE 3.
In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to a drug compound (TA). In one embodiment, TA is selected from the group including, but not limited to, anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, and therapeutic peptides. In one embodiment, the TA is a hydrophobic therapeutic agent. The TA
may also be selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to an imaging probe. In one embodiment, the imaging agent is GBCA for MM. In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as a PET imaging system and combined PET/CT and PET/MRI
systems.
In one embodiment, an imaging probe and/or an additional TA is conjugated to any or both of the domains. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof.
Embodiments Of TREM-1 inhibitory SCHOOL peptides.
Normal transmembrane interactions between the TREM-1 and the DAP-12 dimer forming a functional TREM-1/DAP-12 receptor complex comprise positively charged lysine amino acid within the TREM-1 transmembrane portion and negatively charged aspartic acid pairs in a DAP-12 dimer, thereby allowing subunit association (See FIG. 49).
In one embodiment, the simplest TREM-1 inhibitory SCHOOL agents would be synthetic peptides and their variants (SCHOOL peptides) that correspond to the TREM-1 and/or DAP-12 transmembrane domains or their functionally important minimal protein sequences as disclosed in US 8,513,185, US 9,981,004 and US 20190117725. Although it is not necessary to understand the mechanism of an invention, it is believed that interactions between a lysine residue of SCHOOL peptides that correspond to the TREM-1 transmembrane domain or its functionally important minimal protein sequence and an aspartic acid residue of a DAP-12 dimer disrupt the interactions between TREM-1 and DAP-12 in the membrane, thereby "disconnecting" TREM-1 and resulting in a non-functioning receptor. Accordingly, it is believed that interactions between an aspartic residue of SCHOOL peptides that correspond to the DAP-12 transmembrane domain or its functionally important minimal protein sequence and lysine amino acid residue of the TREM-1 transmembrane domain disrupt the interactions between DAP-12 and TREM-1 in the membrane, thereby "disconnecting" DAP-12 and resulting in a non-functioning receptor. These peptide variants and compositions possess the advantages typically associated with a fully synthetic material and yet possess certain desirable features of materials derived from natural sources.
In some embodiments of the present inventions, TABLE 3 presents a list of the peptides and therapeutic compositions that includes, but is not limited to the SCHOOL
peptide-based TREM-1 inhibitors and their variants that can be designed as disclosed in US
8,513,185, US
9,981,004 and US 20190117725 and used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
In some embodiments, the SCHOOL peptides and their variants that inhibit TREM-transmembrane signaling can be used in a free form. Exemplary TREM-1 inhibitory SCHOOL
peptides include but are not limited to, a 9 amino acid-long peptide TREM-1 inhibitory peptide GF9 (GFLSKSLVF) disclosed in US 8,513,185, US 9,981,004 and US 20190117725 and described in (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Rojas et al. 2018).
Although it is not necessary to understand the mechanism of an invention, it is believed that free SCHOOL peptide self-inserts into the cell membrane from outside the cell, co-localizes with TREM-1/DAP-12 receptor complex and disrupts the protein-protein interactions between TREM-1 and DAP-12, thereby resulting in a non-functional receptor complex that does not provide TREM-1 transmembrane signaling upon binding to a putative TREM-1 ligand(s) (See FIG. 49, Route 1). In one embodiment, FIG. 50 demonstrates colocalization of GF9 with the TREM-1 in the cell membrane. These peptide variants and compositions possess the advantages typically associated with a fully synthetic material and yet possess certain desirable features of materials derived from natural sources.
As described in (Vlieghe et al. 2010, Lau et al. 2018), the main limitations generally attributed to therapeutic peptides are: a short half-life because of their rapid degradation by proteolytic enzymes of the digestive system and blood plasma; rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance);
poor ability to cross physiological barriers because of their general hydrophilicity; high conformational flexibility, resulting sometimes in a lack of selectivity involving interactions with different receptors/targets (poor specific biodistribution), causing activation of several targets and leading to side effects;
eventual risk of immunogenic effects; and high synthetic and production costs (the production cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500 Da molecular mass small molecule by more than 10-fold but clearly not 100-fold).
In some embodiments of the present invention, the SCHOOL peptides and their variants that inhibit TREM-1 transmembrane signaling can be formulated into self-assembling SLP of discoidal (sSLP) or spherical (sSLP) shape that mimic human naturally long half-life high density lipoproteins (HDL) and are disclosed in US 20130045161 and US
20110256224 and described in (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Rojas et al. 2018, Tornai et al. 2019). Although it is not necessary to understand the mechanism of an invention, it is believed that these particles provide targeted delivery of the incorporated SCHOOL peptides to target cells and increase half life of these peptides in circulation. In some embodiments, these SLP contain the modified amphipathic apolipoprotein A-I peptide fragments that not only assist in the self-assembly of SLP but also provide targeted delivery of these particles to target cells in vitro and in vivo. In some embodiments, the modification represents a sulfoxidation of methionine amino acid residue in the apo A-I peptide sequence.
In one embodiment, FIG. 49 presents a schematic representation of targeted delivery of the TREM-1 modulatory SCHOOL peptides by SLP to myeloid cells including but not limited to, macrophages including TAMs. Although it is not necessary to understand the mechanism of an invention, it is believed that SLP that contain TREM-1 modulatory SCHOOL
peptides (exemplary shown for GF9) are endocytosed by macrophages through scavenger receptor(s), and then release the incorporated SCHOOL peptide, which self-inserts into the cell membrane from inside the cell, co-localizes with TREM-1/DAP-12 receptor complex and disrupts the protein-protein interactions between TREM-1 and DAP-12, thereby resulting in a non-functional receptor complex that does not induce TREM-1 transmembrane signaling upon binding to a putative TREM-1 ligand(s) (See FIG. 49, Route 2).
Modulators of TREM-1/DAP-12 signaling pathway.
Modulators (inhibitors) of TREM-1/DAP-12 signaling pathway can be nonexclusively divided into two major categories: those that inhibit TREM-1 transmembrane signaling by blocking binding of TREM-1 to its ligand(s) (type I inhibitors; See FIG. 49) and those that employ a ligand binding-independent mechanism of action and modulate (inhibit) mediated transmembrane signaling by disrupting protein-protein interactions between TREM-1 and DAP-12 in the cell membrane (type II inhibitors; See FIG. 50). Type I
inhibitors can be, in turn, subdivided into two subtypes: those that bind to TREM-1 (type Ia inhibitors) and those that bind to TREM-1 ligand(s) (type lb inhibitors).
Type I TREM-1 inhibitors.
In one embodiment, exemplary TREM-1 type I inhibitors include but not limited to, antagonistic (blocking, inhibiting) anti-TREM-1 antibodies and/or their fragments such as antibodies that block and inhibit TREM-1 disclosed in US 9,000,127 and US
9,550,830 and described in (Brynjolfsson et al. 2016). These TREM-1 inhibitors are believed to block binding of TREM-1 to its ligand(s) by binding to the extracellular domain of TREM-1 (type Ia inhibitors, See FIG. 49).
In one embodiment, exemplary TREM-1 type I inhibitors include but not limited to, synthetic peptides derived from a part of the extracellular domain of either TREM-1 such as Pl, P3 and LP17 peptides disclosed in US 20160193288, US 20150232531, US 8,013,836 and US
9,273,111 and described in (Gibot et al. 2004, Gibot et al. 2006) or the TREM-like transcript-1 (TLT-1) such as LR17 and LR12 peptides disclosed in US 20160193288, US
20160015773, US
20150232531, US 9,255,136; US 9,657,081 and US 9,815.883 and described in (Derive et al.
2012). These TREM-1 inhibitors are believed to act as an endogenous decoy receptor (type lb inhibitors, See FIG. 1) by binding TREM-1 ligands and preventing their engagement to membrane-bound TREM-1 (Pelham et al. 2014).
In some embodiments of the present invention, the TREM-1 type I inhibitors can be used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
In some embodiments of the present inventions, TABLE 3 presents a list of the peptides and peptide analogues that includes, but is not limited to the TREM-1 type lb peptide inhibitors and their variants that can be designed as disclosed in US 20160193288, US
20150232531, US
8,013,836, US 9,273,111, US 20160015773, US 9,255,136; US 9,657,081 and US
9,815.883 and described in (Gibot et al. 2004, Gibot et al. 2006, Derive et al. 2012) and used in order to treat tumors in combinations with other cancer therapies.
Type II TREM-1 inhibitors..
Application of the Signaling Chain HOmoOLigomerization (SCHOOL) model of receptor signaling described in (Sigalov et al. 2004, Sigalov 2004, Sigalov 2006, Sigalov 2018) to the transmembrane signal transduction mediated by a TREM-1 receptor suggested that an inhibition of TREM-1/DAP-12 signaling may be achieved by using transmembrane-targeted agents (SCHOOL agents) which specifically disrupt interactions between TREM-1 and DAP-12 subunits in the cell membrane (See FIG. 2), thereby disconnecting TREM-1 and DAP-12 and .. resulting in a non-functioning TREM-1/DAP-12 receptor complex.
In some embodiments of the present invention, the TREM-1 type II inhibitors can be used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
As described in (Tammaro et al. 2017), although TREM-1 appears to be activated by damage associated molecular patterns (DAMPs) that are shared by other pattern recognition receptors (PRRs), no TREM-1 specific (endogenous) ligand has been discovered to date. It is unknown why these ligands, specifically, share TREM-1 activation. Neither it is known what they have in common, but this information could certainly be of use in the determination of new specific ligands. This makes ligand binding-independent type II TREM-1 inhibitors advantageous compared to type I inhibitors that attempt to block binding TREM-1 to its yet unknown ligand(s).
In some embodiments, type II TREM-1 inhibitors include but are not limited to, the TREM-1 inhibitory SCHOOL peptides. The preferred peptides and compositions of the present invention comprise the TREM-1 modulatory peptide sequences designed using the SCHOOL
.. model of TREM-1 signaling and capable of modulating TREM-1 receptor expressed on myeloid cells as disclosed in US 8,513,185 and US 9,981,004 and described in (Sigalov 2010, Shen and Sigalov 2017).
Listed below in TABLE 2 are reported transmembrane sequences of TREM-1 and DAP-12 in a number of species. These regions are highly conserved and the substitutions between species are very conservative. This suggests a functional role for the transmembrane regions of both, TREM-1 and DAP-12, constituents of the complex. These regions strongly interact between themselves, thus maintaining the integrity of the TREM-1/DAP-12 receptor signaling complex in resting cells. These transmembrane domains are short and should be easily mimicked by synthetic peptides and compounds. In some embodiments, synthetic peptides and compounds .. are contemplated that may provide successful treatment options in the clinical setting.
TABLE 2C Sequence comparison of TREM-1 and DAP-12 transmembrane regions (accession codes are given in parenthesis).
SEQUENCE
SPECIES
HUMAN IVILLAGGFL SKSLVF S VLF A GVLAGIVMGDLVLTVLIALAV
(Q9NP99) (043914) MOUSE VTI S VIC GLL SKSLVF IILF I GVLAGIVLGDLVLTLLIALAV
(Q9JKE2) (054885) BOVIN IIIPAACGLL SKTLVFIGLF A GVLAGIVLGDLMLTLLIALAV
(Q6QUN5) (Q95J79) SHEEP not known GVLAGIVLGDLMLTLLIALAV
(Q95KS5) RAT not known GVLAGIVLGDLVLTLLIALAV
(Q6X9T7) PIG ILPAVCGLLSKSLVFIVLFVV GILAGIVLGDLVLTLLIALAV
(Q6TYI6) (Q9TU45) CLUSTAL W 2.0 multiple sequence alignment:
ILPAVCGLLSKSLVFIVLFVV
*:***:*** ** *:*****:***:**:******
TABLE 3. Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions.
##
Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions 3 (GFLSKSLVF)2 GVLAGIVMGDLVLTVLIALAV
GFL SK SLVF GEEMRDRARAHV
11 GFL SK SLVF GEEM(0)RDRARAHV
13 GFL SK SLVFWQEEM(0)ELYRQKV
GFL SR SL VF GEEM(0)RDRARAHV
17 GFL SRSLVFWQEEM(0)ELYRQKV
19 GLL SK SLVFGEEM(0)RDRARAHV
GLL SK SLVFWQEEMELYRQKV
21 GLL SK SLVFWQEEM(0)ELYRQKV
GFL SK SLVFPYLDDFQKKWQEEMELYRQKVE
26 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
27 GFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
[64C u] GF L SK SLVF GEEM(0)RDRARAHV
31 [64Cu]GFL SK SLVFWQEEM(0)ELYRQKV
34 GFL SK SL VF PL GEEM(0)RDRARAHVDALRTHL A
35 GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
38 [64Cu]GFL SK SL VF PL GEEM(0)RDRARAHVDALRTHL A
39 [64Cu]GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
40 LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
41 LQEEDAGEYGCMPYLDDF QKKWQEEM(0)ELYRQKVE
42 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
43 [64Cu]LQEEDAGEYGCMPYLDDF QKKWQEEM(0)ELYRQKVE
44 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
45 LQVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
46 [64Cu]L QVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
47 [64Cu]L QVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
48 LQEEDAGEYGCMGEEM(0)RDRARAHV
49 LQEEDAGEYGCMWQEEM(0)ELYRQKV
52 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
53 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
54 IIVTDVIATLPLGEEM(0)RDRARAHVDALRTHLA
5 IIVTDVIATLPYLDDF QKKWQEEM(0)ELYRQKVE
56 [64Cu]IIVTDVIATLPLGEEM(0)RDRARAHVDALRTHLA
57 [64Cu]IIVTDVIATLPYLDDF QKKWQEEM(0)ELYRQKVE
58 PtxGFLSKSLVFPLGEEMRDRARAHVDALRTHLA
59 PtxGFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
60 PtxGFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
61 PtxGFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE
64 IVILLAGGFLSKSLVFSVLFAPLGEEM(0)RDRARAHVDALRTHLA
65 IVILLAGGFLSKSLVFSVLFAPYLDDFQKKWQEEM(0)ELYRQKVE
TABLE 3 - Continuted ## Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions 3 (GFLSKSLVF)2 GVLAGIVMGDLVLTVLIALAV
GFLSKSLVFGEEMRDRARAHV
11 GFLSKSLVFGEEM(0)RDRARAHV
13 GFLSKSLVFWQEEM(0)ELYRQKV
15 GFL SR SL VF GEEM(0)RDRARAHV
17 GFL SRSLVFWQEEM(0)ELYRQKV
19 GLL SK SLVFGEEM(0)RDRARAHV
21 GLL SK SLVFWQEEM(0)ELYRQKV
26 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
27 GFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
30 [64C u] GF L SK SLVF GEEM(0)RDRARAHV
31 [64Cu]GFL SK SLVFWQEEM(0)ELYRQKV
34 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
35 GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
38 [64C u] GF L SK SL VF PL GEEM (0)RDRARAHVD ALRTHL A
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
9189746.
.. 102. Zhang Y, Huo M, Zhou J, Xie S. PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel. Comput Methods Programs Biomed 2010;
99:306-14. PMID: 20176408.
103. Scanu A, Hughes WL. Further characterization of the human serum D 1.063-1.21, alpha-lipoprotein. J Clin Invest 1962; 41:1681-9. PMID: 14497795.
104. Furman RH, Sanbar SS, Alaupovic P, Bradford RH, Howard RP. Studies of the Metabolism of Radioiodinated Human Serum Alpha Lipoprotein in Normal and Hyperlipidemic Subjects. J Lab Clin Med 1964; 63:193-204. PMID: 14125106.
105. Shen ZT, Sigalov AB. SARS Coronavirus Fusion Peptide-Derived Sequence Suppresses Collagen-Induced Arthritis in DBA/1J Mice. Sci Rep 2016; 6:28672. PMID:
27349522.
.. 106. Yanachkov I, Zavizion B, Metelev V, Stevens LJ, Tabatadze Y, Yanachkova M, et al.
Self-neutralizing oligonucleotides with enhanced cellular uptake. Org Biomol Chem 2017;
15:1363-80. PMID: 28074950.
107. Huber LC, Distler JH, Moritz F, Hemmatazad H, Hauser T, Michel BA, et al.
Trichostatin A prevents the accumulation of extracellular matrix in a mouse model of bleomycin-induced skin fibrosis. Arthritis Rheum 2007; 56:2755-64. PMID: 17665426.
108. Toyama T, Asano Y, Akamata K, Noda S, Taniguchi T, Takahashi T, et al.
Tamibarotene Ameliorates Bleomycin-Induced Dermal Fibrosis by Modulating Phenotypes of Fibroblasts, Endothelial Cells, and Immune Cells. J Invest Dermatol 2016; 136:387-98. PMID:
26967475.
109. Yamamoto T, Takagawa S, Katayama I, Yamazaki K, Hamazaki Y, Shinkai H, et al.
Animal model of sclerotic skin. I: Local injections of bleomycin induce sclerotic skin mimicking scleroderma. J Invest Dermatol 1999; 112:456-62. PMID: 10201529.
110. Yamamoto T, Takahashi Y, Takagawa S, Katayama I, Nishioka K. Animal model of sclerotic skin. II. Bleomycin induced scleroderma in genetically mast cell deficient WBB6F1-W/W(V) mice. J Rheumatol 1999; 26:2628-34. PMID: 10606374.
111. Avci P, Sadasivam M, Gupta A, De Melo WC, Huang YY, Yin R, et al. Animal models of skin disease for drug discovery. Expert Opin Drug Discov 2013; 8:331-55.
PMID: 23293893.
112. Artlett CM. Animal models of systemic sclerosis: their utility and limitations. Open Access Rheumatol 2014; 6:65-81. PMID: 27790036.
113. Beyer C, Schett G, Distler 0, Distler JH. Animal models of systemic sclerosis: prospects and limitations. Arthritis Rheum 2010; 62:2831-44. PMID: 20617524.
114. Ruzehaji N, Avouac J, Elhai M, Frechet M, Frantz C, Ruiz B, et al.
Combined effect of genetic background and gender in a mouse model of bleomycin-induced skin fibrosis. Arthritis Res Ther 2015; 17:145. PMID: 26025306.
115. Bhattacharyya S, Wang W, Morales-Nebreda L, Feng G, Wu M, Zhou X, et al.
Tenascin-C drives persistence of organ fibrosis. Nat Commun 2016; 7:11703. PMID:
27256716.
116. Hubner RH, Gitter W, El Mokhtari NE, Mathiak M, Both M, Bolte H, et al.
Standardized quantification of pulmonary fibrosis in histological samples. Biotechniques 2008; 44:507-11, 14-7. PMID: 18476815.
117. Lakota K, Hanumanthu VS, Agrawal R, Cams M, Armanios M, Varga J. Short lymphocyte, but not granulocyte, telomere length in a subset of patients with systemic sclerosis.
Ann Rheum Dis 2019. PMID: 30679155.
118. Yamashita T, Lakota K, Taniguchi T, Yoshizaki A, Sato S, Hong W, et al.
An orally-active adiponectin receptor agonist mitigates cutaneous fibrosis, inflammation and microvascular pathology in a murine model of systemic sclerosis. Sci Rep 2018; 8:11843.
PMID: 30087356.
.. 119. Chang HI, Yeh MK. Clinical development of liposome-based drugs:
formulation, characterization, and therapeutic efficacy. Int J Nanomedicine 2012; 7:49-60.
PMID: 22275822.
120. Adler-Moore JP, Gangneux JP, Pappas PG. Comparison between liposomal formulations of amphotericin B. Med Mycol 2016; 54:223-31. PMID: 26768369.
121. Tricoci P, D'Andrea DM, Gurbel PA, Yao Z, Cuchel M, Winston B, et al.
Infusion of Reconstituted High-Density Lipoprotein, CSL112, in Patients With Atherosclerosis: Safety and Pharmacokinetic Results From a Phase 2a Randomized Clinical Trial. J Am Heart Assoc 2015;
4:e002171. PMID: 26307570.
122. Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide inhibitors of TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017. PMID:
28382703.
123. Deshmukh SV, Durston J, Shomer NH. Validation of the use of nonnaive surgically catheterized rats for pharmacokinetics studies. J Am Assoc Lab Anim Sci 2008;
47:41-5. PMID:
19049252.
124. Wang X, Song L, Li N, Qiu Z, Zhou S, Li C, et al. Pharmacokinetics and biodistribution study of paclitaxel liposome in Sprague-Dawley rats and Beagle dogs by liquid chromatography-tandem mass spectrometry. Drug Res (Stuttg) 2013; 63:603-6. PMID: 23842945.
125. Kuwahara Y, Shima Y, Shirayama D, Kawai M, Hagihara K, Hirano T, et al.
Quantification of hardness, elasticity and viscosity of the skin of patients with systemic sclerosis using a novel sensing device (Vesmeter): a proposal for a new outcome measurement procedure.
Rheumatology (Oxford) 2008; 47:1018-24. PMID: 18440998.
Additional Advantages of Using Peptides and Compositions as described herein.
As well-known in the art and described in Irby, et al. Mol Pharm 2017, 14:1325-1338, most anticancer chemotherapeutic agents as well as many other therapeutic agents (TA) are toxic and hydrophobic and cannot be administered by themselves as pure chemicals but have to be included in biocompatible formulations to enhance solubility, increase circulatory residence time of the therapeutic agents, minimize the undesirable side effects and alleviate drug resistance.
Numerous formulation approaches have been developed, including solid lipid particles, emulsions, liposomes, etc., however, the delivery of the poorly water soluble (hydrophobic, or lipophilic) pharmaceuticals remains especially problematic as most of the body compartments, including the blood circulation and intracellular fluids, represent an aqueous environment. As a result, the direct injection of hydrophobic TAs often results in harmful side effects due to hypersensitivity, hemolysis, cardiac and neurological symptoms.
As described in Vlieghe, et al. Drug Discov Today 2010, 15:40-56, the main limitations generally attributed to therapeutic peptides are: a short half-life because of their rapid degradation by proteolytic enzymes of the digestive system and blood plasma;
rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance); poor ability to cross physiological barriers because of their general hydrophilicity; high conformational flexibility, resulting sometimes in a lack of selectivity involving interactions with different receptors/targets (poor specific biodistribution), causing activation of several targets and leading to side effects; eventual risk of immunogenic effects; and high synthetic and production costs (the production cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500 Da molecular mass small molecule by more than 10-fold but clearly not 100-fold).
Consequently, there is need for more effective and low toxic therapies for PC
and other types of cancer as well as new formulations of hydrophobic drugs and therapeutic peptides to improve their targeted delivery, prolonged half-life, biocompatibility and therapeutic efficiency.
As described herein, it is surprisingly found that the peptides and compositions of the present invention capable of modulating the TREM-1 signaling pathway can be synthesized and used for targeted treatment of cancer and imaging. The advantageous trifunctional peptides and compositions are demonstrated by the present invention to solve numerous problems which otherwise are associated with high dosages of TAs and imaging probes required and the lack of control and reproducibility of formulations, especially in large-scale production.
As many other solid tumors, PC is characterized by a marked infiltration of macrophages into the stromal compartment (Shih 2006, Solinas 2009), a process, which is mediated by cancer-associated fibroblasts (CAFs) (FIG. 49) and plays a role in disease progression and its response to therapy. These tumor-associated macrophages (TAMs) secrete a variety of growth factors, cytokines, chemokines, and enzymes that regulate tumor growth, angiogenesis, invasion, and metastasis (Feurino 2006, Lewis and Pollard 2006, Shih 2006). High macrophage infiltration correlates with the promotion of tumor growth and metastasis development (Lin 2006, Lin 2001, Solinas 2009). In patients with PC, macrophage infiltration begins during the pre-invasive stage of the disease and increases progressively (Clark 2007). The number of TAMs is significantly higher in patients with metastases (Gardian 2012). Presence of TAMs in the PC
stroma correlates with increased angiogenesis (Esposito 2004), a known predictor of poor prognosis (Kuwahara 2003). TAM recruitment, activation, growth and differentiation are regulated by macrophage colony-stimulating factor (M-CSF, also known as colony-stimulating factor 1, CSF-1) (Elgert 1998, Varney 2005). High pretreatment serum M-CSF is a strong independent predictor of poor survival in PC patients (Groblewska 2007). In PC mouse models, blockade of M-CSF or its receptor not only suppresses tumor angiogenesis and lymphangiogenesis (Kubota 2009) but also improves response to T-cell checkpoint immunotherapies that target programmed cell death protein 1 (PD-1) and cytotoxic T lymphocyte antigen-4 (CTLA-4) (Zhu 2014).
Importantly, continuous M-CSF inhibition affects pathological angiogenesis but not healthy vascular and lymphatic systems outside tumors (Kubota 2009). In contrast to blockade of vascular endothelial growth factor (VEGF), interruption of M-CSF inhibition does not promote rapid vascular regrowth (Kubota 2009). Collectively, these findings further suggest that targeting TAMs is a promising strategy for treating cancer (Bowman and Joyce 2014, Jinushi and Komohara 2015, Komohara 2016).
Triggering receptor expressed on myeloid cells-1 (TREM-1) amplifies the inflammatory response (Colonna and Facchetti 2003) and is upregulated under inflammatory conditions including including cancer (Ho et al. 2008, Yuan et al. 2014, Nguyen et al.
2015), brain and spinal cord injuries (Li et al 2019) and acute pancreatitis (D. Y. Wang 2004).
For downstream signal transduction, TREM-1 is coupled to the immunoreceptor tyrosine-based activation motif (ITAM)-containing adaptor, DNAX activation protein of 12 kDa (DAP-12).
Activation of TREM-1/DAP-12 receptor complex enhances release of multiple cytokines including monocyte chemoattractant protein-1 (MCP-1; also referred to in the art as CCL2), tumor necrosis factor-a (TNFa), interleukin-la (IL-1a), IL-1I3, IL-6 and macrophage colony-stimulating factor 1 (CSF-1; also referred to in the art as M-CSF) (Schenk et al. 2007, Dower et al.
2008, Sigalov 2014, Shen et al. 2017, Shen et al. 2017, Rojas et al. 2018, Tornai et al. 2019).
Most of these cytokines are increased in cancer patients (Tjomsland et al. 2011, Rossi et al. 2015, Yako et al. 2016, Tsukamoto et al. 2018, Yoshimura 2018)and play a vital role in creating and sustaining inflammation in the tumor favorable microenvironment, thus affecting patient survival.
TREM-1 activation enhances release of multiple cytokines including monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-a (TNFa), interleukin-la (IL-1a), IL-113, IL-6 and M-CSF (Lagler 2009, Schenk 2007, Sigalov 2014). Most of these cytokines are increased in patients with PC (Tjomsland 2011, Yako 2016) and play a vital role in creating and sustaining inflammation in the tumor favorable microenvironment, thus affecting patient survival. Inhibition of TREM-1 lowers levels of proinflammatory cytokines and is a promising approach in a variety of inflammation-associated disorders (Colonna and Facchetti 2003, Pelham and Agrawal 2014, Schenk 2007, Shen and Sigalov 2017, Sigalov 2014).
Importantly, in contrast to cytokine blockers, blockade of TREM-1 can blunt excessive inflammation while preserving the capacity for microbial control (Weber 2014). In vitro silencing of TREM-1 suppresses cancer cell invasion (Ho 2008). In patients with non-small cell lung cancer (NSCLC), expression on TAMs is associated with cancer recurrence and poor survival:
patients with low TREM-1 expression have a 4-year survival rate of over 60%, compared with less than 20% in patients with high TREM-1 expression (Ho 2008).
Inhibition of TREM-1 lowers levels of proinflammatory cytokines and chemokines including CSF-1 (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Rojas et al.
2018, Tornai et al. 2019) and as recently demonstrated in experimental cancer including NSCLC, pancreatic cancer and liver cancer, TREM-1 blockade inhibits tumor growth and improves survival (Wu et al. 2012, Sigalov 2014, Shen and Sigalov 2017, Wu et al.
2019). In vitro silencing of TREM-1 suppresses cancer cell invasion (Ho et al. 2008). In patients with NSCLC, TREM-1 expression on TAMs is associated with cancer recurrence and poor survival: patients with low TREM-1 expression have a 4-year survival rate of over 60%, compared with less than 20% in patients with high TREM-1 expression (Ho et al. 2008). Importantly, in contrast to cytokine blockers, blockade of TREM-1 can blunt excessive inflammation while preserving the capacity for microbial control (Weber et al. 2014). Septic mice lacking DAP-12, a signaling adapter of TREM-1, have improved survival (Turnbull et al. 2005). Humans lacking DAP12 do not have problems resolving infections (Lanier 2009). TREM-1 blockade is safe in healthy and septic subjects (Cuvier et al. 2018, Francois et al. 2018). Taken together, these finding make TREM-1 a promising therapeutic target in oncology.
The present invention provides the low toxic peptides and compositions for targeted treatment of cancer, e.g. PC, and other myeloid cell-related diseases and conditions and the methods for predicting the efficacy of these compositions. The invention further provides a method of using these peptides and compositions. These and other objects and advantages of the invention, as well as additional inventive features, will be apparent from the description of the invention provided herein.
FIG. 14A-C presents the exemplary data showing inhibition of tumor growth (FIG. 14A) and TREM-1 blockade-mediated suppression of intratumoral macrophage infiltration (FIG. 14B, FIG. 14C) in the human pancreatic cancer BxPC-3 xenograft mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 in free form or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. (B) F4/80 staining. Results are expressed as the mean SEM (n = 4 mice per group). *,p < 0.05; **,p < 0.01, ****,p < 0.0001 (versus vehicle). (FIG. 14C) Representative F4/80 images from BxPC-3-bearing mice treated using different free and sSLP-bound SCHOOL TREM-1 inhibitory GF9 sequences including TREM-1/TRIOPEP-sSLP.
Scale bar= 200 pm.
C. Sepsis; Severe Sepsis and Septic Shock.
Sepsis is another disorder with a high mortality rate. Currently, no approved sepsis drugs are available and over 30 drug candidates have failed late-stage clinical trials. Sepsis refers to a potentially life-threatening complication of an infection. Sepsis occurs when endogenous chemicals released into the bloodstream to fight the infection trigger inflammatory responses throughout the body. This inflammation can trigger a cascade of changes that can damage multiple organ systems, causing them to fail. If sepsis progresses to septic shock, blood pressure drops dramatically, which may lead to death.
Anyone can develop sepsis, but it's most common and most dangerous in older adults or those with weakened immune systems. Risk factors include but are not limited to: young or elderly; Have a compromised immune system; Are already very sick, often in a hospital's intensive care unit; Have wounds or injuries, such as burns; Have invasive devices, such as intravenous catheters or breathing tubes; etc.
Early treatment of sepsis, usually with antibiotics and large amounts of intravenous fluids, improves chances for survival. While any type of infection: ncluding bacterial, viral or fungal, can lead to sepsis, the most likely varieties include: Pneumonia;
Abdominal infection;
Kidney infection; Bloodstream infection (bacteremia); etc.
The incidence of sepsis appears to be increasing in the United States. The causes of this increase may include: Aging population. Americans are living longer, which is swelling the ranks of the highest risk age group ¨ people older than 65; Drug-resistant bacteria. Many types of bacteria can resist the effects of antibiotics that once killed them.
These antibiotic-resistant bacteria are often the root cause of the infections that trigger sepsis.; Weakened immune systems. More Americans are living with weakened immune systems, caused by HIV, cancer treatments or transplant drugs.; etc.
Sepsis ranges from less to more severe. As sepsis worsens, blood flow to vital organs, such as brain, heart and kidneys, becomes impaired. Sepsis can also cause blood clots to form in organs and in arms, legs, fingers and toes, leading to varying degrees of organ failure and tissue death (gangrene). Most people recover from mild sepsis, but the mortality rate for septic shock is nearly 50 percent. Also, an episode of severe sepsis may place you at higher risk of future infections.
Sepsis may present as a three-stage syndrome, starting with sepsis and progressing through severe sepsis to septic shock. The goal is to treat sepsis during its early stage, before it becomes more dangerous.the following symptoms, plus a probable or confirmed infection: Body temperature above 101 F (38.3 C) or below 96.8 F (36 C); Heart rate higher than 90 beats a minute; Respiratory rate higher than 20 breaths a minute, etc.
Severe sepsis refers to having at least one of the following signs and symptoms, which indicate an organ may be failing: Significantly decreased urine output; Abrupt change in mental status; Decrease in platelet count; Difficulty breathing; Abnormal heart pumping function;
Abdominal pain; etc.
Septic shock refers to having at least one of the following signs and symptoms of severe sepsis, plus extremely low blood pressure that doesn't adequately respond to simple fluid replacement.
FIG. 15A-B presents the exemplary data showing improved survival of lipopolysaccharide (LPS)-challenged mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA
31 and GE
31 in free form (FIG. 15A) or incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology.
FIG. 15B. **, P = 0.001 to 0.01 as compared with vehicle-treated animals.
FIG. 16 presents exemplary data showing average weights of healthy C57BL/6 mice treated with increasing concentrations of an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE
31 in free form.
D. Rheumatoid arthritis (RA).
Rheumatoid arthritis (RA) refers to a chronic inflammatory disorder that can affect more than just your joints. In some people, the condition also can damage a wide variety of body systems, including the skin, eyes, lungs, heart and blood vessels.
RA affects as much as 1% of the worldwide population. There is no cure for RA
yet and up to 80% or more of RA patients are disabled after 20 years of symptoms.
Unlike the wear-and-tear damage of osteoarthritis, rheumatoid arthritis affects the lining of your joints, causing a painful swelling that can eventually result in bone erosion and joint deformity.
The inflammation associated with rheumatoid arthritis is what can damage other parts of the body as well. While new types of medications have improved treatment options dramatically, severe rheumatoid arthritis can still cause physical disabilities.
Signs and symptoms of rheumatoid arthritis may include: Tender, warm, swollen joints;
Joint stiffness that is usually worse in the mornings and after inactivity;
Fatigue, fever and weight loss; etc.
Early rheumatoid arthritis tends to affect your smaller joints first, particularly the joints that attach your fingers to your hands and your toes to your feet.
As the disease progresses, symptoms often spread to the wrists, knees, ankles, elbows, hips and shoulders. In most cases, symptoms occur in the same joints on both sides of your body.
About 40 percent of the people who have rheumatoid arthritis also experience signs and symptoms that don't involve the joints. Rheumatoid arthritis can affect many nonjoint structures, including: Skin; Eyes; Lungs; Heart; Kidneys; Salivary glands; Nerve tissue;
Bone marrow;
Blood vessels; etc.
Rheumatoid arthritis signs and symptoms may vary in severity and may even come and go. Periods of increased disease activity, called flares, alternate with periods of relative remission ¨ when the swelling and pain fade or disappear. Over time, rheumatoid arthritis can cause joints to deform and shift out of place.
Rheumatoid arthritis increases your risk of developing: Osteoporosis.
Rheumatoid arthritis itself, along with some medications used for treating rheumatoid arthritis, can increase your risk of osteoporosis ¨ a condition that weakens your bones and makes them more prone to fracture. Rheumatoid nodules. These firm bumps of tissue most commonly form around pressure points, such as the elbows. However, these nodules can form anywhere in the body, including the lungs. Dry eyes and mouth. People who have rheumatoid arthritis are much more .. likely to experience Sjogren's syndrome, a disorder that decreases the amount of moisture in your eyes and mouth. Infections. The disease itself and many of the medications used to combat rheumatoid arthritis can impair the immune system, leading to increased infections. Abnormal body composition. The proportion of fat compared to lean mass is often higher in people who have rheumatoid arthritis, even in people who have a normal body mass index (BMI). Carpal .. tunnel syndrome. If rheumatoid arthritis affects your wrists, the inflammation can compress the nerve that serves most of your hand and fingers. Heart problems. Rheumatoid arthritis can increase your risk of hardened and blocked arteries, as well as inflammation of the sac that encloses your heart. Lung disease. People with rheumatoid arthritis have an increased risk of inflammation and scarring of the lung tissues, which can lead to progressive shortness of breath.
Lymphoma. Rheumatoid arthritis increases the risk of lymphoma, a group of blood cancers that develop in the lymph system.
FIG. 17A-B presents the exemplary data showing average clinical arthritis score (FIG. 17A) and mean body weight (BW) changes (FIG. 17B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the collagen-induced arthritis (CIA) mice treated with an equimolar mixture of the sulfoxidized methionine residue-containing TREM-1-related trifunctional peptides (TREM-1/TRIOPEP) GA 31 and GE 31 incorporated into synthetic lipopeptide particles (SLP) particles of discoidal (TREM-1/TRIOPEP-dSLP) and spherical (TREM-1/TRIOPEP-sSLP) morphology. DEX, dexamethasone. *,p <0.05, **, p <0.01;
***,p <0.001 as compared with vehicle-treated or naive animals.
FIG. 42A-B presents exemplary data showing average clinical arthritis score (Collagen-induced arthritis: Score 42A) and Collagen-induced arthritis: Body weight change mean BW changes (42B) calculated as a percentage of the difference between beginning (day 24) and final (day 38) BWs of the CIA mice treated with PBS (vehicle), DEX, TREM-1-related control peptide G-TE21, TCR-related control peptide M-TK32, TCR-related trifunctional peptide M-VE32 or with TREM-1-related trifunctional peptides G-HV21 and G-KV21. In contrast to the relevant control peptides, G-HV21, G-KV21 and M-VE32 all ameliorate the disease (A) and are well-tolerated by arthritic mice (B). *, p <0.05, **,p <0.01; ***,p <0.001 as compared with vehicle-treated animals. Abbreviations: TREM-1, triggering receptor expressed on myeloid cells-1; CIA, collagen-induced arthritis; PBS, phosphate-buffer saline; DEX, dexamethasone;
TCR, T cell receptor; BW, body weight.
E. Retinopathy.
Pathological retinal neovascularization (RNV) causes angiogenesis-related vision impairment in retinopathy of prematurity (ROP), diabetic retinopathy (DR), and retinal vein occlusion (RVO), which are the most common causes of vision loss and blindness in each age group. Conventional therapeutic options include laser ablation and the anti-vascular endothelial growth factor (VEGF) therapy, which both have their limitations and complications. Laser therapy is often accompanied by corneal edema, anterior chamber reaction, intraocular hemorrhage, cataract formation, and intraocular pressure changes, while the VEGF-targeted therapy can be complicated by damage of healthy vessels, potential side effects on neurons, rapid vascular regrowth upon interrupting the VEGF blockade, and limited effectiveness in some patients.
F. Cirrhosis Of The Liver And Alcoholic Liver Disease.
The human liver is located in the upper right side of the abdomen below the ribs. It has many essential body functions. These include: producing bile, which helps your body absorb dietary fats, cholesterol, and vitamins A, D, E, and K; storing sugar and vitamins for later use by the body; removing toxins such as alcohol and bacteria from your system:
creating blood clotting proteins; etc.
Several of the most common causes of cirrhosis of the liver in the United States are long-term viral hepatitis C infection and chronic alcohol abuse. Obesity is also a cause of cirrhosis, although it is not as prevalent as alcoholism or hepatitis C. Obesity can be a risk factor by itself, or in combination with alcoholism and hepatitis C.
According to the The National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) and other components of the National Institutes of Health (NIH), cirrhosis can develop in women who drink more than two alcoholic drinks per day (including beer and wine) for many years. For men, drinking more than three drinks a day for years can put them at risk for cirrhosis.
However, the amount is different for every person, and this doesn't mean that everyone who has ever drunk more than a few drinks will develop cirrhosis. Cirrhosis caused by alcohol is usually the result of regularly drinking more than these amounts over the course of 10 or 12 years.
Cirrhosis causes the liver to shrink and harden. This makes it difficult for nutrient-rich blood to flow into the liver from the portal vein. The portal vein carries blood from the digestive organs to the liver. The pressure in the portal vein rises when blood can't pass into the liver. The end result is a serious condition called portal hypertension, in which the vein develops high blood pressure.
The unfortunate consequence of portal hypertension is that this high-pressure system causes a backup, which leads to esophageal varices (like varicose veins), which can then burst and bleed.
Cirrhosis of the liver refers to severe scarring of the liver and poor liver function seen at the terminal stages of chronic liver disease. The scarring is most often caused by long-term exposure to toxins such as alcohol or viral infections.
Alcoholic liver cirrhosis is directly related to alcohol intake and is the final phase of alcoholic liver disease. Symptoms including but not limited to: anemia (low blood levels due to too little iron); high blood ammonia level); high blood sugar levels;
leukocytosis (large amount of white blood cells) ; unhealthy liver tissue when a sample is removed from a biopsy and studied in a laboratory; liver enzyme blood tests that show the level of aspartate aminotransferase (AST) is two times that of alanine aminotransferase (ALT); low blood magnesium levels; low blood potassium levels; low blood sodium levels; portal hypertension; etc.
Alcoholic liver cirrhosis can cause serious complications. This is known as decompensated cirrhosis. Examples of these complications include: ascites, or a buildup of fluid in the stomach; encephalopathy, or mental confusion; internal bleeding, known as bleeding varices; jaundice, which makes the skin and eyes have a yellow tint; etc.
Those with this the more severe form of cirrhosis often require a liver transplant to survive.; etc.
According to the Cleveland Clinic, patients with decompensated alcoholic liver cirrhosis who receive a liver transplant have a five-year survival rate of 70 percent.
Alcoholic liver disease (ALD) occurs after years of heavy drinking. The chances of getting liver disease go up the longer you have been drinking and more alcohol you consume.
Typically, a person has drank heavily for at least eight years. The National Institute on Alcohol Abuse and Alcoholism defines heavy drinking as drinking five or more drinks in one day on at least five of the past 30 days.
Symptoms of alcoholic liver cirrhosis typically develop when a person is between the ages of 30 and 40. A human body will be able to compensate for it's liver's limited function in the early stages of the disease. As the disease progresses, symptoms will become more noticeable. The disease is common in people between 40 and 50 years of age.
Men are more likely to have this problem. However, Women are also more at-risk for alcoholic liver disease.
Women don't have as many enzymes in their stomachs to break down alcohol particles. Because of this, more alcohol is able to reach the liver and make scar tissue.
Alcoholic liver disease can also have some genetic factors. For example, some people are born with a deficiency in enzymes that help to eliminate alcohol. Obesity, a high-fat diet, and having hepatitis C can also increase a person's likelihood they will have alcoholic liver disease.
women may develop the disease after less exposure to alcohol than men. Some people may have an inherited risk for the disease. The disease is part of a progression. It may start with fatty liver disease, then progress to alcoholic hepatitis, and then to alcoholic cirrhosis. However, it's possible a person can develop alcoholic liver cirrhosis without ever having alcoholic hepatitis.
When a person drinks alcohol heavily over the course of decades, the body starts to replace the liver's healthy tissue with scar tissue. Doctors call this condition alcoholic liver cirrhosis.
Alcoholic liver disease affects millions of people globally and often leads to fibrosis and cirrhosis. Liver cirrhosis is the 12th leading cause of death in the United States and costs society more than $15 billions annually. Despite this profound economic and health impact, there are currently no approved drugs to treat ALD. Current treatments including corticosteroids, immunosuppressants, and antioxidants have multiple shortcomings including a high level of serious side effects and insufficient efficacy.
slow the disease's progress and reduce your symptoms.
In some emboidments, either or both of the TREM-1 rHDLS and TREM-1 trifunctional peptides may be used in combination with treatments including but not limited to: Medications:
including but not limited to corticosteroids, calcium channel blockers, insulin, antioxidant supplements, and S-adenosyl-L-methionine (SAMe).; Nutritional Counseling:
Alcohol abuse can lead to malnutrition.; Extra protein: Patients often require extra protein in certain forms to help reduce the likelihood for developing brain disease (encephalopathy).; Liver Transplant.; etc.
investigated the role of TREM-1 in ALD and the potential therapeutic effect of the TREM-1 inhibitory GF9-HDL and GA/ E31-HDL formulations in the Lieber-DeCarli ALD
mouse model.
1. TREM-1 BLOCKADE AMELIORATES EXPRESSION OF EARLY
FIBROSIS MARKER GENES INDUCED BY CHRONIC ALCOHOL CONSUMPTION.
The clinical progression of ALD is associated with liver fibrosis.27 Our mouse model of ALD mimics the early phase of the human disease, yet mRNA levels of early fibrosis markers Pro-Colla and a-SMA were significantly increased in alcohol-fed mice compared to PF controls in the whole-liver samples (FIG. 20A-B). Induction of these makers was remarkably attenuated in the vehicle-treated group and further decreased by the TREM-1 inhibitory formulations used (FIG. 20A-B).
FIG. 20A-B presents exemplary data showing TREM-1/TRIOPEP-sSLP suppresses the expression of fibrinogenesis marker molecules, FIG. 20A Pro-Collagen la and FIG. 20B a-Smooth Muscle Actin, at the RNA level, as measured in whole-liver lysates of mice with (alcohol-fed) and without (pair-fed) alcoholic liver disease (ALD).
* indicates significance level compared to the non-treated pair-fed (PF) group; # indicates significance level compared to the non-treated alcohol-fed group. o indicates significance level compared to the vehicle-treated alcohol-fed group. The significant levels are as follows: *, 0.05 > P> 0.01; **, 0.01 > P> 0.001; ***, 0.001 > P > 0.0001; ****, P <0.0001.
2. TREM-1 INHIBITORY FORMULATIONS AND HDL
AMELIORATE CHRONIC ALCOHOL-INDUCED LIVER INJURY AND
STEATOSIS.
We evaluated the impact of the TREM-1 inhibitors on hepatocyte damage and steatosis in liver. Serum ALT levels obtained during week 5 of the alcohol feeding showed significant increases in alcohol-fed mice compared to PF controls. This ALT
increase was attenuated in both TREM-1 inhibitor-treated groups, indicating attenuation of liver injury (Fig. 21A). Interestingly, vehicle treatment (HDL) also showed a similar protective effect (Fig. 21A).
Consistent with steatosis, we found a significant increase in Oil Red 0 staining in livers of alcohol-fed mice compared to PF controls (Fig. 21C). Oil Red 0 (Fig.
21B-D) and H& (Fig. 21D) staining revealed attenuation of steatosis in the alcohol-fed inhibitor-treated mice compared to both untreated and vehicle (HDL)-treated alcohol-fed groups (Fig. 21B-D).
FIG. 21A-D presents exemplary data showing that TREM-1/TRIOPEP-sSLP suppresses the production of alanine aminotransferase (ALT) in mice with alcoholic liver disease (ALD), as measured in serum of mice with (alcohol-fed) and without (pair-fed) ALD, in addition to improving indicators of liver disease and inflammation. * indicates significance level compared to the alcohol-fed group treated with vehicle ¨ synthetic lipopeptide particles of spherical morphology that contained an equimolar mixture of PE22 and PA22 (sSLP) but no inhibitory peptide GF9. # indicates significance level compared to the non-treated alcohol-fed group. Liver damage after 5 weeks of alcohol feeding and effect of TREM-1 pathway inhibition in a mouse model of ALD. sSLP, 5mg/kg treatement of TREM-1 peptide vs. TREM-1/TRIOPEP-sSLP. Cheek blood and livers were harvested at death. (FIG. 21A) Serum ALT
levels were measured using a kinetic method. Exemplary data showing TREM-sSLP suppresses alanine aminotransferase in serum of alcohol fed mice over TREM-1 peptide alone. (FIG. 21B-D) Liver sections were stained with (B,C) Oil Red 0 and (FIG.
21D) H&E
staining, and the lipid content was analyzed by ImageJ (FIG. 21B). * indicates significance level compared to the nontreated PF group; * indicates significance level compared to the nontreated alcohol-fed group; indicates significance level compared to the vehicle-treated alcohol-fed group. The numbers of the symbols sign the significant levels as the following: **OP < 0.05;
WooP < 0.01;*"/###P <0.001; ****P < 0.0001. ***, 0.001 > P> 0.0001; ##, 0.01 >
P> 0.001.
3. BLOCKADE OF TREM-1 SIGNALING REDUCES THE EXPRESSION
OF INFLAMMATION-ASSOCIATED GENES IN ALD IN MICE.
Previous reports showed that TREM-1 activation leads to the expression and release of proinflammatory cytokines and chemokines through nuclear factor kB activation, which also regulates the expression of TREM-1, providing a positive feedback loop on the expression of the receptor.4 Proinflammatory cytokine expression is increased in ALDA1-3123124, therefore, we hypothesized that TREM-1 signaling contributes to the amplification of proinflammatory pathways in ALD.
To evaluate this hypothesis, first we tested whole-liver mRNAs of Et0H-fed and PF mice with or without treatment with two different TREM-1 inhibitory formulations and a vehicle control in a 5-week alcohol administration model of ALD in mice.(25) We found that mRNA
levels of TREM-1 and MCP-1 were significantly increased in livers of alcohol-fed mice compared to PF controls (Fig. 1A,B).
In contrast, in mice treated with the TREM-1 inhibitors, both GF9-HDL and HDL inhibited alcohol-related changes in TREM-1; in addition, MCP-1 mRNA
levels corresponded to those of the PF controls (Fig. 1A,B). Although induction of TNF-a and IL-11s in alcohol-fed mice did not reach statistical significance compared to PF
controls, TREM-1 block-ade by GF9-HDL resulted in a significant inhibition of TNF-a mRNA in the alcohol-fed mice compared to vehicle treatment (Fig. 1C), while IL-lfi mRNA expression was also significantly attenuated by both the GF9-HDL and GA/E31-HDL formulations in the alcohol-fed as well as in the PF groups (Fig. ID). MIP-la mRNA levels were increased in alcohol-fed mice, but TREM-1 blockade with GF9-HDL or GA/E31-HDL significantly attenuated this increase compared to the vehicle control (Fig. IE). Regulated on activation, normal T cell expressed, and secreted (RANTES) mRNA levels did not change regardless of alcohol feeding or TREM-1 treatment (Fig. IF).
FIG. 45A-E TREM-1 pathway inhibition. TREM-1 pathway inhibition suppresses the expression of (FIG. 45A) TREM-1 and inflammatory cytokines (FIG. 45B) MCP-1, (FIG. 45C) TNF-a, (FIG. 45D) IL-113, and (FIG. 45E) MIP-la but not (F) RANTES at the mRNA level as measured in whole-liver lysates by real-time quantitative PCR. * indicates significance level compared to nontreated PF group; # indicates significance level compared to nontreated alcohol-fed group; o indicates significance level compared to vehicle-treated alcohol-fed group.
Significance levels are as follows: * /#/o P <0.05; ** /##/oo P <0.01; *** /000 P <0.001; ****P
<0.0001.
Abbreviation: CCL, chemokine (C-C motif) ligand.
Next, we used specific ELISA kits to assess the protein levels of cytokines in the serum and in whole-liver lysates (Fig. 2). We found a significant increase in MCP-1 level in the serum and liver and TNF-a in the liver of alcohol-fed mice compared to PF controls (Fig. 2A-D). All these alcohol-induced increases were prevented both in the serum and liver by administration of either TREM-1 inhibitor. Interestingly, we found attenuation of alcohol-induced liver MCP-1 and TNF-a induction even in the vehicle-treated (HDL only) groups (Fig. 2A-C).
The increase in total IL-lfs levels after alcohol feeding and its attenuation by TREM-1 inhibition did not reach statistical significance (Fig. 2D).
Because TREM-1 is a membrane-associated molecule that triggers SYK activation as one of its proximal signaling molecules and we previously found increased SYK
phosphorylation in liver in ALD/24" we EBtested the levels of total and activated phospho-SYK
(p_syKY525/526) in the livers. We found significantly increased total and p-SYKY525/s26 levels after alcohol feeding .. (Fig. 2E-G). Treatment with GA/E31-HDL significantly decreased the p-levels in alcohol-fed mice compared to the untreated and vehicle-treated alcohol-fed groups, while GF9-HDL decreased p-SYKYs25/526 levels compared to the vehicle-treated group.
(Fig. 2E,F).
FIG. 46AE-G TREM-1 blockade and inflammatory cytokine levels. TREM-1 blockade reduces inflammatory cytokine levels in (FIG. 46A) serum and (FIG. 46B-D) whole-liver lysates as .. measured with specific ELISA kits. (FIG. 46E-G) Total liver protein was analyzed for total SYK
and activated p-SYK Y525/526 expression by western blotting using 13-actin as a loading control.
Statistical analysis was performed by evaluating two blots (n = 4/group).*
indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##
P < 0.01; ***P <
0.001; **** /
5. BLOCKADE OF TREM-1 ACTIVATION REDUCES EXPRESSION OF
MACROPHAGE AND NEUTROPHIL MARKERS IN LIVER
In agreement with previous studies indicating that chronic alcohol use causes hepatic macrophage infiltration and activation/1'3'26^ we found increased expression of the Kupffer cell/macrophage markers F4/80 and CD68 at the mRNA level. Treatment with the inhibitors significantly attenuated alcohol-induced expression of both F4/80 and CD68 in the liver, indicating anti-significant decrease in F4/80 expression on paraffin-embedded liver sections by IHC in alcohol-fed mice treated with either GF9-HDL or GA/E31-HDL
compared to the Et0H-fed vehicle-treated group (Fig. 3C,D).
Neutrophil infiltration of the liver is a characteristic of alcoholic hepatitis; therefore, we investigated markers associated with this cell population. Expression of the neutrophil markers Ly6G and MPO were significantly increased in livers of alcohol-fed mice compared to PF
controls. This was fully prevented by TREM-1 blockade (Fig. 3E,F).
Interestingly, the HDL
vehicle alone also resulted in a decreasing trend of Ly6G and MPO expression in alcohol-fed mice; however, the GF9-HDL and GA/E31-HDL TREM-1 inhibitors significantly attenuated Ly6G and MPO levels even when compared to the vehicle-treated alcohol-fed mice (Fig. 3E,F).
MPO staining on IHC confirmed that both TREM-1 inhibitors significantly reduced WO-positive cell numbers compared to the untreated alcohol-fed control group (Fig. 3G,H).
FIG. 47A-H. Effects of TREM-1 inhibition. (FIG. 47A, FIG. 47B) TREM-1 inhibition suppresses the mRNA expression of macrophage cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47C, FIG. 47D) Both TREM-1 inhibitors attenuated F4/80 as shown by IHC. (FIG. 47E, FIG. 47F) TREM-1 inhibition suppresses the mRNA
expression of neutrophil cell markers in the liver as measured by real-time quantitative PCR. (FIG. 47G, H) Both TREM-1 inhibitors attenuated MPO-positive cell infiltration as shown by IHC. * indicates significance level compared to the nontreated PF group; # indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P <
0.05; ** /## P < 0.01;
### P < 0.001; **** /
6. TREM-1 INHIBITORY FORMULATIONS AND HDL AMELIORATE
CHRONIC ALCOHOL-INDUCED LIVER INJURY AND STEATOSIS
To further assess the effects of the TREM-1 inhibitors on mechanisms of lipid metabolism, we tested genes involved in lipid synthesis (sterol regulatory element binding transcription factor 1 [SREBF1] and acetyl-coenzyme A carboxylase 1 [ACC1]) along with the lipid accumulation marker perilipin-2 (ADRP) (FIG. 48A-C). Both TREM-1 inhibitors but not vehicle treatment prevented alcohol-induced up-regulation of SREBF1, ACC1, and ADRP at the mRNA level (FIG. 48A-C). To assess lipid oxidation, we tested peroxisome proliferator-activated receptor a (PPARa), carnitine palmitoyl transferase 1A (CPT1A), and medium-chain acyl-coenzyme A dehydrogenase (MCAD) mRNA levels in whole-liver samples (FIG.
48D-F).
Alcohol feeding significantly reduced mRNA expression of PPARa and CPT1A, while MCAD
had a decreasing trend. Both TREM-1 inhibitors as well as the vehicle treatment significantly increased PPARa and MCAD levels compared to the untreated alcohol-fed controls (FIG. 48D-F).
FIG. 48A-F Measurement of mRNA expression. mRNA expression of genes involved in (FIG.
48A, FIG. 48B) lipid synthesis (SERBF1, ACC1), (FIG. 48C) the lipid accumulation marker (ADRP), and (FIG. 48D-F) lipid oxidation (PPARa, CPT1a, MCAD) were measured in whole liver.
* indicates significance level compared to the nontreated PF group; #
indicates significance level compared to the nontreated alcohol-fed group; o indicates significance level compared to the vehicle-treated alcohol-fed group. Significance levels are as follows: * /#/o P < 0.05; ** /##/oo P
<0.01; ### P <0.001; ****P <0.0001.
7. GF9HDL AND GA/E31HDL IS MAINLY MEDIATED BY S R A
We studied the uptake of GF9-HDL and GA/ E31-HDL in vitro in order to evaluate potential mechanisms of targeted delivery of GF9 (GA/E31). Kupffer cells and recruited hepatic macrophages express high levels of SRs, including SR-A, that are involved in phagocytosis and removal of oxidatively damaged lipoproteins and cells from the blood circulation.28'29 We previously demonstrated intracellu-SR lar macrophage delivery of GF9, GA31, and GE31 by macrophage-targeted GF9-HDL and GA/E31-HDL, respectively, and hypothesized that the observed macrophage endocytosis of these complexes is SR mediated.16'17 See, FIG 9A1 and 9A2. To further investigate the molecular mechanisms involved in this process, we used J774 macrophages as a model for Kupffer cells and incubated them with rho B-labeled GF9-HDL or GA/ E31-HDL in the presence or absence of cytochalasin D, fucoidan, or BLT-1, which are known to inhibit all SRs,(30) SR-A,(31) or SR-BI,(32) respectively.
In the presence of cytochalasin D, which inhibits both SR-A and SR-BI, the macrophage uptake of both TREM-1 inhibitor complexes was significantly inhibited, suggesting that this uptake is SR mediated. Fucoidan, an SR-A inhibitor, substantially suppressed endocytosis of TREM-1 inhibitor complexes at 22 hours but not at 4 hours, indicating time-dependent mechanisms of SR-A-mediated endocytosis (Fig. 9B). In contrast, BLT-1, which inhibits SR-BI, similarly inhibited the uptake of the complexes at both time points but to a lesser extent compared with that of fucoidan (Fig. 9C), presumably because of lower expression of SR-BI on J774 macrophages(33'1) These findings suggest that SR-A is the main contributor in SR-mediated endocytosis of both GF9-HDL and GA/E31-HDL.
Interestingly, quantitatively determined macrophage uptake levels in the presence or absence of fucoidan or BLT-1 were similar for GF9-HDL and GA/E31-HDL (Fig.
7B). This suggests that the combination of GF9 and apo AT peptide sequences in GA31 and sequences does not change the level and mechanisms of macrophage endocytosis of GA/E31-HDL compared with those of GF9-HDL.
8. Summary of TREM-1 in ALD.
Using a mouse model, significant up-regulation of TREM-1 was measured in livers of mice following chronic alcohol feeding. Treatment with novel ligand-independent TREM-1 inhibitors reduced the expression of the TREM-1 molecule itself, attenuated or fully prevented alcohol-induced increases in proinflammatory cytokines at the mRNA level, and inhibited SYK
activation. TREM-1 blockade provided by trifunctional peptides described herein, results in reduced macrophage and neutrophil infiltration and activation indicated by reduced F4/80, .. CD68, Ly6G, and MPO expression in the liver. These findings complement data demonstrating that TREM-1 blockade using GF9-HDL and GA/ E31-HDL suppresses macrophage infiltration of the tumor in cancer mice.(Reference 17) The TREM-1 inhibitors attenuated alcohol-induced liver steatosis. HDL and the TREM-1 inhibitors also attenuated liver injury and markers of early fibrosis in alcohol-fed mice. Interestingly, the HDL vehicle control showed similar efficiency as .. the inhibitory formulations at the protein level of the proinflammatory cytokines. Thereforeitwas also discovered that rHDL itself has some protective effects on ALD at the level of ALT and lipid oxidation.
While the ligand of TREM-1 is still unknown, it has been shown that TREM-1 activation amplifies inflammation and synergizes with TLR signaling pathways.(34) It was also observed that bacterial infection and challenge with LPS or lipoteichoic acid increase expression,(7) indicating a positive feedback loop among PAMP exposure, TREM-1 expression, and inflammatory cytokine induction. Different DAMPs, such as 3-hydroxy-3-methyl-glutaryl Bl and heat shock protein 70, have been suggested to stimulate TREM-1,(35)' while other studies found cell (granulocyte and platelet)-surface-associated activators as well.(35,36) Both PAMPs and DAMPs are present in ALD, providing potential mechanisms for TREM-1 up-regulation in this disease. Alcohol induces changes in the gut microbiome and disrupts the gut barrier function, resulting in increased levels of endotoxin and microbial PAMPs in circulation.(1,37) Alcohol also causes hepatocyte damage that leads to the release of DAMP5,(23) and these processes contribute to TREM-1 activation.
TREM-1 signaling leads to phosphorylation and activation of SYK, which has been indicated as a major regulator in inflammatory processes in ALD.(38) TREM-1 also amplifies TLR4 signaling that involves activation of SYK, which has been indicated as a downstream SYK
activation and phosphorylation.(38) Indeed, we found increased total and phosphorylated SYK
levels in the livers of alcohol-fed mice that was attenuated by TREM-1 inhibitor administration.
A previous study showed that inhibition of SYK activation attenuates alcohol-induced liver inflammation, cell death, and steatosis, suggesting that the SYK pathway could be a feasible therapeutic target in ALD.(24) SYK is expressed in a wild spectrum of cells, while TREM-1 inhibition may specifically modulate macrophages, neutrophils, and stellate cells that each play a role in ALD. Another advantage of TREM-1 inhibition is that it likely attenuates signaling from a broader spectrum ofTLRs, in addition toTLR4.
TREM-1 activation alone has been shown to increase the production of proinflammatory chemo-kines and cytokines.(39) Furthermore, simultaneous stimulation of TREM-1 and TLRs by an agonistic anti-TREM-1 antibody and different TLR ligands synergized in the induction of these proinflammatory molecules. TREM-1 and TLR4 costimulated monocytes showed increased production of MCP-1, IL-113, and IL-8. In contrast, the level of the anti-inflammatory cytokine IL-10 decreased when anti-TREM-1 antibody and the TLR3 ligand poly(LC) or the TLR4 ligand LPS simultaneously attached to their receptors.(40) Because self-perpetuating proinflammatory pathways are present in alcoholic hepatitis, interruption of these pathways using TREM-1 inhibition seems attractive.
By inducing TNF-a, IL-6, MCP-1, IL-8, and granulocyte-macrophage colony-stimulating factor and inhibiting IL-10 production, TREM-1 is involved in activation and recruitment of monocytes and modulation of inflammatory responses.(40) Furthermore, TREM-1 expression was highly up-regulated on the surface of infiltrating monocytes and neutrophils in human tissues infected by bacteria, highlighting the importance of this receptor in these processes.(7) In alcoholic hepatitis, neutrophils infiltrate the liver, inducing oxidative stress and cytotoxicity that contributes to the high mortality of the disease.(2) We showed that these processes can be attenuated by TREM-1 inhibitors. Mechanistically, the GF9-HDL and GA/E31-HDL
formulations target the liver more efficiently than peptides alone and release the TREM-1 inhibitory sequences inside the target cells where these peptides likely inhibit TREM-1 signaling by disrupting the intramembrane interactions of the TREM-1 receptor and its signaling adaptor molecule death-associated protein 12 (Fig. 27).(15-17) It was contemplated that observed preferential endocytosis of GF9-HDL and HDL by macrophages and hepatic clearance of these complexes is mediated by SR
recognition of putative epitopes in the modified apo A-I peptide constituents of GF9-HDL
and GA/E31-HDL.(16,17,19) Findings described herein indicate that GF9-HDL and GA/ E31-HDL
are largely recognized by SR-A on macrophages (Fig. 9A-B). We also observed SR-BI-mediated uptake, which likely explains the previously observed hepatic clearance for these complexes in another animal model.(19) While these data confirm our hypothesis, future studies are needed to determine the clearance properties for GF9-HDL and GA/E31-HDL in ALD.
Further, our present study demonstrates that GF9-HDL and GA/E31-HDL exhibit not only similar macrophage uptake in vitro largely driven by SR-A (Fig. 9A1) but also similar therapeutic effect in a mouse model of ALD (Figs. 20-21). This is in line with our previous studies where GF9-HDL and GA/E31-HDL exhibited similar therapeutic activities in cancer and arthritic mice. (16,17) We suggest that SR-A epitopes are similarly exposed on GA31 and GE31 in GA/ E31-HDL and on PA22 and PE22 in GF9-HDL, providing similar uptake of these complexes and as a result delivery of TREM-1 inhibitory GF9 peptide sequences in vivo. The use of GA/E31-HDL in the further development of effective and low-toxicity therapy for ALD is advantageous because it makes the entire manufacturing process easier and less expensive. We also suggest that the in vitro macrophage uptake assay can be potentially used to predict the outcomes for macrophage-targeted TREM-1 therapy in vivo.
In addition to attenuating inflammatory processes, the TREM-1 inhibitory formulations also ameliorated hepatocyte damage and steatosis. Serum ALT and liver triglyceride levels were both decreased in the GF9-HDL, GA/E31-HDL, and HDL-vehicle treated groups. The vehicle also had an inhibitory effect on TNF-a and MCP-1 protein levels as well as on mRNA expression of neutrophil and fibrosis markers, indicating that the HDL vehicle formulation can attenuate inflammation to a moderate extent. A previous study found evidence that HDL
can protect hepatocytes from endoplasmic reticulum stress, (41) while other publications reported a scavenger function of HDL for LPS and lipoteichoic acid(42,43) that could prevent immune cells from being activated by those molecules.(42,43) Further, the observed moderate beneficial effect of HDL treatment alone on fatty acid oxidation markers in alcohol-exposed mice (Fig. 47A-C) is in line with data that demonstrate infusion of reconstituted HDL reduces fatty acid oxidation in patients with type 2 diabetes mellitus. (44) In human and rat plasma, apo A-I, the major protein of HDL, has been shown to inhibit lipid peroxidation. (45) These data might provide an explanation for our findings of the hepatoprotective effects of HDL.
Our study shows that TREM-1 inhibitors with HDL formulation exerted significant inhibition on early signaling events of proinflammatory processes at ^the level of cytokine mRNA and the activated p-SYK protein levels compared to the HDL vehicle alone in a mouse model of ALD. This effect presumably would be even more obvious at the protein level of cytokines in a more severe liver injury. However, in mice, the most commonly used 5-week alcohol feeding that weused resulted in moderate liver damage and minimal(25) inflammation, which is a limitation of our study. As shown on the stained liver sections, the GF9-HDL and GA/E31-HDL formulations significantly inhibited immune cell infiltration and steatosis compared to the HDL vehicle only in mice with ALD. Thus, in some emboidments, inhibitors, such as the trifuncitonal peptides described herein, are contemplate for administration to patients showing at least one sympton, or at risk of developing a sympton, for ALD for decreasing inflammation in liver tissue for reducing said symptom or delaying/preventing said sympton.
Materials and Methods, for example, in relation to experiments assocated with treating ALD.
REAGENTS AND CELLS
The murine macrophage J774A.1 cell line was purchased from ATCC (Manassas, VA).
Cytochalasin D was purchased from MP Biomedicals (Solon, OH). Blocker of lipid transport 1 (BLT-1) was purchased from Calbiochem (Torrey Pines, CA). Sodium cho-late, cholesteryl oleate, fucoidan, and other chemicals were purchased from Sigma-Aldrich (St.
Louis, MO). 1-Palmitoy1-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-dimyristoy1-0T-glycero-3-phosphoeth-anolamine-N-(lissamine rhodamine B sulfonyl) (rho EEB-PE), and cholesterol were purchased from Avanti Polar Lipids (Alabaster, AL).
PEPTIDE SYNTHESIS
The following synthetic peptides were ordered from Bachem (Torrance, CA): one 9-mer peptide, GFLSKSLVF (human TREM-1213- 221, GF9); two 22-mer methionine sulfoxidized peptides, PYLDDFQKKWQEEM(0)ELYRQKVE (H4) and PLG
EEM(0)RDRARAHVDALRTHLA (H6), which correspond to human apo A-I helices 4 (apo A-1123.144) and 6 (apo A-I167-188), respectively; and two 31-mer methionine sulfoxidized peptides, GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE (GE31) and GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (GA31).
LIPOPEPTIDE COMPLEXES
HDL-mimicking lipopeptide complexes of spherical morphology that contained either GF9 and an equimolar mixture of PE22 and PA22 (GF9-HDL) or an equimolar mixture of GA31 and GE31 (GA/ E31-HDL) were synthesized using the sodium cholate dialysis procedure, purified, and characterized as described.(16-18, 22) For GF9-HDL, the initial molar ratio was 125:6:2:3:1:210, corresponding to POPC:cholesterol:cholesteryl oleate:GF9:apo A-I:sodium cholate, respectively, where apo A-I was an equimolar mixture of PE22 and PA22. For GA/E31-HDL, the initial molar ratio was 125:6:2:1:210, corresponding to POPC:cholesterol:cholesteryl oleate:GA/ E31: sodium cholate, respectively, where GA/E31 was an equimolar mixture of GA31 and GE31.
A quantitative in vitro macrophage assay of endo-cytosis of rho B-labeled HDL-mimicking lipopeptide complexes by J774 macrophage was performed as described.(18-20) Briefly, BALB/c murine macrophage J774A.1 cells (ATCC) were cultured at 37 C
with 5% CO2 in Dulbecco's modified Eagle's medium (Cellgro Mediatech, Manassas, VA) with 2 mM
glutamine, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 10% heat-inactivated fetal bovine serum (Cellgro Mediatech) and grown to approximately 90% confluency in 12-well tissue culture plates (Corning Costar, Corning, NY). After reaching target confluency, cells were incubated for 1 hour in medium with or without fucoidan (400 ug/mL), BLT-1 ((1011M), or cytochalasin D (4011M). Cells were subsequently incubated for 4 hours and 22 hours at 37 C in medium containing 2 [EM of rho B-labeled GF9-HDL or GA/E31-HDL (as calculated for rho B).
Cells were washed twice using phosphate-buffered saline and lysed using Passive Lysis Buffer (Promega, Madison, WI). Rho B fluorescence was measured in the lysates with 544-nm excitation and 590-nm emission filters, using a Fluoroscan Ascent CF
fluorescence microplate reader (Thermo Labsystems, Vantaa, Finland). Protein concentrations in the lysates were measured using Bradford reagent (Sigma-Aldrich) and an MRX microplate reader (Dynex Technologies, Chantilly, VA) according to the manufacturer's recommended protocol.
ANIMALS
C57BL/6 female mice (10- to 12-week-old) were purchased from the Jackson Laboratory (Bar Harbor, ME) and housed at the University of Massachusetts Medical School (UMMS) animal facility. Animals received humane care in accordance with protocols approved by the UMMS Institutional Animal Use and Care Committee. Mice (n = 6-9/group) were acclimated to a Lieber-DeCarli liquid diet of 5% eth-anol (Et0H) (volume [vol]/vol) over a period of 1 week, then maintained on the 5% diet for 4 weeks. Pair-fed (PF) control mice were fed a calorie-matched dextran-maltose diet. Animals had unrestricted access to water throughout the entire experimental period. In treated groups, mice were intraperitone-ally treated 5 days/week with vehicle (empty HDL) or the TREM-1 inhibitory formulations GF9-HDL (2.5 mg of GF9/kg) or GA/E31-HDL (4 mg equivalent of GF9/kg) (SignaBlok, Shrewsbury, MA) from the first day on a 5% Et0H diet. At the end of all animal experiments, cheek blood samples were collected in serum collection tubes (BD Biosciences, San Jose, CA) and processed within an hour. After blood collections, mice were euthanized and liver samples were harvested and stored at -80 C
until further analysis.
TOTAL PROTEIN ISOLATION FROM LIVER
Total protein was extracted from liver samples using radio immunoprecipitation assay buffer (BP-115; Boston BioProducts) supplemented with protease inhibitor cocktail tablets (11836153001; Roche) and Phospho Stop phosphatase inhibitor (04906837001;
Roche). Cell debris was removed from cell lysates by 10 minutes centrifugation at 2,000 rpm.
BIOCHEMICAL ASSAYS AND CYTOKINES
Serum ALT levels were determined by the kinetic method using commercially available reagents from Teco Diagnostics (Anaheim, CA). Cytokine levels were measured in serum samples, and whole-liver lysates were diluted in assay diluent following the manufacturer's instructions. Specific anti-mouse enzyme-linked immunosorbent assay (ELISA) kits were used for the quantification of MCP-1, TNF-a (BioLegend Inc., San Diego, CA), and IL-ip (R&D
Systems, Minneapolis, MN) levels. For normalization, the total protein concentration of the whole-liver lysate was determined using the Pierce bicinchoninic acid protein assay.
WESTERN BLOT ANALYSIS
Whole-liver proteins were boiled in Laemmli's buffer. Samples were resolved in 10%
sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel under reducing conditions, using a Tris-glycine buffer system; resolved proteins were transferred onto a nitrocellulose membrane.
SYK proteins were detected by specific primary antibodies (SYK, 2712 [Cell Signaling];
phospho-SYKY525/526, ab58575 [Abeam]) followed by an appropriate secondary horseradish peroxidase-conjugated immunoglobulin G antibody from Santa Cruz Biotechnology, p-actin, detected by an ab49900 antibody (Abeam), was used as a loading control. The specific immunoreactive bands of interest were visualized by chemiluminescence (Bio-Rad Laboratories) using the Fujifilm LAS-4000 luminescent image analyzer.
RNA EXTRACTION AND QUANTITATIVE REALTIME POLYMERASE
CHAIN REACTION ANALYSIS
Total RNA was extracted using the Qiagen RNeasy kit (Qiagen) according to the manufacturer's instructions with on-column deoxyribonuclease treatment. RNA
was quantified using a Nanodrop 2000 spectrophotometer (Thermo Fisher Scientific), and complementary DNA
synthesis was performed using the iScript Reverse Transcription Supermix (Bio-Rad Laboratories) and 1 ug total RNA. Real-time quantitative polymerase chain reaction (PCR) was performed using Bio-Rad iTaq Universal SYBR Green Supermix and a CFX96 real-time detection system (Bio-Rad Laboratories). Relative gene expression was calculated by the comparative AAACt method. The expression level of target genes was normalized to the housekeeping gene 18S ribosomal RNA in each sample, and the fold change in the target gene expression among experimental groups was expressed as a ratio. Primers were synthesized by IDT, Inc.; exemplary sequences are listed in Table 1.
LIVER HISTOPATHOLOGY
Sections of formalin-fixed paraffin-embedded liver specimens from mice were stained with hematoxylin and eosin (H&E) or F4/80 (MF48000; Thermo Fisher Scientific) and MPO
(ab9535; Abeam) antibodies for immunohistochemistry (IHC). The fresh-frozen samples were stained with Oil Red 0 at the UMMS Diabetes and Endocrinology Research Center histology core facility.
STATISTICAL ANALYSIS
Statistical analyses were performed using GraphPad Prism 7.02 (GraphPad Software Inc.). Significance levels were determined using one-way analysis of variance followed by a post-hoc test for multiple comparisons. Data are shown as mean SEM, and differences were considered statistically significant when P < 0.05.
REFERENCES related to sections on ALD.
1) Szabo G, Bala S, Petrasek J, Gattu A. Gut-liver axis and sensing microbes.
Dig Dis 2010;28:737-744.
2) Bautista AP. Neutrophilic infiltration in alcoholic hepatitis. Alcohol 2002;27:17-21.
3) SzaboG, PetrasekJ, BalaS. Innate immunity and alcoholic liver disease. Dig Dis 2012;30(Suppl 1.):55-60.
4) Tessarz AS, Cerwenka A. The TREM-1/DAP12 pathway. Immunol Lett 2008;116:111-116.
5) Arts RJ, Joosten LA, Dinarello CA, Kullberg BJ, van der Meer JW, Netea MG.
interaction with the LPS/TLR4 receptor complex. Eur Cytokine Netw 2011;22:11-14.
6) Campanholle G, Mittelsteadt K, Nakagawa S, Kobayashi A, Lin SL, Gharib SA, et al. TLR-2/TLR-4 TREM-1 signaling pathway is dispensable in inflammatory myeloid cells during sterile kidney injury. PLoS One 2013;8:e68640.
7) Bouchon A, Facchetti F, Weigand MA, Colonna M. TREM-1 amplifies inflammation and is a crucial mediator of septic shock. Nature 2001;410:1103-1107.
8) Zysset D, Weber B, Rihs S, Brasseit J, Freigang S, Riether C, et al. TREM-1 links dyslipidemia to inflammation and lipid deposition in atherosclerosis. Nat Commun 2016;7:13151.
9) Schenk M, Bouchon A, Seibold F, Mueller C. TREM-1¨expressing intestinal macrophages crucially amplify chronic inflammation in experimental colitis and inflammatory bowel diseases. J Clin Invest 2007;117:3097-3106.
10) Liao R, Sun TW, Yi Y, Wu H, Li YW, Wang FX, et al. Expression of TREM-1 in hepatic stellate cells and prognostic value in hepatitis B-related hepatocellular carcinoma. Cancer Sci 2012;103:984-992.
11) Wu J, Li J, Salcedo R, Mivechi NF, Trinchieri G, Horuzsko A. The proinflammatory myeloid cell receptor TREM-1 controls Kupffer cell activation and development of hepatocellular carcinoma. Cancer Res 2012;72:3977-3986.
12) ReadCB, KuijperJL,Hjorth SA, HeipelMD,TangX,Fleetwood AJ, et al. Cutting edge:
identification of neutrophil PGLYRP1 as a ligand for TREM-1.J Immunol 2015;194:1417-1421.
13) Sigalov AB. Multichain immune recognition receptor signaling: different players, same game? Trends Immunol 2004;25:583-589.
14) Sigalov AB. Immune cell signaling: a novel mechanistic model reveals new therapeutic targets. Trends Pharmacol Sci 2006;27:518-524.
15) Rojas MA, Shen ZT, Caldwell RB, Sigalov AB. Blockade of TREM-1 prevents vitreoretinal neovascularization in mice with oxygen-induced retinopathy. Biochim Biophys Acta Mol Basis Dis 2018;1864:2761-2768.
16) Shen ZT, Sigalov AB. Rationally designed ligand-independent peptide inhibitors of TREM-1 ameliorate collagen-induced arthritis. J Cell Mol Med 2017;21:2524-2534.
17) Shen ZT, Sigalov AB. Novel TREM-1 inhibitors attenuate tumor growth and prolong survival in experimental pancreatic cancer. Mol Pharm 2017;14:4572-4582.
18) Sigalov AB. A novel ligand-independent peptide inhibitor of TREM-1 suppresses tumor growth in human lung cancer xenografts and prolongs survival of mice with lipopolysaccharide-in-duced septic shock. Int Immunopharmacol 2014;21:208-219.
19) Shen ZT, Zheng S, Gounis MJ, Sigalov AB. Diagnostic magnetic resonance imaging of atherosclerosis in apolipoprotein E knockout mouse model using macrophage-targeted gadolinium-containing synthetic lipopeptide nanoparticles. PLoS One 2015;10:e0143453.
20) Sigalov AB. Nature-inspired nanoformulations for contrast-enhanced in vivo MR imaging of macrophages. Contrast Media Mol Imaging 2014;9:372-382.
21) Lieber CS, DeCarli LM. The feeding of alcohol in liquid diets: two decades of applications and 1982 update. Alcohol Clin Exp Res 1982;6:523-531.
22) Shen ZT, Sigalov AB. SARS coronavirus fusion peptide-derived sequence suppresses collagen-induced arthritis in DBA/If mice. Sci Rep 2016;6:28672.
23) Iracheta-Vellve A, Petrasek J, Satishchandran A, Gyongyosi B, Saha B, Kodys K, et al.
Inhibition of sterile danger signals, uric acid and ATP, prevents inflammasome activation and protects from alcoholic steatohepatitis in mice. J Hepatol 2015;63:1147-1155.
24) Bukong TN, Iracheta-Vellve A, Saha B, Ambade A, Satishchandran A, Gyongyosi B, et al.
Inhibition of spleen tyrosine kinase activation ameliorates inflammation, cell death, and steatosis in alcoholic liver disease. Hepatology 2016;64:1057-1071.
25) Bertola A, Mathews S, Ki SH, Wang H, Gao B. Mouse model of chronic and binge ethanol feeding (the NIAAA model). Nat Protoc 2013;8:627-637.
26) Ju C, Mandrekar P. Macrophages and alcohol-related liver inflammation.
Alcohol Res 2015;37:251-262.
27) Teli MR, Day CP, Burt AD, Bennett MK, James OF. Determinants of progression to cirrhosis or fibrosis in pure alcoholic fatty liver. Lancet 1995;346:987-990.
28) Terpstra V, van Berkel TJ. Scavenger receptors on liver Kupffer cells mediate the in vivo uptake of oxidatively damaged red blood cells in mice. Blood 2000;95:2157-2163.
29) Zingg JIM, Ricciarelli R, Azzi A. Scavenger receptors and modified lipoproteins: fatal attractions? ITJBMB Life 2000;49:397-403.
30) Gu BJ, Saunders BM, Jursik C, Wiley JS. The P2X7-nonmuscle myosin membrane complex regulates phagocytosis of nonop-sonized particles and bacteria by a pathway attenuated by extracellular ATP. Blood 2010;115:1621-1631.
31) Sigola LB, Fuentes AL, Millis LM, Vapenik J, Murira A. Effects of Toll-like receptor ligands on RAW 264.7 macrophage morphology and zymosan phagocytosis. Tissue Cell 2016;48:389-396.
32) Yu M, Romer KA, Nieland TJ, Xu S, Saenz-Vash V, Penman M, et al.
Exoplasmic cysteine Cys384 of the HDL receptor SR-BI is critical for its sensitivity to a small-molecule inhibitor and normal lipid transport activity. Proc Natl Acad Sci USA 2011;108:12243-12248.
33) Dong P, Xie T, Zhou X, Hu W, Chen Y, Duan Y, et al. Induction of macrophage scavenger receptor type BI expression by tamoxifen and 4-hydroxytamoxifen.
Atherosclerosis 2011;218:435-442.
34) Tammaro A, Derive M, Gibot S, Leemans JC, Florquin S, Dessing MC. TREM-1 and its potential ligands in non-infectious diseases: from biology to clinical perspectives. Pharmacol Ther 2017;177:81-95.
35) El Mezayen R, El Gazzar M, Seeds MC, McCall CE, Dreskin SC, Nicolls MR.
Endogenous signals released from necrotic cells augment inflammatory responses to bacterial endotoxin.
Immunol Lett 2007;111:36-44.
36) Gibot S, Buonsanti C, Massin F, Romano M, Kolopp-Sarda MN, Benigni F, et al.
Modulation of the triggering receptor expressed on the myeloid cell type 1 pathway in murine septic shock. Infect Immun 2006;74:2823-2830.
37) Bala S, Marcos M, Gattu A, Catalano D, Szabo G. Acute binge drinking increases serum endotoxin and bacterial DNA levels in healthy individuals. PLoS One 2014;9:e96864.
38) Arts RJ, Joosten LA, van der Meer JW, Netea MG. TREM-1: intracellular signaling pathways and interaction with pattern recognition receptors. J Leukoc Biol 2013;93:209-215.
39) Klesney-Tait J, Turnbull IR, Colonna M. The TREM receptor family and signal integration.
Nat Immunol 2006;7:1266-1273.
40) Bleharski JR, Kiessler V, Buonsanti C, Sieling PA, Stenger S, Colonna M, et al. A role for triggering receptor expressed on myeloid cells-1 in host defense during the early-induced and adaptive phases of the immune response. J Immunol 2003;170:3812-3818.
41) Hong D, Li LF, Gao HC, Wang X, Li CC, Luo Y, et al. High-density lipoprotein prevents endoplasmic reticulum stress-induced downregulation of liver LOX-1 expression.
PLoS One 2015;10:e0124285.
42) Feingold KR, Grunfeld C. The role of HDL in innate immunity. J Lipid Res 2011;52:1-3.
43) Tobias PS, Ulevitch RJ. Control of lipopolysaccharide-high density lipoprotein binding by acute phase protein(s). J Immunol 1983;131:1913-1916.
44) Drew BG, Carey AL, Natoli AK, Formosa MF, Vizi D, Reddy-Luthmoodoo M, et al.
Reconstituted high-density lipoprotein infusion modulates fatty acid metabolism in patients with type 2 diabetes mellitus. J Lipid Res 2011;52:572-581.
45) Mashima R, Yamamoto Y, Yoshimura S. Reduction of phosphatidylcholine hydroperoxide by apolipoprotein A-I: purification of the hydroperoxide-reducing proteins from human blood plasma. J Lipid Res 1998;39:1133-1140.
Taken together, this highlights the urgent need for novel approaches to prevent, treat and/or diagnose these diseases. However, it should be noted that the techniques and compositions listed and described herein are applicable to a broad range of disease states including, but not limiting to, cardiovascular disease, bacterial infectious diseases, diabetes, and autoimmune diseases. Other features and advantages of the invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration, because various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
VI. Imaging probes.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to an imaging probe. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof In one embodiment, the present invention relates to the targeted treatment, prevention and/or detection of cancer including but not limited to lung, pancreatic, breast, stomach, prostate, colon, brain and skin cancers, cancer cachexia, atherosclerosis, allergic diseases, acute radiation syndrome, inflammatory bowel disease, empyema, acute mesenteric ischemia, hemorrhagic shock, multiple sclerosis, liver diseases, autoimmune diseases, including but not limited to, atopic dermatitis, lupus, scleroderma, rheumatoid arthritis, psoriatic arthritis and other rheumatic diseases, sepsis and other inflammatory diseases or other condition involving myeloid cell activation and, more particularly, TREM receptor-mediated cell activation, including but not limited to diabetic retinopathy and retinopathy of prematurity, Alzheimer's, Parkinson's and Huntington's diseases.
VII. Exempalry Methods Of Providing Synthetic (recombinant) Lipopeptide Particles (SLPs or rHDLs) And Synthetic Peptides.
In one embodiment, the invention provides methods for making SLPs. The method comprises co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids.
The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof. The co-dissolving is conducted for a time period sufficient to allow the mixture to self-assemble into structures whereby particles are formed. The method further comprises isolating particles that have a size of between about 5 to about 200 nm diameter.
The lipid of the method may include PC, PE, PS, PI, PG, CL, SM, DOTAP or PA.
In certain embodiments, the invention provides a method for making SLP comprising co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with a predetermined amount of cholesterol, a predetermined amount of triglycerides and/or cholesteryl ester. The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of sodium cholate and a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof The co-dissolving is conducted for a time period sufficient to allow the components to coalesce into particles. The method still further comprises removing sodium cholate from the mixture, and isolating particles that have a size of between about 5 to about 200 nm diameter. The lipid of the method may include PC, PE, PS, PI, PG, CL, SM, DOTAP, or PA.
In one embodiment, in the methods of the present disclosure, the peptides and compounds of the invention are pre-formulated into synthetic lipopeptide particles (SLP). In one embodiment, SLPs are discoidal in shape. In one embodiment, SLPs are spherical in shape.
While the size of the particles is preferably between 5 nm and 50 nm, the diameter may be up to 200 nm. In one embodiment, the lipid of the particles may include cholesterol, a cholesteryl ester, a phospholipid, a glycolipid, a sphingolipid, a cationic lipid, a diacylglycerol, or a triacylglycerol. And further, the phospholipid may include phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), cardiolipin (CL), sphingomyelin (SM), or phosphatidic acid (PA), and any combinations thereof.
And even further, the cationic lipid can be 1,2-dioleoy1-3-trimethylammonium-propane (DOTAP). The lipid of the synthetic nanoparticle may be polyethylene glycol(PEG)ylated. In one embodiment, lipid is conjugated to at least one imaging probe.
In certain embodiments, an imaging probe is selected from the group comprising Gd(III), Mn(II), Mn(III), Cr(II), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb(III) Dy(III), Ho(III), Eu(II), Eu(III), and Er(III), T1201, K42, In", Fe.59, TC99111, Cr51, Ga67, Ga68, Cu64, Rb82,m099, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, xe133, 1125, 1131, 1123, P32, Cll, N13, 015, Br76, Kr81, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol, or a combination thereof.
In one embodiment, the imaging agent is a GBCA for MM. In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as PET imaging systems (and combined PET/CT and PET/MRI systems). In one embodiment, the peptides and compositions of the invention are used in combinations thereof. In one embodiment, the peptides and compositions of the invention are used in combinations with other anticancer therapeutic agents. In certain embodiments, the modulators and compositions described herein are incorporated into long half-life SLP. In certain embodiments, the modulators and compositions described herein may incorporate into lipopeptide particles (LP) in vivo upon administration to the individual. In certain embodiments, the peptides and compositions of the invention can cross the blood-brain barrier (BBB), blood-retinal barrier (BRB) and blood-tumor barrier (BTB). Thus, in one aspect, the invention provides for a method for suppressing tumor growth in an individual in need thereof by administering to the individual an amount of a TREM-1 inhibitor that is effective for suppressing tumor growth.
A. Discoidal SLP (dSLP).
In one embodiment, the invention provides a method for making discoidal SLP
(dSLP).
The method comprises co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids. The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof The co-dissolving is conducted for a time period sufficient to allow the mixture to self-assemble into structures whereby particles are formed. The method further comprises isolating particles that have a size of between about 5 to about 200 nm diameter.
B. Spherical SLP (sSLP).
In one embodiment, the invention provides a method for making spherical SLP
(sSLP) comprising co-dissolving a predetermined amount of a mixture of neutral and/or charged lipids with a predetermined amount of cholesterol, a predetermined amount of triglycerides and/or cholesteryl ester. The method further comprises drying the mixture under nitrogen. The method even further comprises co-dissolving the dried mixture with a predetermined amount of sodium cholate and a predetermined amount of a trifunctional peptide or compound of the present invention or combinations thereof The co-dissolving is conducted for a time period sufficient to allow the components to coalesce into particles. The method still further comprises removing sodium cholate from the mixture, and isolating particles that have a size of between about 5 to about 200 nm diameter.
From second prov C. Peptides.
Synthetic peptides, including trifunctional peptides of the present invention may include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH2(CH2)õCOOH
wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe;
citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.
Naturally occurring residues are divided into groups based on common side chain properties:
(1) hydrophobic: norleucine, methioninc (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile), Histidine (His), Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe);
(2) neutral hydrophilic: Cysteine (Cys), Serine (Ser), Threonine (Thr);
(3) acidic/negatively charged: Aspartic acid (Asp), Glutamic acid (Glu);
(4) basic: Asparagine (Asn), Glutamine (Gin), Histidine (His), Lysine (Lys), Arginine (Arg);
(5) residues that influence chain orientation: Glycine (Gly), Proline (Pro);
(6) aromatic: Tryptophan (Trp), Tyrosine (Tyr), Phenylalanine (Phe), Histidine (His);
(7) polar: Ser, Thr, Asn, Gln;
(8) basic positively charged: Arg, Lys, His; and;
(9) charged: Asp, Glu, Arg, Lys, His Analogues may be generated by substitutional mutagenesis and retain the biological activity of the original trifunctional peptides. Examples of substitutions identified as "conservative substitutions" are shown in TABLE 1. If such substitutions result in a change not desired, then other type of substitutions, denominated "exemplary substitutions" in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened for their capability of executing three functions.
TABLE 1. Amino acid substitutions.
Amino acid substitution Original residue Exemplary substitution Conservative substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gin (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gin, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gin, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu TABLE 2A. Exemplary Trifunctional Peptides and Compositions.
## Exemplary Trifunctional Peptides and Compositions 2 GFL SKSLVFGEEM(0)RDRARAHV
4 GFL SKSLVFWQEEM(0)ELYRQKV
GFL SR SLVF GEEMRDRARAHV
6 GFL SR SLVF GEEM(0)RDRARAHV
8 GFL SRSLVFWQEEM(0)ELYRQKV
GLLSKSLVFGEEM(0)RDRARAHV
12 GLL SKSLVFWQEEM(0)ELYRQKV
GFL SKSLVFPLGEEMRDRARAHVDALRTHLA
17 GFL SKSLVFPLGEEM(0)RDRARAHVDALRTHLA
18 GFL SK SLVFPYLDDFQKKWQEEM(0)ELYRQKVE
GFL SKSLVFPYLDDFQKKWQEEMELYRQKVERGD
21 [64Cu]GFL SKSLVFGEEM(0)RDRARAHV
22 [64Cu]GFLSKSLVFWQEEM(0)ELYRQKV
23 [64Cu]GFL SK SLVFPLGEEM(0)RDRARAHVDALRTHLA
24 [64Cu]GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE
LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
26 LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
27 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
28 [64Cu]LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
29 LQEEDAGEYGCMGEEM(0)RDRARAHV
LQEEDAGEYGCMWQEEM(0)ELYRQKV
31 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
32 LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
33 [64Cu]L QVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
34 [64Cu]LQVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
35 LQVTD SGLYRCVIYHPPGEEM(0)RDRARAHV
36 LQVTD SGLYRCVIYHPPWQEEM(0)ELYRQKV
41 [64Cu]MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
42 [64Cu]MWKTPTLKYFPYLDDF QKKWQEEMELYRQKVE
45 [64Cu] GAR SMTL TVQARQLPLGEEMRDRARAHVDALRTHLA
46 [64Cu]GARSMTLTVQARQLPYLDDF QKKWQEEMELYRQKVE
49 [64Cu]GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
50 [64Cu]GVLRLLLFKLPYLDDF QKKWQEEMELYRQKVE
53 [64Cu]LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
54 [64Cu]LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
57 [64Cu]YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
58 [64C u] YLLD GILF IYP YLDDF QKKWQEEMELYRQKVE
61 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
62 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
65 [64Cu]FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
66 [64Cu]FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
69 [64Cu]IVIVDIC IT GPL GEEMRDRARAHVDALRTHLA
70 [64Cu]IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
75 [64C u] GNL VRICL GAPL GEEMRDRARAHVD ALRTHL A
76 [64C u] GNL VRICL GAP YLDDF QKKWQEEMELYRQKVE
79 [64Cu]VMGDLVLTVLPLGEEMRDRARAHVDALRTHLA
80 [64C u] VIVIGDL VL T VLP YLDDF QKKWQEEMELYRQKVE
83 [64Cu]LVAADAVASLPLGEEMRDRARAHVDALRTHLA
84 [64Cu]LVAADAVASLPYLDDF QKKWQEEMELYRQKVE
91 PtxGFL SK SLVFPLGEEMRDRARAHVDALRTHLA
92 PtxGFL SK SLVFPYLDDF QKKWQEEMELYRQKVE
93 PtxGFL SK SLVFPLGEEM(0)RDRARAHVDALRTHLA
94 PtxGFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
TABLE 2B. Exemplary Trifunctional Peptides and Compositions.
## Exemplary Trifunctional Peptides and Compositions 3 GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
4 GFLSKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
GFLSKSLVFPLGEEMRDRARAHVDALRTHLARGD
7 [64C u] GF L SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
8 [64Cu]GFL SKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
9 LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
11 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
12 [64Cu]LQEEDAGEYGCMPYLDDFQKKWQEEM(0)ELYRQKVE
13 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
14 LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
[64Cu]LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
16 [64Cu]LQVTD SGLYRCVIYHPPPYLDDFQKKWQEEM(0)ELYRQKVE
19 [64Cu]MWKTPTLKYFPLGEEMRDRARAHVDALRTHLA
[64Cu]MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE
23 [64Cu] GAR SMTL TVQARQLPLGEEMRDRARAHVDALRTHLA
24 [64Cu]GARSMTLTVQARQLPYLDDFQKKWQEEMELYRQKVE
GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
27 [64Cu]GVLRLLLFKLPLGEEMRDRARAHVDALRTHLA
28 [64Cu]GVLRLLLFKLPYLDDFQKKWQEEMELYRQKVE
LGKATLYAVLPYLDDFQKKWQEEMELYRQKVE
31 [64Cu]LGKATLYAVLPLGEEMRDRARAHVDALRTHLA
32 [64Cu]LGKATLYAVLPYLDDF QKKWQEEMELYRQKVE
35 [64Cu]YLLDGILFIYPLGEEMRDRARAHVDALRTHLA
36 [64Cu]YLLDGILFIYPYLDDF QKKWQEEMELYRQKVE
39 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
40 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
43 [64Cu]FLFAEIVSIFPLGEEMRDRARAHVDALRTHLA
44 [64Cu]FLFAEIVSIFPYLDDF QKKWQEEMELYRQKVE
47 [64Cu]IVIVDIC IT GPL GEEMRDRARAHVDALRTHLA
48 [64Cu]IVIVDIC IT GPYLDDF QKKWQEEMELYRQKVE
53 [64Cu]GNLVRICLGAPLGEEMRDRARAHVDALRTHLA
54 [64Cu]GNLVRICLGAPYLDDF QKKWQEEMELYRQKVE
57 [64C1.1]VMGDLVLTVLPLGEEIVIRDRARAHVDALRTHLA
58 [64CU]VMGDLVLTVLPYLDDFQKKWQEEMELYRQKVE
61 [64Cu]LVAADAVASLPLGEEMRDRARAHVDALRTHLA
62 [64Cu]LVAADAVASLPYLDDFQKKWQEEMELYRQKVE
69 Ptx-GFLSKSLVFPLGEEMRDRARAHVDALRTHLA
70 Ptx-GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
71 Ptx-GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
72 Ptx-GFLSKSLVFPYLDDFQKKWQEEM(0)ELRQKVE
TREM-1 inhibitory trifunctional SCHOOL peptides In certain embodiments, the present invention relates to amphipathic TREM-1 inhibitory trifunctional peptides and therapeutic compositions comprising such trifunctional peptides for use in treating cancer in combination with other cancer therapies. In one embodiment, these peptides may possess the antitumor activity. In one embodiment, these peptides may not possess the antitumor activity.
In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating formation of naturally long half-life lipopeptide/lipoprotein particles upon interaction with lipoproteins, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In some embodiments, each trifunctional peptide is capable of at least three functions: 1) mediating the self-assembly of naturally long half-life lipopeptide particles upon binding to lipid or lipid mixtures, 2) facilitation of the targeted delivery to cells of interest and/or sites of disease, and 3) treatment, prevention, and/or detection of a disease or condition. In certain embodiments, the present invention relates to amphipathic trifunctional peptides consisting of two amino acid domains, wherein upon interaction with plasma lipoproteins, one amino acid domain mediates formation of naturally long half-life lipopeptide/lipoprotein particles and targets these particles to macrophages, whereas the other amino acid domain inhibits the TREM-1/DAP-12 receptor signaling complex expressed on myeloid cells including but not limited to, macrophages.
In one embodiment, the TREM-1 inhibitory trifunctional SCHOOL peptides (TRIOPEPs) of the present invention form self-assembling SLP in vitro. In one embodiment, TRIOPEPs are incorporated into self-assembled nanosized SLP of discoidal or spherical morphology (dSLP and sSLP, respectively) that contain apo A-I peptide fragments comprising 22 amino acid residue-long peptide sequences of the apo A-I helix 4 and/or helix 6. In one embodiment, the TREM-1 inhibitory trifunctional SCHOOL peptides described herein form naturally long half life lipopeptide particles in vivo. In certain embodiments, the present invention relates to peptides consisting of two amino acid domains, wherein upon binding to lipid or lipid mixtures, one amino acid domain assists in the self-assembly of naturally long half-life lipopeptide particles and targets these particles to macrophages, whereas another amino acid domain inhibits TREM-1/DAP-12 receptor complex expressed on macrophages.
In some embodiments of the present inventions, TABLE 3 presents a list of the peptides and therapeutic compositions that includes, but is not limited to the trifunctional SCHOOL
peptide-based TREM-1 inhibitors and therapeutic compositions that can be used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
Exemplary TREM-1 inhibitory trifunctional SCHOOL peptides include but are not limited to, 31 amino acid-long peptide TREM-1 inhibitory peptides GA31 (GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA, M(0), methionine sulfoxide) (SEQ ID
NO. 26) and GE31 (GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE, M(0), methionine sulfoxide) (SEQ ID NO. 27). In one embodiment, methionine residues of the peptides GE31 (GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE) (SEQ ID NO. 25) and GA31 (GFLSKSLVFPLGEEMRDRARAHVDALRTHLA) (SEQ ID NO. 24) are unmodified. See TABLE 3.
In one embodiment, any or both the domains comprise minimal biologically active amino acid sequence. In one embodiment, the peptide variant comprises a cyclic peptide sequence. In one embodiment, the peptide variant comprises a disulfide-linked dimer. In one embodiment, the peptide variant includes amino acids selected from the group of natural and unnatural amino acids including, but not limited to, L-amino acids, or D-amino acids.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to a drug compound (TA). In one embodiment, TA is selected from the group including, but not limited to, anticancer, antibacterial, antiviral, autoimmune, anti-inflammatory and cardiovascular agents, antioxidants, and therapeutic peptides. In one embodiment, the TA is a hydrophobic therapeutic agent. The TA
may also be selected from the group comprising paclitaxel, valrubicin, doxorubicin, taxotere, campotechin, etoposide, and any combination thereof.
In one embodiment, one or both amino acid domains of the peptides and compounds of the present invention are conjugated to an imaging probe. In one embodiment, the imaging agent is GBCA for MM. In one embodiment, the imaging agent is a [64Cu]-containing imaging probe for imaging systems such as a PET imaging system and combined PET/CT and PET/MRI
systems.
In one embodiment, an imaging probe and/or an additional TA is conjugated to any or both of the domains. In one embodiment, the peptides and compounds of the present invention are used in combinations thereof.
Embodiments Of TREM-1 inhibitory SCHOOL peptides.
Normal transmembrane interactions between the TREM-1 and the DAP-12 dimer forming a functional TREM-1/DAP-12 receptor complex comprise positively charged lysine amino acid within the TREM-1 transmembrane portion and negatively charged aspartic acid pairs in a DAP-12 dimer, thereby allowing subunit association (See FIG. 49).
In one embodiment, the simplest TREM-1 inhibitory SCHOOL agents would be synthetic peptides and their variants (SCHOOL peptides) that correspond to the TREM-1 and/or DAP-12 transmembrane domains or their functionally important minimal protein sequences as disclosed in US 8,513,185, US 9,981,004 and US 20190117725. Although it is not necessary to understand the mechanism of an invention, it is believed that interactions between a lysine residue of SCHOOL peptides that correspond to the TREM-1 transmembrane domain or its functionally important minimal protein sequence and an aspartic acid residue of a DAP-12 dimer disrupt the interactions between TREM-1 and DAP-12 in the membrane, thereby "disconnecting" TREM-1 and resulting in a non-functioning receptor. Accordingly, it is believed that interactions between an aspartic residue of SCHOOL peptides that correspond to the DAP-12 transmembrane domain or its functionally important minimal protein sequence and lysine amino acid residue of the TREM-1 transmembrane domain disrupt the interactions between DAP-12 and TREM-1 in the membrane, thereby "disconnecting" DAP-12 and resulting in a non-functioning receptor. These peptide variants and compositions possess the advantages typically associated with a fully synthetic material and yet possess certain desirable features of materials derived from natural sources.
In some embodiments of the present inventions, TABLE 3 presents a list of the peptides and therapeutic compositions that includes, but is not limited to the SCHOOL
peptide-based TREM-1 inhibitors and their variants that can be designed as disclosed in US
8,513,185, US
9,981,004 and US 20190117725 and used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
In some embodiments, the SCHOOL peptides and their variants that inhibit TREM-transmembrane signaling can be used in a free form. Exemplary TREM-1 inhibitory SCHOOL
peptides include but are not limited to, a 9 amino acid-long peptide TREM-1 inhibitory peptide GF9 (GFLSKSLVF) disclosed in US 8,513,185, US 9,981,004 and US 20190117725 and described in (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Rojas et al. 2018).
Although it is not necessary to understand the mechanism of an invention, it is believed that free SCHOOL peptide self-inserts into the cell membrane from outside the cell, co-localizes with TREM-1/DAP-12 receptor complex and disrupts the protein-protein interactions between TREM-1 and DAP-12, thereby resulting in a non-functional receptor complex that does not provide TREM-1 transmembrane signaling upon binding to a putative TREM-1 ligand(s) (See FIG. 49, Route 1). In one embodiment, FIG. 50 demonstrates colocalization of GF9 with the TREM-1 in the cell membrane. These peptide variants and compositions possess the advantages typically associated with a fully synthetic material and yet possess certain desirable features of materials derived from natural sources.
As described in (Vlieghe et al. 2010, Lau et al. 2018), the main limitations generally attributed to therapeutic peptides are: a short half-life because of their rapid degradation by proteolytic enzymes of the digestive system and blood plasma; rapid removal from the circulation by the liver (hepatic clearance) and kidneys (renal clearance);
poor ability to cross physiological barriers because of their general hydrophilicity; high conformational flexibility, resulting sometimes in a lack of selectivity involving interactions with different receptors/targets (poor specific biodistribution), causing activation of several targets and leading to side effects;
eventual risk of immunogenic effects; and high synthetic and production costs (the production cost of a 5000 Da molecular mass peptide exceeds the production cost of a 500 Da molecular mass small molecule by more than 10-fold but clearly not 100-fold).
In some embodiments of the present invention, the SCHOOL peptides and their variants that inhibit TREM-1 transmembrane signaling can be formulated into self-assembling SLP of discoidal (sSLP) or spherical (sSLP) shape that mimic human naturally long half-life high density lipoproteins (HDL) and are disclosed in US 20130045161 and US
20110256224 and described in (Sigalov 2014, Shen and Sigalov 2017, Shen and Sigalov 2017, Rojas et al. 2018, Tornai et al. 2019). Although it is not necessary to understand the mechanism of an invention, it is believed that these particles provide targeted delivery of the incorporated SCHOOL peptides to target cells and increase half life of these peptides in circulation. In some embodiments, these SLP contain the modified amphipathic apolipoprotein A-I peptide fragments that not only assist in the self-assembly of SLP but also provide targeted delivery of these particles to target cells in vitro and in vivo. In some embodiments, the modification represents a sulfoxidation of methionine amino acid residue in the apo A-I peptide sequence.
In one embodiment, FIG. 49 presents a schematic representation of targeted delivery of the TREM-1 modulatory SCHOOL peptides by SLP to myeloid cells including but not limited to, macrophages including TAMs. Although it is not necessary to understand the mechanism of an invention, it is believed that SLP that contain TREM-1 modulatory SCHOOL
peptides (exemplary shown for GF9) are endocytosed by macrophages through scavenger receptor(s), and then release the incorporated SCHOOL peptide, which self-inserts into the cell membrane from inside the cell, co-localizes with TREM-1/DAP-12 receptor complex and disrupts the protein-protein interactions between TREM-1 and DAP-12, thereby resulting in a non-functional receptor complex that does not induce TREM-1 transmembrane signaling upon binding to a putative TREM-1 ligand(s) (See FIG. 49, Route 2).
Modulators of TREM-1/DAP-12 signaling pathway.
Modulators (inhibitors) of TREM-1/DAP-12 signaling pathway can be nonexclusively divided into two major categories: those that inhibit TREM-1 transmembrane signaling by blocking binding of TREM-1 to its ligand(s) (type I inhibitors; See FIG. 49) and those that employ a ligand binding-independent mechanism of action and modulate (inhibit) mediated transmembrane signaling by disrupting protein-protein interactions between TREM-1 and DAP-12 in the cell membrane (type II inhibitors; See FIG. 50). Type I
inhibitors can be, in turn, subdivided into two subtypes: those that bind to TREM-1 (type Ia inhibitors) and those that bind to TREM-1 ligand(s) (type lb inhibitors).
Type I TREM-1 inhibitors.
In one embodiment, exemplary TREM-1 type I inhibitors include but not limited to, antagonistic (blocking, inhibiting) anti-TREM-1 antibodies and/or their fragments such as antibodies that block and inhibit TREM-1 disclosed in US 9,000,127 and US
9,550,830 and described in (Brynjolfsson et al. 2016). These TREM-1 inhibitors are believed to block binding of TREM-1 to its ligand(s) by binding to the extracellular domain of TREM-1 (type Ia inhibitors, See FIG. 49).
In one embodiment, exemplary TREM-1 type I inhibitors include but not limited to, synthetic peptides derived from a part of the extracellular domain of either TREM-1 such as Pl, P3 and LP17 peptides disclosed in US 20160193288, US 20150232531, US 8,013,836 and US
9,273,111 and described in (Gibot et al. 2004, Gibot et al. 2006) or the TREM-like transcript-1 (TLT-1) such as LR17 and LR12 peptides disclosed in US 20160193288, US
20160015773, US
20150232531, US 9,255,136; US 9,657,081 and US 9,815.883 and described in (Derive et al.
2012). These TREM-1 inhibitors are believed to act as an endogenous decoy receptor (type lb inhibitors, See FIG. 1) by binding TREM-1 ligands and preventing their engagement to membrane-bound TREM-1 (Pelham et al. 2014).
In some embodiments of the present invention, the TREM-1 type I inhibitors can be used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
In some embodiments of the present inventions, TABLE 3 presents a list of the peptides and peptide analogues that includes, but is not limited to the TREM-1 type lb peptide inhibitors and their variants that can be designed as disclosed in US 20160193288, US
20150232531, US
8,013,836, US 9,273,111, US 20160015773, US 9,255,136; US 9,657,081 and US
9,815.883 and described in (Gibot et al. 2004, Gibot et al. 2006, Derive et al. 2012) and used in order to treat tumors in combinations with other cancer therapies.
Type II TREM-1 inhibitors..
Application of the Signaling Chain HOmoOLigomerization (SCHOOL) model of receptor signaling described in (Sigalov et al. 2004, Sigalov 2004, Sigalov 2006, Sigalov 2018) to the transmembrane signal transduction mediated by a TREM-1 receptor suggested that an inhibition of TREM-1/DAP-12 signaling may be achieved by using transmembrane-targeted agents (SCHOOL agents) which specifically disrupt interactions between TREM-1 and DAP-12 subunits in the cell membrane (See FIG. 2), thereby disconnecting TREM-1 and DAP-12 and .. resulting in a non-functioning TREM-1/DAP-12 receptor complex.
In some embodiments of the present invention, the TREM-1 type II inhibitors can be used in order to treat tumors in combinations with other cancer therapies or to predict response of the subject to the treatment by using the modulators of TREM-1/DAP-12 signaling pathway in combination-therapy regiment.
As described in (Tammaro et al. 2017), although TREM-1 appears to be activated by damage associated molecular patterns (DAMPs) that are shared by other pattern recognition receptors (PRRs), no TREM-1 specific (endogenous) ligand has been discovered to date. It is unknown why these ligands, specifically, share TREM-1 activation. Neither it is known what they have in common, but this information could certainly be of use in the determination of new specific ligands. This makes ligand binding-independent type II TREM-1 inhibitors advantageous compared to type I inhibitors that attempt to block binding TREM-1 to its yet unknown ligand(s).
In some embodiments, type II TREM-1 inhibitors include but are not limited to, the TREM-1 inhibitory SCHOOL peptides. The preferred peptides and compositions of the present invention comprise the TREM-1 modulatory peptide sequences designed using the SCHOOL
.. model of TREM-1 signaling and capable of modulating TREM-1 receptor expressed on myeloid cells as disclosed in US 8,513,185 and US 9,981,004 and described in (Sigalov 2010, Shen and Sigalov 2017).
Listed below in TABLE 2 are reported transmembrane sequences of TREM-1 and DAP-12 in a number of species. These regions are highly conserved and the substitutions between species are very conservative. This suggests a functional role for the transmembrane regions of both, TREM-1 and DAP-12, constituents of the complex. These regions strongly interact between themselves, thus maintaining the integrity of the TREM-1/DAP-12 receptor signaling complex in resting cells. These transmembrane domains are short and should be easily mimicked by synthetic peptides and compounds. In some embodiments, synthetic peptides and compounds .. are contemplated that may provide successful treatment options in the clinical setting.
TABLE 2C Sequence comparison of TREM-1 and DAP-12 transmembrane regions (accession codes are given in parenthesis).
SEQUENCE
SPECIES
HUMAN IVILLAGGFL SKSLVF S VLF A GVLAGIVMGDLVLTVLIALAV
(Q9NP99) (043914) MOUSE VTI S VIC GLL SKSLVF IILF I GVLAGIVLGDLVLTLLIALAV
(Q9JKE2) (054885) BOVIN IIIPAACGLL SKTLVFIGLF A GVLAGIVLGDLMLTLLIALAV
(Q6QUN5) (Q95J79) SHEEP not known GVLAGIVLGDLMLTLLIALAV
(Q95KS5) RAT not known GVLAGIVLGDLVLTLLIALAV
(Q6X9T7) PIG ILPAVCGLLSKSLVFIVLFVV GILAGIVLGDLVLTLLIALAV
(Q6TYI6) (Q9TU45) CLUSTAL W 2.0 multiple sequence alignment:
ILPAVCGLLSKSLVFIVLFVV
*:***:*** ** *:*****:***:**:******
TABLE 3. Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions.
##
Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions 3 (GFLSKSLVF)2 GVLAGIVMGDLVLTVLIALAV
GFL SK SLVF GEEMRDRARAHV
11 GFL SK SLVF GEEM(0)RDRARAHV
13 GFL SK SLVFWQEEM(0)ELYRQKV
GFL SR SL VF GEEM(0)RDRARAHV
17 GFL SRSLVFWQEEM(0)ELYRQKV
19 GLL SK SLVFGEEM(0)RDRARAHV
GLL SK SLVFWQEEMELYRQKV
21 GLL SK SLVFWQEEM(0)ELYRQKV
GFL SK SLVFPYLDDFQKKWQEEMELYRQKVE
26 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
27 GFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
[64C u] GF L SK SLVF GEEM(0)RDRARAHV
31 [64Cu]GFL SK SLVFWQEEM(0)ELYRQKV
34 GFL SK SL VF PL GEEM(0)RDRARAHVDALRTHL A
35 GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
38 [64Cu]GFL SK SL VF PL GEEM(0)RDRARAHVDALRTHL A
39 [64Cu]GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
40 LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
41 LQEEDAGEYGCMPYLDDF QKKWQEEM(0)ELYRQKVE
42 [64Cu]LQEEDAGEYGCMPLGEEM(0)RDRARAHVDALRTHLA
43 [64Cu]LQEEDAGEYGCMPYLDDF QKKWQEEM(0)ELYRQKVE
44 LQVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
45 LQVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
46 [64Cu]L QVTD SGLYRCVIYHPPPLGEEM(0)RDRARAHVDALRTHLA
47 [64Cu]L QVTD SGLYRCVIYHPPPYLDDF QKKWQEEM(0)ELYRQKVE
48 LQEEDAGEYGCMGEEM(0)RDRARAHV
49 LQEEDAGEYGCMWQEEM(0)ELYRQKV
52 [64Cu]IIVTDVIATLPLGEEMRDRARAHVDALRTHLA
53 [64Cu]IIVTDVIATLPYLDDF QKKWQEEMELYRQKVE
54 IIVTDVIATLPLGEEM(0)RDRARAHVDALRTHLA
5 IIVTDVIATLPYLDDF QKKWQEEM(0)ELYRQKVE
56 [64Cu]IIVTDVIATLPLGEEM(0)RDRARAHVDALRTHLA
57 [64Cu]IIVTDVIATLPYLDDF QKKWQEEM(0)ELYRQKVE
58 PtxGFLSKSLVFPLGEEMRDRARAHVDALRTHLA
59 PtxGFLSKSLVFPYLDDFQKKWQEEMELYRQKVE
60 PtxGFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA
61 PtxGFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE
64 IVILLAGGFLSKSLVFSVLFAPLGEEM(0)RDRARAHVDALRTHLA
65 IVILLAGGFLSKSLVFSVLFAPYLDDFQKKWQEEM(0)ELYRQKVE
TABLE 3 - Continuted ## Exemplary TREM-1/DAP-12 Pathway Modulatory Peptide Sequences and Compositions 3 (GFLSKSLVF)2 GVLAGIVMGDLVLTVLIALAV
GFLSKSLVFGEEMRDRARAHV
11 GFLSKSLVFGEEM(0)RDRARAHV
13 GFLSKSLVFWQEEM(0)ELYRQKV
15 GFL SR SL VF GEEM(0)RDRARAHV
17 GFL SRSLVFWQEEM(0)ELYRQKV
19 GLL SK SLVFGEEM(0)RDRARAHV
21 GLL SK SLVFWQEEM(0)ELYRQKV
26 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
27 GFL SK SLVFPYLDDF QKKWQEEM(0)ELYRQKVE
30 [64C u] GF L SK SLVF GEEM(0)RDRARAHV
31 [64Cu]GFL SK SLVFWQEEM(0)ELYRQKV
34 GFL SK SL VF PL GEEM(0)RDRARAHVD ALRTHL A
35 GFL SK SLVFPYLDDF QKKWQEEM(0)ELRQKVE
38 [64C u] GF L SK SL VF PL GEEM (0)RDRARAHVD ALRTHL A
DEMANDE OU BREVET VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVET COMPREND
PLUS D'UN TOME.
NOTE : Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets JUMBO APPLICATIONS/PATENTS
THIS SECTION OF THE APPLICATION/PATENT CONTAINS MORE THAN ONE
VOLUME
NOTE: For additional volumes, please contact the Canadian Patent Office NOM DU FICHIER / FILE NAME:
NOTE POUR LE TOME / VOLUME NOTE:
Claims (56)
1. A method for treating cancer in a subject in need thereof, said method comprising administering to said subject a therapeutically effective amount of at least one peptide inhibitor for inhibiting activity of the TREM-1/DAP-12 signaling pathway.
2. The method of Claim 1, wherein said therapeutically effective amount comprises one dose of said at least one peptide inhibitor.
3. The method of Claim 2, wherein said therapeutically effective amount comprises between two to ten doses of said at least one peptide inhibitor.
4. The method of Claim 1, wherein said at least one peptide inhibitor is the amino acid sequence GFLSKSLVF (GF9).
5. The method of Claim 4, wherein said at least one peptide inhibitor is administered without recombinant high-density lipoprotein carriers.
6. The method of Claim 1, wherein said at least one peptide inhibitor has a methionine sulfoxide M(0) modified amino acid residue.
7. The method of Claim 1, wherein said at least peptide inhibitor is administered without recombinant high-density lipoprotein carriers.
8. The method of Claim 1, wherein said at least one peptide inhibitor is administered with recombinant high-density lipoprotein carriers.
9. The method of Claim 1, wherein said at least one peptide inhibitor has an amino acid sequence selected from the group consisting of GFLSKSLVFPYLDDFQKKWQEEMELYRQKVE and GFLSKSLVFPLGEEMRDRARAHVDALRTHLA.
AMENDED SHEET
PCT Application
AMENDED SHEET
PCT Application
10. The method of Claim 1, wherein said at least one peptide inhibitor has an amino acid sequence selected from the group consisting of GFLSKSLVFPYLDDFQKKWQEEM(0)ELYRQKVE (GA31) and GFLSKSLVFPLGEEM(0)RDRARAHVDALRTHLA (GE31).
11. The method of Claim 10, wherein said peptide inhibitor has equimolar amounts of peptide GA31 and peptide GE31.
12. The method of Claim 1, wherein said at least one peptide inhibitor has an amino acid sequence selected from the group consisting of GFLSKSLVFGEEMRDRARAHV (G-HV21), GFLSKSLVFWQEEMELYRQKV (G-KV21), MWKTPTLKYFPYLDDFQKKWQEEMELYRQKVE (M-VE32), and mixtures thereof.
13. The method of Claim 1, wherein said at least one peptide inhibitor has an amino acid sequence selected from the group consisting of GFLSKSLVFGEEM(0)RDRARAHV (G-HV21), GFLSKSLVFWQEEM(0)ELYRQKV (G-KV21), (M(0)WKTPTLKYFPYLDDFQKKWQEEM(0)ELYRQKVE (M-VE32), and mixtures thereof.
14. The method of Claim 1, wherein said at least one peptide inhibitor is administered together with a therapeutically effective amount of a therapeutic selected from the group consisting of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery, and any combination thereof.
15. The method of Claim 1, comprising administering said at least one peptide inhibitor together with a pharmaceutically acceptable carrier selected from the group consisting of an excipient, diluent, and any combination thereof.
16. The method of Claim 15, wherein said carrier is selected from the group consisting of lipids, proteins or polypeptides, and mixtures thereof.
AMENDED SHEET
PCT Application
AMENDED SHEET
PCT Application
17. The method of Claim 2, wherein, prior to administering said first dose of said peptide inhibitor, said subject received a prior therapy selected from thc group consisting of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof.
18. The method of Claim 17, wherein said cancer recurred or progressed after said prior therapy.
19. The method of Claim 1, wherein said administering is continued as a maintenance treatment for duration between two weeks to five years.
20. The method of Claim 1, wherein said administration is continued for a duration of up to one year.
21. The method of Claim 14, wherein said anticancer vaccine is selected from the group consisting of Gardasil, Cervarix, and Sipuleucel-T/Provenge.
22. The method of Claim 14, wherein the anticancer immunotherapy agent is selected from the group consisting of Alemtuzumab, Ipilimumah, Ofatumurnab, Nivolumab, Pembrolizumab, Rituximab, Blinatumomab, Daratumumab, Trastuzumab, Cetuximab, Elotuzumab, adoptive T-cell therapy, T-Vec, Interferon, Interleukin, and any combination thereof.
23. The method of Claim 14 wherein the anticancer immunomodulatory agent is selected from the group consisting of thalidomide, lenolidomide, pomalidomide, and any combination thereof.
24. The method of Claim 14, wherein the additional anticancer therapeutic is selected from the group consisting of an alkylating agent, a tubulin inhibitor, a topoisomerase inhibitor, proteasome inhibitor, a CHK I inhibitor, a CHK2 inhibitor, a PARP inhibitor, a tyrosine kinase AMENDED SHEET
PCT Application inhibitor, CSF-1/CSF-1R inhibitor, doxorubicin, gemcitabine, entrectinib, epirubicin, vinblastine, etoposide, topotecan, bleomycin, andinytomycin c.
PCT Application inhibitor, CSF-1/CSF-1R inhibitor, doxorubicin, gemcitabine, entrectinib, epirubicin, vinblastine, etoposide, topotecan, bleomycin, andinytomycin c.
25. The method of Claim 24, wherein said alkylating agent is selected from the group consisting of Dacarbazine, Procarbazine, Carmustine, Lomustine, Uramustine, BuSulfan, Streptozocin, Altreamine, Ifosfamine, Chrormethine, Cyclophasphamide, Cyclophosphamide, Chlorambucil, Fluorouracil (5-Fu), Melphalan, Triplatin tetranitrate, Satraplatin, Nedaplatin, Cisplatin, Carboplatin, and_Oxaliplatin.
26. The method of Claim 24, wherein said tubulin inhibitor is selected from the group consisting of Taxol, Docetaxel, Abraxane, Vinblastin, Epothilone, Colchicine, Cryptophycin, BMS 347550, Rhizoxin, Ecteinascidin, Dolastin 10, Cryptophycin 52,_and_IDN-5109.
27. The method of Claim 24, wherein said topoisomerase inhibitor is a topoisomerase inhibitor selected from the group consisting of Irinotecan, Topotecan, and_Camptothecins (CPT).
28. The method of Claim 24, wherein said topoisomerase inhibitor is a topoisomerase II
inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, andsllipticine.
inhibitor selected from the group consisting of Amsacrine, Etoposide, Teniposide, Epipodophyllotoxins, andsllipticine.
29. The method of Claim 24, wherein said proteasome inhibitor is selected from the group consisting of Velcade (bortezomib), and Kyprolis (carfilzomib).
30. The method of Claim 24, wherein said CHK1 inhibitor is selected from the group consisting of TCS2312, PF-0047736, AZ07762, A-69002, and A-641397.
31. The method of Claim 24, wherein the PARP inhibitor is selected from the group consisting of Olaparib, Talazoparib, ABT-888, (veliparib), KU-59436, AZD-2281, AG-014699, BSI-201, BGP-15, INO-1001, and ONO-2231.
AMENDED SHEET
PCT Application
AMENDED SHEET
PCT Application
32. The method of Claim 24, wherein the tyrosine kinase inhibitor is selected from the group consisting of pexidartinib, entrectinib, matinib mesylate (ST1571; Gleevec), gefitinib (Iressa), erlotinib (OSI-1774; Tarceva), lapatinib (GW-572016), canertinib (CI-1033), semaxinib (SU5416), vatalanib (PTK787/ZK222584), sorafenib (BAY 43-9006), sutent (SU11248), and leflunomide (SU101).
33. The method of Claim 24, wherein the CSF-1/CSF-1R inhibitor is selected from the group consisting of CSF-1R kinase inhibitor, an antibody that binds CSF-1R and is capable of blocking binding of CSF-1 to CSF-1R and IL-34 to CSF-1R.
34. The method of Claim 24, wherein the CSF-1R kinase inhibitor is selected from the group consisting of imatinib, nilotinib and PLX3397.
35. The method of Claim 14, wherein said radiation therapy is selected from the group consisting of X-rays, ion beams, electron beams, gamma-rays, UV-rays, and decay of a radioactive isotope, and any combination thereof.
36. The method of CI ai m 14, wherein said surgery is a tumor resection.
37. The method of Claim 1, wherein said cancer is lung cancer selected from the group consisting of non-small cell lung cancer, pancreatic cancer, breast cancer, liver cancer, multiple myeloma, melanoma, leukemia, central nervous system cancer, stomach cancer, prostate, colon cancer, colorectal cancer, brain cancer, gastrointestinal cancer, gastric cancer, ovarian cancer, renal cancer, skin cancer, osteosarcoma, endometrial cancer, esophageal cancer, kidney cancer, prostate cancer, thyroid cancer, neuroblastoma, neurofibroma, glioma, glioblastoma, glioblastoma multiforme, stomach cancer, bladder cancer, head and neck cancer, cervical cancer, giant cell tumor of the tendon sheath, tenosynovial giant cell tumor, pigmented villonodular synovitis, cancers in which myeloid cells are involved, cancers in which myeloid cells are recruited and cancer cachexia.
AMENDED SHEET
PCT Application
AMENDED SHEET
PCT Application
38. The method of Claim 1, wherein said at least one peptide inhibitor comprises a variant peptide sequence.
39. The method of Claim 1, wherein said TREM-1/DAP-12 activity is selected group the group consisting of signaling and activation.
40. The method of Claim 38, wherein said variant peptide sequence cornprises at least one D-amino acid.
41. The method of Claim 38, wherein said variant peptide sequence is a cyclic peptide.
42. The method of Claim 38, wherein said variant peptide sequence is derived from transmembrane domain sequences of human or animal TREM-1 and/or its signaling subunit, DAP-12, and any corribination thereof.
43. The method of Claim 39, wherein said variant peptide sequence is_selected group the group consisting of LR12, LP17 and a combination thereof.
44. The method of Claim 1, wherein said method further_comprises administering to said subject_at least one antibody or fragment thereof, that specifically binds to TREM-1/DAP-12.
45. The method of claim 44, wherein said antibody or fragment thereof reduces TREM-1/DAP-12 activity.
46. The method of Claim 2, wherein said subject is diagnosed prior to said_administering said first dose.
47. The method of Claim 46, wherein said subject is diagnosed after said administering said first dose.
AMENDED SHEET
PCT Application
AMENDED SHEET
PCT Application
48. The method of Claim 47, wherein said diagnosis is selected from the group consisting of determining cancer progression, determining a result of cancer treatment.
determining results of inhibiting TREM-1-mediated cell activation and reducing tumor growth.
determining results of inhibiting TREM-1-mediated cell activation and reducing tumor growth.
49. The method of Claim 46, wherein said diagnosiis comprises isolating a biological sample from said subject.
50. The method of Claim 49, wherein said diagnosis is based on expression levels of a marker selected from the group consisting of CSF-1, CSF-1R, IL-6, TREM-1, CD68 or any combination thereof.
51. The method of Claim 50, wherein said diagnosis is based on the number of CD68 positive cells in said sample.
52. The method of Claim 50, wherein said diagnosis is based on a response to said at least one peptide inhibitor selected from the group consisting of a higher expression level of a marker selected from the group consisting of CSF-1, CSF-1R, IL-6, TREM-1, CD68, a higher number of CD68-positive cells, and any combination thereof.
53. The method of Claim 1, wherein said method further comprises:
administering to said subject an amount of said at least one said peptide inhibitor that binds TREM-1 and is conjugated to at least one imaging probe; imaging at least a portion of said subject;
detecting said imaging probe, wherein the location and amount of said imaging probe correlates with the TREM-1 expression levels in said cancer.
administering to said subject an amount of said at least one said peptide inhibitor that binds TREM-1 and is conjugated to at least one imaging probe; imaging at least a portion of said subject;
detecting said imaging probe, wherein the location and amount of said imaging probe correlates with the TREM-1 expression levels in said cancer.
54. The method of Claim 52, wherein higher TREM-1 expression levels predict a better response to said peptide inhibitor.
55. The method of Claim 53, wherein said an imaging probe is selected from the group consisting of Gd(III), Mn(II), Mn(III), Cr(III), Cu(II), Fe (III), Pr(III), Nd(III) Sm(III), Tb(III), Yb (III) Dy(III), Flo(III), Eu(II), Eu(III), and Er(III), T1201, -42, K In1", Fe.59, Tc99"1, Cr51, AMENDED SHEET
PCT Application Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 11.31, 1123, 1332, Cll, N13, 015, Br76, Krsl, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol, or a combination thereof.
PCT Application Ga67, Ga68, Cu64, Rb82,Mo99, Dy165, Fluorescein, Carboxyfluorescein, Calcein, F18, Xe133, 1125, 11.31, 1123, 1332, Cll, N13, 015, Br76, Krsl, Diatrizoate, Metrizoate, Isopaque, Ioxaglate, Iopamidol, Iohexol, Iodixanol, or a combination thereof.
56. A method for treating cancer in a subject, said method comprising administering to said subject a therapeutically effective amount of at least one isolated antibody or fragment thereof, that specifically binds TREM-1/DAP-1 2 for inhibiting the TREM-1/DAP-1 2 signaling pathway together with a therapeutically amount of a therapeutic selected from the group consisting of an anticancer vaccine, an anticancer immunotherapy agent, anti-cancer immunomodulatory agent, an additional anticancer therapeutic, radiation therapy, surgery or a combination thereof.
AMENDED SHEET
AMENDED SHEET
Applications Claiming Priority (11)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201862717929P | 2018-08-13 | 2018-08-13 | |
US62/717,929 | 2018-08-13 | ||
US201862751303P | 2018-10-26 | 2018-10-26 | |
US62/751,303 | 2018-10-26 | ||
US201962836823P | 2019-04-22 | 2019-04-22 | |
US62/836,823 | 2019-04-22 | ||
US201962843835P | 2019-05-06 | 2019-05-06 | |
US62/843,835 | 2019-05-06 | ||
US201962875287P | 2019-07-17 | 2019-07-17 | |
US62/875,287 | 2019-07-17 | ||
PCT/US2019/046392 WO2020036987A1 (en) | 2018-08-13 | 2019-08-13 | Peptides and compositions for targeted treatment and imaging |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3109702A1 true CA3109702A1 (en) | 2020-02-20 |
Family
ID=67957375
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3109702A Pending CA3109702A1 (en) | 2018-08-13 | 2019-08-13 | Peptides and compositions for targeted treatment and imaging |
Country Status (3)
Country | Link |
---|---|
EP (1) | EP3836951A1 (en) |
CA (1) | CA3109702A1 (en) |
WO (1) | WO2020036987A1 (en) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA3218327A1 (en) * | 2021-05-19 | 2022-11-24 | Alexander Sigalov | Trem-2/dap-12 inhibitors for treating lung disease and injury and combinations thereof |
Family Cites Families (43)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4427660A (en) | 1982-03-03 | 1984-01-24 | Research Corporation | Formyl-methionyl chemotatic peptide antibiotic conjugates useful in treating infections |
AU685470B2 (en) | 1993-06-11 | 1998-01-22 | Merrell Pharmaceuticals Inc. | Trifunctional antithrombin and antiplatelet peptides |
JP3665342B2 (en) | 1995-01-16 | 2005-06-29 | ノーザン シドニー エリア ヘルス サービス | New peptide |
AU739130B2 (en) | 1996-06-11 | 2001-10-04 | Northern Sydney And Central Coast Area Health Service | T cell antigen receptor peptides |
US20050070478A1 (en) | 1996-06-11 | 2005-03-31 | Northern Sydney Area Health Services | T cell antigen receptor peptides |
CA2431177C (en) * | 2000-12-08 | 2015-04-14 | Baylor College Of Medicine | Trem-1 splice variant for use in modifying immune responses |
CA2342376C (en) * | 2001-03-20 | 2013-11-12 | Marco Colonna | A receptor trem (triggering receptor expressed on myeloid cells) and uses thereof |
IL158532A0 (en) | 2001-05-02 | 2004-05-12 | Purdue Research Foundation | Treatment and diagnosis of macrophage mediated disease |
AU2004296829A1 (en) | 2003-12-05 | 2005-06-23 | The Cleveland Clinic Foundation | Risk markers for cardiovascular disease |
GB0401730D0 (en) | 2004-01-27 | 2004-03-03 | Bioxell Spa | Diagnosis method |
GB0426146D0 (en) | 2004-11-29 | 2004-12-29 | Bioxell Spa | Therapeutic peptides and method |
TW200744603A (en) | 2005-08-22 | 2007-12-16 | Chugai Pharmaceutical Co Ltd | Novel anticancer concomitant drug |
ES2531142T3 (en) | 2005-10-17 | 2015-03-11 | Sloan Kettering Inst Cancer | Class II HLA binding WT1 peptides, and compositions and methods comprising them |
US20080293795A1 (en) | 2006-01-17 | 2008-11-27 | Abbott Laboratories | Combination therapy with parp inhibitors |
JP4087420B2 (en) | 2006-06-28 | 2008-05-21 | シャープ株式会社 | Image display device, image data transmission device, image display system, image display method, image display program and recording medium thereof, and image data transmission program and recording medium thereof |
US7972794B2 (en) | 2006-07-18 | 2011-07-05 | Quest Diagnostics Investments Incorporated | Oxidized apoA-I determination by mass spectrometry |
US7811995B2 (en) | 2006-12-13 | 2010-10-12 | Susavion Biosciences, Inc. | Therapeutic and diagnostic peptides |
US8496942B2 (en) | 2006-12-13 | 2013-07-30 | Susavion Biosciences, Inc. | Therapeutic peptides and uses thereof |
US8278271B2 (en) | 2006-12-13 | 2012-10-02 | University Of Massachusetts | Inhibiting collagen-induced platelet aggregation and activation with peptide variants |
BRPI0806680A2 (en) | 2007-01-16 | 2015-06-16 | Wyeth Corp | Treatment, detection and monitoring of train-1 inflammation |
US8415453B2 (en) | 2007-02-13 | 2013-04-09 | Academia Sinica | Lung cancer-targeted peptides and applications thereof |
AU2008337517B2 (en) | 2007-12-14 | 2014-06-26 | Novo Nordisk A/S | Antibodies against human NKG2D and uses thereof |
WO2010014784A2 (en) | 2008-08-01 | 2010-02-04 | Bristol-Myers Squibb Company | Combination of anti-ctla4 antibody with diverse therapeutic regimens for the synergistic treatment of proliferative diseases |
US8921314B2 (en) | 2008-10-15 | 2014-12-30 | Angiochem, Inc. | Conjugates of GLP-1 agonists and uses thereof |
EP2358740A4 (en) | 2008-11-24 | 2012-04-25 | Sydney West Area Health Service | Cyclic peptides and uses thereof |
US8680139B2 (en) | 2009-04-01 | 2014-03-25 | Progenra | Anti-neoplastic compounds, compositions and methods |
CN102510759A (en) | 2009-04-20 | 2012-06-20 | 安吉奥开米公司 | Treatment of ovarian cancer using an anticancer agent conjugated to an angiopep-2 analog |
US9161988B2 (en) | 2009-07-02 | 2015-10-20 | Angiochem Inc. | Multimeric peptide conjugates and uses thereof |
RU2012100903A (en) | 2009-07-13 | 2013-08-20 | Зе Юниверсити Оф Саррей | THERAPEUTIC PEPTIDES, POLYPEPTIDES AND SEQUENCES OF NUCLEIC ACIDS |
US10138276B2 (en) | 2009-09-30 | 2018-11-27 | Signablok, Inc. | Inhibition of TCR signaling with peptide variants |
WO2011044545A2 (en) | 2009-10-09 | 2011-04-14 | Sigalov Alexander B | Methods and compositions for targeted imaging |
WO2011047097A2 (en) | 2009-10-13 | 2011-04-21 | Sigalov Alexander B | Inhibition of trem receptor signaling with peptide variants |
AU2011237851B2 (en) | 2010-04-08 | 2015-02-05 | Inserm (Institut National De La Sante Et De La Recherche Medicale) | Inhibiting peptides derived from TREM-Like Transcript 1 (TLT-1) and uses thereof |
CA2834696C (en) | 2011-05-05 | 2019-07-23 | Novartis Ag | Csf-1r inhibitors for treatment of brain tumors |
US9550830B2 (en) | 2012-02-15 | 2017-01-24 | Novo Nordisk A/S | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (TREM-1) |
TWI605061B (en) | 2012-02-15 | 2017-11-11 | 諾佛 儂迪克股份有限公司 | Antibodies that bind and block triggering receptor expressed on myeloid cells-1 (trem-1) |
WO2014037565A2 (en) | 2012-09-07 | 2014-03-13 | INSERM (Institut National de la Santé et de la Recherche Médicale) | Inhibiting peptides derived from triggering receptor expressed on myeloid cells-1 (trem-1) trem-like transcript 1 (tlt-1) and uses thereof |
SG11201507383WA (en) | 2013-03-12 | 2015-10-29 | Abbvie Inc | Dihydro-pyrrolopyridinone bromodomain inhibitors |
US9387257B2 (en) | 2014-01-17 | 2016-07-12 | Academia Sinica | Lung cancer specific peptides for targeted drug delivery and molecular imaging |
JP6797801B2 (en) | 2014-12-22 | 2020-12-09 | ファイヴ プライム セラピューティクス インク | Anti-CSF1R antibody for treating PVNS |
UY36471A (en) | 2014-12-23 | 2016-06-30 | Bristol Myers Squibb Company Una Corporación Del Estado De Delaware | ANTIBODIES AGAINST THE IMMUNORRECEPTOR (TIGIT) OF T LYMPHOCYTES WITH IG DOMAINS AND REASONS FOR INHIBITION OF THE TYMOSINE-BASED IMMUNORRECEPTOR (ITIM) |
US9717717B1 (en) | 2015-07-31 | 2017-08-01 | Progenra, Inc. | Methods of treating cancer through the inhibition of USP7 and immune system modulation |
US11213598B2 (en) | 2015-11-12 | 2022-01-04 | The Board Of Trustees Of The Leland Stanford Junior University | Labeled probe and methods of use |
-
2019
- 2019-08-13 EP EP19769270.0A patent/EP3836951A1/en active Pending
- 2019-08-13 CA CA3109702A patent/CA3109702A1/en active Pending
- 2019-08-13 WO PCT/US2019/046392 patent/WO2020036987A1/en active Search and Examination
Also Published As
Publication number | Publication date |
---|---|
EP3836951A1 (en) | 2021-06-23 |
WO2020036987A1 (en) | 2020-02-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5474535B2 (en) | FKBP-L and its use | |
ES2928111T3 (en) | Inhibition of AXL signaling in antimetastatic therapy | |
EP3813881A2 (en) | Compositions and methods for modulating monocyte and macrophage inflammatory phenotypes and immunotherapy uses thereof | |
US20230256053A1 (en) | Methods and compositions for immunomodulation | |
KR20090071598A (en) | Treatment of liver disorders by administration of receptor-associated protein(rap)-conjugates | |
CN111718424A (en) | Single-chain TRAIL receptor agonist proteins | |
AU2018283161A1 (en) | Renal-homing peptide conjugates and methods of use thereof | |
US20210363240A1 (en) | Compositions and methods for modulating lair signal transduction | |
Park et al. | PEGylated TRAIL ameliorates experimental inflammatory arthritis by regulation of Th17 cells and regulatory T cells | |
JP2023504286A (en) | Dendrimer compositions and methods for drug delivery | |
WO2020052570A1 (en) | Method and composition for preventing and treating atherosclerosis and related diseases | |
CA3109702A1 (en) | Peptides and compositions for targeted treatment and imaging | |
WO2017082186A1 (en) | New use for npr-a agonist | |
WO2012122941A1 (en) | Polypeptide drug against hepatitis b virus x protein | |
US20210322508A1 (en) | Peptides and compositions for targeted treatment and imaging | |
KR20240056684A (en) | Vasoactive intestinal peptide (VIP) receptor antagonist | |
EP1501535A1 (en) | Antagonists of megalin or cubilin for use in preventing organ damage induced by therapeutic agents | |
JP2003525628A (en) | Protein tyrosine phosphatase polynucleotides, polypeptides, and antibodies | |
US7345018B2 (en) | Method of treating side effects induced by therapeutic agents | |
TW202132334A (en) | Cxcr4 antagonist peptides | |
Li et al. | T cell/Macrophage Dual-Targeting Biomimetic Triptolide Self-Assembly Nanodrugs For Rheumatoid Arthritis Therapy by Inflammatory Microenvironment Remodeling | |
CA3218327A1 (en) | Trem-2/dap-12 inhibitors for treating lung disease and injury and combinations thereof | |
Wagner | Investigation of a novel small molecule TRAIL inducer, ONC201: pre-clinical anti-cancer efficacy, anti-metastasis effects, tumor immunity; and the structure-activity relationships (SAR) and mechanism of action of potential analogues | |
WO2021021276A1 (en) | Multi-targeted, tunable, sustained delivery of payloads to charged avascular tissues | |
WO2022076932A1 (en) | Compositions and methods of treating a pi3k mediated disease |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20220614 |
|
EEER | Examination request |
Effective date: 20220614 |
|
EEER | Examination request |
Effective date: 20220614 |
|
EEER | Examination request |
Effective date: 20220614 |
|
EEER | Examination request |
Effective date: 20220614 |
|
EEER | Examination request |
Effective date: 20220614 |