CA3127903A1 - Bi-ligand drug conjugate and use thereof - Google Patents
Bi-ligand drug conjugate and use thereof Download PDFInfo
- Publication number
- CA3127903A1 CA3127903A1 CA3127903A CA3127903A CA3127903A1 CA 3127903 A1 CA3127903 A1 CA 3127903A1 CA 3127903 A CA3127903 A CA 3127903A CA 3127903 A CA3127903 A CA 3127903A CA 3127903 A1 CA3127903 A1 CA 3127903A1
- Authority
- CA
- Canada
- Prior art keywords
- conjugate compound
- acceptable salt
- pharmaceutically acceptable
- cells
- molecule
- 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
- 239000003446 ligand Substances 0.000 title claims description 131
- 239000003814 drug Substances 0.000 title claims description 68
- 229940079593 drug Drugs 0.000 title description 64
- 150000001875 compounds Chemical class 0.000 claims abstract description 286
- 150000003839 salts Chemical class 0.000 claims abstract description 140
- 230000008685 targeting Effects 0.000 claims abstract description 121
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 31
- 238000000034 method Methods 0.000 claims abstract description 31
- 201000010099 disease Diseases 0.000 claims abstract description 26
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 26
- 206010028980 Neoplasm Diseases 0.000 claims description 124
- 102100041003 Glutamate carboxypeptidase 2 Human genes 0.000 claims description 103
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 90
- -1 Claudin18.2 Chemical compound 0.000 claims description 71
- 230000002195 synergetic effect Effects 0.000 claims description 58
- 108090000369 Glutamate Carboxypeptidase II Proteins 0.000 claims description 57
- 102100035139 Folate receptor alpha Human genes 0.000 claims description 49
- 101001023230 Homo sapiens Folate receptor alpha Proteins 0.000 claims description 49
- 101000892862 Homo sapiens Glutamate carboxypeptidase 2 Proteins 0.000 claims description 46
- 230000012202 endocytosis Effects 0.000 claims description 41
- 239000000203 mixture Substances 0.000 claims description 36
- 235000001014 amino acid Nutrition 0.000 claims description 35
- 150000001413 amino acids Chemical class 0.000 claims description 34
- 206010058467 Lung neoplasm malignant Diseases 0.000 claims description 31
- 201000005202 lung cancer Diseases 0.000 claims description 31
- 208000020816 lung neoplasm Diseases 0.000 claims description 31
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims description 30
- 229940014144 folate Drugs 0.000 claims description 28
- 235000019152 folic acid Nutrition 0.000 claims description 28
- 239000011724 folic acid Substances 0.000 claims description 28
- 206010060862 Prostate cancer Diseases 0.000 claims description 24
- 208000000236 Prostatic Neoplasms Diseases 0.000 claims description 24
- 201000011510 cancer Diseases 0.000 claims description 24
- 101150096736 TRPV6 gene Proteins 0.000 claims description 19
- 102000003569 TRPV6 Human genes 0.000 claims description 18
- 125000006850 spacer group Chemical group 0.000 claims description 18
- 206010006187 Breast cancer Diseases 0.000 claims description 17
- 208000026310 Breast neoplasm Diseases 0.000 claims description 17
- KLWPJMFMVPTNCC-UHFFFAOYSA-N Camptothecin Natural products CCC1(O)C(=O)OCC2=C1C=C3C4Nc5ccccc5C=C4CN3C2=O KLWPJMFMVPTNCC-UHFFFAOYSA-N 0.000 claims description 17
- 102100033851 Gonadotropin-releasing hormone receptor Human genes 0.000 claims description 17
- 229940127093 camptothecin Drugs 0.000 claims description 17
- 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 claims description 17
- 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 claims description 16
- 239000004472 Lysine Substances 0.000 claims description 15
- 206010061902 Pancreatic neoplasm Diseases 0.000 claims description 14
- 208000015486 malignant pancreatic neoplasm Diseases 0.000 claims description 14
- 201000002528 pancreatic cancer Diseases 0.000 claims description 14
- 208000008443 pancreatic carcinoma Diseases 0.000 claims description 14
- 108090000623 proteins and genes Proteins 0.000 claims description 13
- 108010044540 auristatin Proteins 0.000 claims description 12
- 239000003255 cyclooxygenase 2 inhibitor Substances 0.000 claims description 12
- 239000000427 antigen Substances 0.000 claims description 11
- 108091007433 antigens Proteins 0.000 claims description 11
- 102000036639 antigens Human genes 0.000 claims description 11
- 235000018102 proteins Nutrition 0.000 claims description 11
- 102000004169 proteins and genes Human genes 0.000 claims description 11
- 239000004220 glutamic acid Substances 0.000 claims description 10
- JFINOWIINSTUNY-UHFFFAOYSA-N pyrrolidin-3-ylmethanesulfonamide Chemical compound NS(=O)(=O)CC1CCNC1 JFINOWIINSTUNY-UHFFFAOYSA-N 0.000 claims description 9
- 108091005804 Peptidases Proteins 0.000 claims description 8
- 239000004365 Protease Substances 0.000 claims description 8
- 230000001419 dependent effect Effects 0.000 claims description 8
- 206010009944 Colon cancer Diseases 0.000 claims description 7
- 229940093444 Cyclooxygenase 2 inhibitor Drugs 0.000 claims description 7
- 239000003937 drug carrier Substances 0.000 claims description 7
- 201000007270 liver cancer Diseases 0.000 claims description 7
- 208000014018 liver neoplasm Diseases 0.000 claims description 7
- 208000023275 Autoimmune disease Diseases 0.000 claims description 6
- 102100035943 HERV-H LTR-associating protein 2 Human genes 0.000 claims description 6
- 102100034458 Hepatitis A virus cellular receptor 2 Human genes 0.000 claims description 6
- 101001021491 Homo sapiens HERV-H LTR-associating protein 2 Proteins 0.000 claims description 6
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 claims description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 6
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 6
- 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 claims description 6
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 claims description 6
- 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 claims description 6
- 208000010125 myocardial infarction Diseases 0.000 claims description 6
- 208000024172 Cardiovascular disease Diseases 0.000 claims description 5
- 208000012902 Nervous system disease Diseases 0.000 claims description 5
- 208000025966 Neurological disease Diseases 0.000 claims description 5
- 206010033128 Ovarian cancer Diseases 0.000 claims description 5
- 206010061535 Ovarian neoplasm Diseases 0.000 claims description 5
- 102100026144 Transferrin receptor protein 1 Human genes 0.000 claims description 5
- VLXBWPOEOIIREY-UHFFFAOYSA-N dimethyl diselenide Natural products C[Se][Se]C VLXBWPOEOIIREY-UHFFFAOYSA-N 0.000 claims description 5
- WQOXQRCZOLPYPM-UHFFFAOYSA-N dimethyl disulfide Chemical group CSSC WQOXQRCZOLPYPM-UHFFFAOYSA-N 0.000 claims description 5
- 208000016097 disease of metabolism Diseases 0.000 claims description 5
- 208000026278 immune system disease Diseases 0.000 claims description 5
- 208000030159 metabolic disease Diseases 0.000 claims description 5
- QYNUQALWYRSVHF-OLZOCXBDSA-N (6R)-5,10-methylenetetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1C1)N)N1C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 QYNUQALWYRSVHF-OLZOCXBDSA-N 0.000 claims description 4
- 208000001333 Colorectal Neoplasms Diseases 0.000 claims description 4
- 108010001517 Galectin 3 Proteins 0.000 claims description 4
- 208000008839 Kidney Neoplasms Diseases 0.000 claims description 4
- 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 claims description 4
- 229930126263 Maytansine Natural products 0.000 claims description 4
- 229930012538 Paclitaxel Natural products 0.000 claims description 4
- 206010038389 Renal cancer Diseases 0.000 claims description 4
- 208000005718 Stomach Neoplasms Diseases 0.000 claims description 4
- 150000001732 carboxylic acid derivatives Chemical class 0.000 claims description 4
- 206010017758 gastric cancer Diseases 0.000 claims description 4
- 201000010982 kidney cancer Diseases 0.000 claims description 4
- ZNOVTXRBGFNYRX-ABLWVSNPSA-N levomefolic acid Chemical compound C1NC=2NC(N)=NC(=O)C=2N(C)C1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 ZNOVTXRBGFNYRX-ABLWVSNPSA-N 0.000 claims description 4
- 235000007635 levomefolic acid Nutrition 0.000 claims description 4
- 239000011578 levomefolic acid Substances 0.000 claims description 4
- WKPWGQKGSOKKOO-RSFHAFMBSA-N maytansine Chemical compound CO[C@@H]([C@@]1(O)C[C@](OC(=O)N1)([C@H]([C@@H]1O[C@@]1(C)[C@@H](OC(=O)[C@H](C)N(C)C(C)=O)CC(=O)N1C)C)[H])\C=C\C=C(C)\CC2=CC(OC)=C(Cl)C1=C2 WKPWGQKGSOKKOO-RSFHAFMBSA-N 0.000 claims description 4
- 229960000485 methotrexate Drugs 0.000 claims description 4
- 229960001592 paclitaxel Drugs 0.000 claims description 4
- 201000011549 stomach cancer Diseases 0.000 claims description 4
- 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 4
- 229940124597 therapeutic agent Drugs 0.000 claims description 4
- ALBODLTZUXKBGZ-JUUVMNCLSA-N (2s)-2-amino-3-phenylpropanoic acid;(2s)-2,6-diaminohexanoic acid Chemical compound NCCCC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CC1=CC=CC=C1 ALBODLTZUXKBGZ-JUUVMNCLSA-N 0.000 claims description 3
- HOZBSSWDEKVXNO-BXRBKJIMSA-N (2s)-2-azanylbutanedioic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O.OC(=O)[C@@H](N)CC(O)=O HOZBSSWDEKVXNO-BXRBKJIMSA-N 0.000 claims description 3
- VVIAGPKUTFNRDU-STQMWFEESA-N (6S)-5-formyltetrahydrofolic acid Chemical compound C([C@H]1CNC=2N=C(NC(=O)C=2N1C=O)N)NC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 VVIAGPKUTFNRDU-STQMWFEESA-N 0.000 claims description 3
- 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 claims description 3
- FMJUDUJLTNVWCH-UHFFFAOYSA-N 2-ethoxy-3-(4-hydroxyphenyl)propanoic acid Chemical group CCOC(C(O)=O)CC1=CC=C(O)C=C1 FMJUDUJLTNVWCH-UHFFFAOYSA-N 0.000 claims description 3
- 101150058497 ANPEP gene Proteins 0.000 claims description 3
- BYXHQQCXAJARLQ-ZLUOBGJFSA-N Ala-Ala-Ala Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O BYXHQQCXAJARLQ-ZLUOBGJFSA-N 0.000 claims description 3
- 102100026882 Alpha-synuclein Human genes 0.000 claims description 3
- 208000024827 Alzheimer disease Diseases 0.000 claims description 3
- 102100022749 Aminopeptidase N Human genes 0.000 claims description 3
- 206010002383 Angina Pectoris Diseases 0.000 claims description 3
- 102100029822 B- and T-lymphocyte attenuator Human genes 0.000 claims description 3
- 108010074708 B7-H1 Antigen Proteins 0.000 claims description 3
- 108010006654 Bleomycin Proteins 0.000 claims description 3
- 102100031650 C-X-C chemokine receptor type 4 Human genes 0.000 claims description 3
- 108091005932 CCKBR Proteins 0.000 claims description 3
- 102100024263 CD160 antigen Human genes 0.000 claims description 3
- 102100027207 CD27 antigen Human genes 0.000 claims description 3
- 102100038078 CD276 antigen Human genes 0.000 claims description 3
- 101710185679 CD276 antigen Proteins 0.000 claims description 3
- 101150013553 CD40 gene Proteins 0.000 claims description 3
- 102100032912 CD44 antigen Human genes 0.000 claims description 3
- 102100025221 CD70 antigen Human genes 0.000 claims description 3
- 108010021064 CTLA-4 Antigen Proteins 0.000 claims description 3
- 229940045513 CTLA4 antagonist Drugs 0.000 claims description 3
- 102100025475 Carcinoembryonic antigen-related cell adhesion molecule 5 Human genes 0.000 claims description 3
- 208000031229 Cardiomyopathies Diseases 0.000 claims description 3
- ZEOWTGPWHLSLOG-UHFFFAOYSA-N Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F Chemical compound Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F ZEOWTGPWHLSLOG-UHFFFAOYSA-N 0.000 claims description 3
- 206010010071 Coma Diseases 0.000 claims description 3
- 208000002330 Congenital Heart Defects Diseases 0.000 claims description 3
- 102100039498 Cytotoxic T-lymphocyte protein 4 Human genes 0.000 claims description 3
- 108010092160 Dactinomycin Proteins 0.000 claims description 3
- 102100036466 Delta-like protein 3 Human genes 0.000 claims description 3
- 208000032928 Dyslipidaemia Diseases 0.000 claims description 3
- 101150029707 ERBB2 gene Proteins 0.000 claims description 3
- 206010014733 Endometrial cancer Diseases 0.000 claims description 3
- 206010014759 Endometrial neoplasm Diseases 0.000 claims description 3
- 208000000461 Esophageal Neoplasms Diseases 0.000 claims description 3
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 claims description 3
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 claims description 3
- 102100023600 Fibroblast growth factor receptor 2 Human genes 0.000 claims description 3
- 101710182389 Fibroblast growth factor receptor 2 Proteins 0.000 claims description 3
- 102100027844 Fibroblast growth factor receptor 4 Human genes 0.000 claims description 3
- 102100021261 Frizzled-10 Human genes 0.000 claims description 3
- 102100038407 G-protein coupled receptor 87 Human genes 0.000 claims description 3
- 102100031351 Galectin-9 Human genes 0.000 claims description 3
- 101710121810 Galectin-9 Proteins 0.000 claims description 3
- 102100036016 Gastrin/cholecystokinin type B receptor Human genes 0.000 claims description 3
- 102000058063 Glucose Transporter Type 1 Human genes 0.000 claims description 3
- ZWZWYGMENQVNFU-UHFFFAOYSA-N Glycerophosphorylserin Natural products OC(=O)C(N)COP(O)(=O)OCC(O)CO ZWZWYGMENQVNFU-UHFFFAOYSA-N 0.000 claims description 3
- 201000005569 Gout Diseases 0.000 claims description 3
- 108010017213 Granulocyte-Macrophage Colony-Stimulating Factor Proteins 0.000 claims description 3
- 102100039620 Granulocyte-macrophage colony-stimulating factor Human genes 0.000 claims description 3
- 206010019196 Head injury Diseases 0.000 claims description 3
- 101710083479 Hepatitis A virus cellular receptor 2 homolog Proteins 0.000 claims description 3
- 102100026122 High affinity immunoglobulin gamma Fc receptor I Human genes 0.000 claims description 3
- 101000834898 Homo sapiens Alpha-synuclein Proteins 0.000 claims description 3
- 101000864344 Homo sapiens B- and T-lymphocyte attenuator Proteins 0.000 claims description 3
- 101000922348 Homo sapiens C-X-C chemokine receptor type 4 Proteins 0.000 claims description 3
- 101000761938 Homo sapiens CD160 antigen Proteins 0.000 claims description 3
- 101000914511 Homo sapiens CD27 antigen Proteins 0.000 claims description 3
- 101000868273 Homo sapiens CD44 antigen Proteins 0.000 claims description 3
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 claims description 3
- 101000914324 Homo sapiens Carcinoembryonic antigen-related cell adhesion molecule 5 Proteins 0.000 claims description 3
- 101000928513 Homo sapiens Delta-like protein 3 Proteins 0.000 claims description 3
- 101100066427 Homo sapiens FCGR1A gene Proteins 0.000 claims description 3
- 101000917134 Homo sapiens Fibroblast growth factor receptor 4 Proteins 0.000 claims description 3
- 101000819451 Homo sapiens Frizzled-10 Proteins 0.000 claims description 3
- 101001033052 Homo sapiens G-protein coupled receptor 87 Proteins 0.000 claims description 3
- 101001068133 Homo sapiens Hepatitis A virus cellular receptor 2 Proteins 0.000 claims description 3
- 101001019455 Homo sapiens ICOS ligand Proteins 0.000 claims description 3
- 101000945371 Homo sapiens Killer cell immunoglobulin-like receptor 2DL2 Proteins 0.000 claims description 3
- 101000945333 Homo sapiens Killer cell immunoglobulin-like receptor 2DL3 Proteins 0.000 claims description 3
- 101000984186 Homo sapiens Leukocyte immunoglobulin-like receptor subfamily B member 4 Proteins 0.000 claims description 3
- 101001137987 Homo sapiens Lymphocyte activation gene 3 protein Proteins 0.000 claims description 3
- 101001109501 Homo sapiens NKG2-D type II integral membrane protein Proteins 0.000 claims description 3
- 101001023705 Homo sapiens Nectin-4 Proteins 0.000 claims description 3
- 101000896414 Homo sapiens Nuclear nucleic acid-binding protein C1D Proteins 0.000 claims description 3
- 101000611936 Homo sapiens Programmed cell death protein 1 Proteins 0.000 claims description 3
- 101001043564 Homo sapiens Prolow-density lipoprotein receptor-related protein 1 Proteins 0.000 claims description 3
- 101000633784 Homo sapiens SLAM family member 7 Proteins 0.000 claims description 3
- 101000652359 Homo sapiens Spermatogenesis-associated protein 2 Proteins 0.000 claims description 3
- 101000835093 Homo sapiens Transferrin receptor protein 1 Proteins 0.000 claims description 3
- 101000801234 Homo sapiens Tumor necrosis factor receptor superfamily member 18 Proteins 0.000 claims description 3
- 101000851370 Homo sapiens Tumor necrosis factor receptor superfamily member 9 Proteins 0.000 claims description 3
- 101000666896 Homo sapiens V-type immunoglobulin domain-containing suppressor of T-cell activation Proteins 0.000 claims description 3
- 208000023105 Huntington disease Diseases 0.000 claims description 3
- 208000013016 Hypoglycemia Diseases 0.000 claims description 3
- 102100034980 ICOS ligand Human genes 0.000 claims description 3
- 102100026879 Interleukin-2 receptor subunit beta Human genes 0.000 claims description 3
- 101150069255 KLRC1 gene Proteins 0.000 claims description 3
- 102100033599 Killer cell immunoglobulin-like receptor 2DL2 Human genes 0.000 claims description 3
- 102100033634 Killer cell immunoglobulin-like receptor 2DL3 Human genes 0.000 claims description 3
- 102000017578 LAG3 Human genes 0.000 claims description 3
- 102100025578 Leukocyte immunoglobulin-like receptor subfamily B member 4 Human genes 0.000 claims description 3
- 208000017170 Lipid metabolism disease Diseases 0.000 claims description 3
- 206010025323 Lymphomas Diseases 0.000 claims description 3
- 101100404845 Macaca mulatta NKG2A gene Proteins 0.000 claims description 3
- 108010061593 Member 14 Tumor Necrosis Factor Receptors Proteins 0.000 claims description 3
- 208000034578 Multiple myelomas Diseases 0.000 claims description 3
- 101100341510 Mus musculus Itgal gene Proteins 0.000 claims description 3
- 101000597780 Mus musculus Tumor necrosis factor ligand superfamily member 18 Proteins 0.000 claims description 3
- 102100022682 NKG2-A/NKG2-B type II integral membrane protein Human genes 0.000 claims description 3
- 102100022680 NKG2-D type II integral membrane protein Human genes 0.000 claims description 3
- UISKUVAEXDIKMS-KOKHDOHPSA-N N[C@@H](CCCCN)C(=O)O.N[C@@H](CC(=O)O)C(=O)O.N[C@@H](CC(=O)O)C(=O)O Chemical compound N[C@@H](CCCCN)C(=O)O.N[C@@H](CC(=O)O)C(=O)O.N[C@@H](CC(=O)O)C(=O)O UISKUVAEXDIKMS-KOKHDOHPSA-N 0.000 claims description 3
- 102100035487 Nectin-3 Human genes 0.000 claims description 3
- 102100035486 Nectin-4 Human genes 0.000 claims description 3
- 208000008589 Obesity Diseases 0.000 claims description 3
- 206010030155 Oesophageal carcinoma Diseases 0.000 claims description 3
- 208000018737 Parkinson disease Diseases 0.000 claims description 3
- 206010035226 Plasma cell myeloma Diseases 0.000 claims description 3
- 102100029740 Poliovirus receptor Human genes 0.000 claims description 3
- 102100024216 Programmed cell death 1 ligand 1 Human genes 0.000 claims description 3
- 102100021923 Prolow-density lipoprotein receptor-related protein 1 Human genes 0.000 claims description 3
- 102100029198 SLAM family member 7 Human genes 0.000 claims description 3
- 108091006296 SLC2A1 Proteins 0.000 claims description 3
- 208000000453 Skin Neoplasms Diseases 0.000 claims description 3
- 208000006011 Stroke Diseases 0.000 claims description 3
- 201000009594 Systemic Scleroderma Diseases 0.000 claims description 3
- 206010042953 Systemic sclerosis Diseases 0.000 claims description 3
- 229940126547 T-cell immunoglobulin mucin-3 Drugs 0.000 claims description 3
- 208000024770 Thyroid neoplasm Diseases 0.000 claims description 3
- 102100035283 Tumor necrosis factor ligand superfamily member 18 Human genes 0.000 claims description 3
- 102100028785 Tumor necrosis factor receptor superfamily member 14 Human genes 0.000 claims description 3
- 102100033728 Tumor necrosis factor receptor superfamily member 18 Human genes 0.000 claims description 3
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 3
- 102100036856 Tumor necrosis factor receptor superfamily member 9 Human genes 0.000 claims description 3
- 208000002495 Uterine Neoplasms Diseases 0.000 claims description 3
- 108010079206 V-Set Domain-Containing T-Cell Activation Inhibitor 1 Proteins 0.000 claims description 3
- 102100038929 V-set domain-containing T-cell activation inhibitor 1 Human genes 0.000 claims description 3
- 102100038282 V-type immunoglobulin domain-containing suppressor of T-cell activation Human genes 0.000 claims description 3
- 208000012886 Vertigo Diseases 0.000 claims description 3
- 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 claims description 3
- 206010003119 arrhythmia Diseases 0.000 claims description 3
- 230000006793 arrhythmia Effects 0.000 claims description 3
- 229960001561 bleomycin Drugs 0.000 claims description 3
- 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 3
- 229960002173 citrulline Drugs 0.000 claims description 3
- 238000003776 cleavage reaction Methods 0.000 claims description 3
- 208000029742 colonic neoplasm Diseases 0.000 claims description 3
- 208000028831 congenital heart disease Diseases 0.000 claims description 3
- 208000018631 connective tissue disease Diseases 0.000 claims description 3
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 claims description 3
- 235000018417 cysteine Nutrition 0.000 claims description 3
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 3
- 229960000640 dactinomycin Drugs 0.000 claims description 3
- 229940127276 delta-like ligand 3 Drugs 0.000 claims description 3
- 206010012601 diabetes mellitus Diseases 0.000 claims description 3
- 201000003914 endometrial carcinoma Diseases 0.000 claims description 3
- 206010015037 epilepsy Diseases 0.000 claims description 3
- 229930013356 epothilone Natural products 0.000 claims description 3
- 150000003883 epothilone derivatives Chemical class 0.000 claims description 3
- 201000004101 esophageal cancer Diseases 0.000 claims description 3
- 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 claims description 3
- 201000001421 hyperglycemia Diseases 0.000 claims description 3
- 208000015210 hypertensive heart disease Diseases 0.000 claims description 3
- 230000002218 hypoglycaemic effect Effects 0.000 claims description 3
- 208000032839 leukemia Diseases 0.000 claims description 3
- 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 claims description 3
- 229960004857 mitomycin Drugs 0.000 claims description 3
- 201000006417 multiple sclerosis Diseases 0.000 claims description 3
- 239000002773 nucleotide Substances 0.000 claims description 3
- 125000003729 nucleotide group Chemical group 0.000 claims description 3
- 235000020824 obesity Nutrition 0.000 claims description 3
- 229960003171 plicamycin Drugs 0.000 claims description 3
- 230000009467 reduction Effects 0.000 claims description 3
- 208000004124 rheumatic heart disease Diseases 0.000 claims description 3
- 206010039073 rheumatoid arthritis Diseases 0.000 claims description 3
- 230000007017 scission Effects 0.000 claims description 3
- 201000000849 skin cancer Diseases 0.000 claims description 3
- 201000000596 systemic lupus erythematosus Diseases 0.000 claims description 3
- 201000002510 thyroid cancer Diseases 0.000 claims description 3
- 206010046766 uterine cancer Diseases 0.000 claims description 3
- 231100000889 vertigo Toxicity 0.000 claims description 3
- KLALXKYLOMZDQT-ZLUOBGJFSA-N Ala-Ser-Asn Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(N)=O KLALXKYLOMZDQT-ZLUOBGJFSA-N 0.000 claims description 2
- OMLWNBVRVJYMBQ-YUMQZZPRSA-N Arg-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O OMLWNBVRVJYMBQ-YUMQZZPRSA-N 0.000 claims description 2
- 101000709472 Homo sapiens Sialic acid-binding Ig-like lectin 15 Proteins 0.000 claims description 2
- HMNSRTLZAJHSIK-YUMQZZPRSA-N Pro-Arg Chemical compound NC(=N)NCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1 HMNSRTLZAJHSIK-YUMQZZPRSA-N 0.000 claims description 2
- FXGIMYRVJJEIIM-UWVGGRQHSA-N Pro-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FXGIMYRVJJEIIM-UWVGGRQHSA-N 0.000 claims description 2
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 claims description 2
- 102100034361 Sialic acid-binding Ig-like lectin 15 Human genes 0.000 claims description 2
- 108010017893 alanyl-alanyl-alanine Proteins 0.000 claims description 2
- 108010068380 arginylarginine Proteins 0.000 claims description 2
- 108700042769 prolyl-leucyl-glycine Proteins 0.000 claims description 2
- 108010004914 prolylarginine Proteins 0.000 claims description 2
- 101000996727 Homo sapiens Gonadotropin-releasing hormone receptor Proteins 0.000 claims 4
- 101000829127 Homo sapiens Somatostatin receptor type 2 Proteins 0.000 claims 3
- 102100023802 Somatostatin receptor type 2 Human genes 0.000 claims 3
- 102100039558 Galectin-3 Human genes 0.000 claims 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 1
- 208000024313 Testicular Neoplasms Diseases 0.000 claims 1
- 206010057644 Testis cancer Diseases 0.000 claims 1
- 201000003120 testicular cancer Diseases 0.000 claims 1
- 210000004027 cell Anatomy 0.000 description 462
- 239000000562 conjugate Substances 0.000 description 235
- 230000014509 gene expression Effects 0.000 description 117
- 235000002639 sodium chloride Nutrition 0.000 description 101
- 241000282414 Homo sapiens Species 0.000 description 85
- 102000005962 receptors Human genes 0.000 description 71
- 108020003175 receptors Proteins 0.000 description 71
- 230000002401 inhibitory effect Effects 0.000 description 66
- 239000011347 resin Substances 0.000 description 53
- 229920005989 resin Polymers 0.000 description 53
- 239000000243 solution Substances 0.000 description 53
- 230000000694 effects Effects 0.000 description 49
- 239000000523 sample Substances 0.000 description 49
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 44
- 229940024606 amino acid Drugs 0.000 description 44
- 230000005764 inhibitory process Effects 0.000 description 44
- 230000027455 binding Effects 0.000 description 43
- 238000002474 experimental method Methods 0.000 description 43
- 241000699670 Mus sp. Species 0.000 description 41
- 210000004881 tumor cell Anatomy 0.000 description 41
- 238000006243 chemical reaction Methods 0.000 description 40
- 230000004614 tumor growth Effects 0.000 description 38
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 36
- IEDXPSOJFSVCKU-HOKPPMCLSA-N [4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[6-(2,5-dioxopyrrolidin-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate Chemical compound CC[C@H](C)[C@@H]([C@@H](CC(=O)N1CCC[C@H]1[C@H](OC)[C@@H](C)C(=O)N[C@H](C)[C@@H](O)c1ccccc1)OC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCc1ccc(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN2C(=O)CCC2=O)C(C)C)cc1)C(C)C IEDXPSOJFSVCKU-HOKPPMCLSA-N 0.000 description 35
- JFCQEDHGNNZCLN-UHFFFAOYSA-N anhydrous glutaric acid Natural products OC(=O)CCCC(O)=O JFCQEDHGNNZCLN-UHFFFAOYSA-N 0.000 description 35
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 35
- RTBFRGCFXZNCOE-UHFFFAOYSA-N 1-methylsulfonylpiperidin-4-one Chemical compound CS(=O)(=O)N1CCC(=O)CC1 RTBFRGCFXZNCOE-UHFFFAOYSA-N 0.000 description 34
- 108010093470 monomethyl auristatin E Proteins 0.000 description 30
- 238000002347 injection Methods 0.000 description 28
- 239000007924 injection Substances 0.000 description 28
- 238000006467 substitution reaction Methods 0.000 description 24
- 241001465754 Metazoa Species 0.000 description 23
- 239000002904 solvent Substances 0.000 description 23
- 210000001519 tissue Anatomy 0.000 description 23
- 229910002092 carbon dioxide Inorganic materials 0.000 description 22
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 21
- 239000013642 negative control Substances 0.000 description 21
- DTQVDTLACAAQTR-UHFFFAOYSA-N trifluoroacetic acid Substances OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 21
- 230000012010 growth Effects 0.000 description 20
- 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 20
- 239000007787 solid Substances 0.000 description 20
- 239000007788 liquid Substances 0.000 description 19
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 19
- 239000002609 medium Substances 0.000 description 18
- 238000002360 preparation method Methods 0.000 description 18
- 102000004196 processed proteins & peptides Human genes 0.000 description 18
- 239000000047 product Substances 0.000 description 17
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 16
- 239000003153 chemical reaction reagent Substances 0.000 description 16
- 238000012258 culturing Methods 0.000 description 16
- 125000006239 protecting group Chemical group 0.000 description 16
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 229910001868 water Inorganic materials 0.000 description 16
- 102000018697 Membrane Proteins Human genes 0.000 description 15
- 108010052285 Membrane Proteins Proteins 0.000 description 15
- 238000004128 high performance liquid chromatography Methods 0.000 description 15
- 239000012139 lysis buffer Substances 0.000 description 15
- UZOVYGYOLBIAJR-UHFFFAOYSA-N 4-isocyanato-4'-methyldiphenylmethane Chemical compound C1=CC(C)=CC=C1CC1=CC=C(N=C=O)C=C1 UZOVYGYOLBIAJR-UHFFFAOYSA-N 0.000 description 14
- 238000010790 dilution Methods 0.000 description 14
- 239000012895 dilution Substances 0.000 description 14
- 239000003550 marker Substances 0.000 description 14
- 229920001184 polypeptide Polymers 0.000 description 14
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 13
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 13
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 13
- 108010021290 LHRH Receptors Proteins 0.000 description 13
- 125000003275 alpha amino acid group Chemical group 0.000 description 13
- 239000012470 diluted sample Substances 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 229930182816 L-glutamine Natural products 0.000 description 12
- 230000000259 anti-tumor effect Effects 0.000 description 12
- 230000010261 cell growth Effects 0.000 description 12
- 238000010828 elution Methods 0.000 description 12
- 239000012091 fetal bovine serum Substances 0.000 description 12
- 238000011729 BALB/c nude mouse Methods 0.000 description 11
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 239000000872 buffer Substances 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 229960003646 lysine Drugs 0.000 description 11
- 239000000463 material Substances 0.000 description 11
- 230000035755 proliferation Effects 0.000 description 11
- 150000003384 small molecules Chemical class 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 210000003462 vein Anatomy 0.000 description 11
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 10
- 102000000844 Cell Surface Receptors Human genes 0.000 description 10
- 108010001857 Cell Surface Receptors Proteins 0.000 description 10
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 10
- 241000700159 Rattus Species 0.000 description 10
- 125000003917 carbamoyl group Chemical group [H]N([H])C(*)=O 0.000 description 10
- 230000000875 corresponding effect Effects 0.000 description 10
- 238000011161 development Methods 0.000 description 10
- 230000018109 developmental process Effects 0.000 description 10
- 125000000524 functional group Chemical group 0.000 description 10
- 230000003834 intracellular effect Effects 0.000 description 10
- 238000003786 synthesis reaction Methods 0.000 description 10
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 9
- 102000020313 Cell-Penetrating Peptides Human genes 0.000 description 9
- 108010051109 Cell-Penetrating Peptides Proteins 0.000 description 9
- 108090000631 Trypsin Proteins 0.000 description 9
- 102000004142 Trypsin Human genes 0.000 description 9
- 150000001408 amides Chemical class 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000037406 food intake Effects 0.000 description 9
- 229960002989 glutamic acid Drugs 0.000 description 9
- 231100000419 toxicity Toxicity 0.000 description 9
- 230000001988 toxicity Effects 0.000 description 9
- 239000012588 trypsin Substances 0.000 description 9
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-Diisopropylethylamine (DIPEA) Chemical compound CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 description 8
- 238000010171 animal model Methods 0.000 description 8
- 229940049595 antibody-drug conjugate Drugs 0.000 description 8
- 230000006399 behavior Effects 0.000 description 8
- 239000006143 cell culture medium Substances 0.000 description 8
- 230000035622 drinking Effects 0.000 description 8
- 235000012631 food intake Nutrition 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 210000000056 organ Anatomy 0.000 description 8
- 238000002953 preparative HPLC Methods 0.000 description 8
- 238000007920 subcutaneous administration Methods 0.000 description 8
- 239000000725 suspension Substances 0.000 description 8
- 230000004584 weight gain Effects 0.000 description 8
- 235000019786 weight gain Nutrition 0.000 description 8
- 230000004580 weight loss Effects 0.000 description 8
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 7
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 7
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 7
- 102000035195 Peptidases Human genes 0.000 description 7
- 108010087230 Sincalide Proteins 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- 230000003321 amplification Effects 0.000 description 7
- 239000002246 antineoplastic agent Substances 0.000 description 7
- 230000008901 benefit Effects 0.000 description 7
- 238000010609 cell counting kit-8 assay Methods 0.000 description 7
- 230000004663 cell proliferation Effects 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 238000007865 diluting Methods 0.000 description 7
- 239000000706 filtrate Substances 0.000 description 7
- 235000013922 glutamic acid Nutrition 0.000 description 7
- 238000003199 nucleic acid amplification method Methods 0.000 description 7
- 239000012071 phase Substances 0.000 description 7
- 238000007747 plating Methods 0.000 description 7
- 239000000843 powder Substances 0.000 description 7
- 238000012545 processing Methods 0.000 description 7
- 238000001228 spectrum Methods 0.000 description 7
- NWZSZGALRFJKBT-KNIFDHDWSA-N (2s)-2,6-diaminohexanoic acid;(2s)-2-hydroxybutanedioic acid Chemical compound OC(=O)[C@@H](O)CC(O)=O.NCCCC[C@H](N)C(O)=O NWZSZGALRFJKBT-KNIFDHDWSA-N 0.000 description 6
- JOAQINSXLLMRCV-UHFFFAOYSA-N 4-{[(2-amino-4-hydroxypteridin-6-yl)methyl]amino}benzoic acid Chemical compound C1=NC2=NC(N)=NC(O)=C2N=C1CNC1=CC=C(C(O)=O)C=C1 JOAQINSXLLMRCV-UHFFFAOYSA-N 0.000 description 6
- 208000010507 Adenocarcinoma of Lung Diseases 0.000 description 6
- 108091023037 Aptamer Proteins 0.000 description 6
- 101150079449 Folh1 gene Proteins 0.000 description 6
- BZLVMXJERCGZMT-UHFFFAOYSA-N Methyl tert-butyl ether Chemical compound COC(C)(C)C BZLVMXJERCGZMT-UHFFFAOYSA-N 0.000 description 6
- 238000004115 adherent culture Methods 0.000 description 6
- 238000010276 construction Methods 0.000 description 6
- 230000002596 correlated effect Effects 0.000 description 6
- IKDUDTNKRLTJSI-UHFFFAOYSA-N hydrazine monohydrate Substances O.NN IKDUDTNKRLTJSI-UHFFFAOYSA-N 0.000 description 6
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 6
- 239000005457 ice water Substances 0.000 description 6
- 201000005249 lung adenocarcinoma Diseases 0.000 description 6
- 239000010413 mother solution Substances 0.000 description 6
- 230000001225 therapeutic effect Effects 0.000 description 6
- 210000002700 urine Anatomy 0.000 description 6
- KSDTXRUIZMTBNV-INIZCTEOSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)butanedioic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)O)C(O)=O)C3=CC=CC=C3C2=C1 KSDTXRUIZMTBNV-INIZCTEOSA-N 0.000 description 5
- GOPWHXPXSPIIQZ-FQEVSTJZSA-N (4s)-4-(9h-fluoren-9-ylmethoxycarbonylamino)-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCC(O)=O)C(=O)OC(C)(C)C)C3=CC=CC=C3C2=C1 GOPWHXPXSPIIQZ-FQEVSTJZSA-N 0.000 description 5
- FJHBVJOVLFPMQE-QFIPXVFZSA-N 7-Ethyl-10-Hydroxy-Camptothecin Chemical compound C1=C(O)C=C2C(CC)=C(CN3C(C4=C([C@@](C(=O)OC4)(O)CC)C=C33)=O)C3=NC2=C1 FJHBVJOVLFPMQE-QFIPXVFZSA-N 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 5
- PLXLYXLUCNZSAA-QLXKLKPCSA-N CC[C@@]1(O)C(=O)OCC2=C1C=C1N(CC3=C1N=C1C=C(F)C(C)=C4CC[C@H](NC(=O)CO)C3=C14)C2=O Chemical compound CC[C@@]1(O)C(=O)OCC2=C1C=C1N(CC3=C1N=C1C=C(F)C(C)=C4CC[C@H](NC(=O)CO)C3=C14)C2=O PLXLYXLUCNZSAA-QLXKLKPCSA-N 0.000 description 5
- 201000009030 Carcinoma Diseases 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 101150095463 Folr1 gene Proteins 0.000 description 5
- 239000004471 Glycine Substances 0.000 description 5
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 5
- 239000002202 Polyethylene glycol Substances 0.000 description 5
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- 239000002738 chelating agent Substances 0.000 description 5
- 238000004140 cleaning Methods 0.000 description 5
- 229910001873 dinitrogen Inorganic materials 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 210000003608 fece Anatomy 0.000 description 5
- 102000006815 folate receptor Human genes 0.000 description 5
- 108020005243 folate receptor Proteins 0.000 description 5
- 238000011534 incubation Methods 0.000 description 5
- 230000003285 pharmacodynamic effect Effects 0.000 description 5
- 229920001223 polyethylene glycol Polymers 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 238000003746 solid phase reaction Methods 0.000 description 5
- FODJWPHPWBKDON-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-[(2-methylpropan-2-yl)oxy]-4-oxobutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(=O)OC(C)(C)C)C(O)=O)C3=CC=CC=C3C2=C1 FODJWPHPWBKDON-IBGZPJMESA-N 0.000 description 4
- 101710160107 Outer membrane protein A Proteins 0.000 description 4
- 206010041067 Small cell lung cancer Diseases 0.000 description 4
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- NLMBVBUNULOTNS-HOKPPMCLSA-N [4-[[(2s)-5-(carbamoylamino)-2-[[(2s)-2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl n-[(2s)-1-[[(2s)-1-[[(3r,4s,5s)-1-[(2s)-2-[(1r,2r)-3-[[(1s,2r)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-o Chemical compound C1([C@H](O)[C@@H](C)NC(=O)[C@H](C)[C@@H](OC)[C@@H]2CCCN2C(=O)C[C@H]([C@H]([C@@H](C)CC)N(C)C(=O)[C@@H](NC(=O)[C@H](C(C)C)N(C)C(=O)OCC=2C=CC(NC(=O)[C@H](CCCNC(N)=O)NC(=O)[C@@H](NC(=O)CCCCCN3C(C=CC3=O)=O)C(C)C)=CC=2)C(C)C)OC)=CC=CC=C1 NLMBVBUNULOTNS-HOKPPMCLSA-N 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- 230000017531 blood circulation Effects 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000012043 crude product Substances 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 239000003995 emulsifying agent Substances 0.000 description 4
- 238000011010 flushing procedure Methods 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 210000003734 kidney Anatomy 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 206010041823 squamous cell carcinoma Diseases 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- 239000012224 working solution Substances 0.000 description 4
- MFRNYXJJRJQHNW-DEMKXPNLSA-N (2s)-2-[[(2r,3r)-3-methoxy-3-[(2s)-1-[(3r,4s,5s)-3-methoxy-5-methyl-4-[methyl-[(2s)-3-methyl-2-[[(2s)-3-methyl-2-(methylamino)butanoyl]amino]butanoyl]amino]heptanoyl]pyrrolidin-2-yl]-2-methylpropanoyl]amino]-3-phenylpropanoic acid Chemical compound CN[C@@H](C(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(O)=O)CC1=CC=CC=C1 MFRNYXJJRJQHNW-DEMKXPNLSA-N 0.000 description 3
- DVBUCBXGDWWXNY-SFHVURJKSA-N (2s)-5-(diaminomethylideneamino)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CCCN=C(N)N)C(O)=O)C3=CC=CC=C3C2=C1 DVBUCBXGDWWXNY-SFHVURJKSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- ITWBWJFEJCHKSN-UHFFFAOYSA-N 1,4,7-triazonane Chemical compound C1CNCCNCCN1 ITWBWJFEJCHKSN-UHFFFAOYSA-N 0.000 description 3
- SYFGLWDDLZQFNI-UHFFFAOYSA-N 2-[4-(carboxymethyl)-1,4,8,11-tetrazabicyclo[6.6.2]hexadecan-11-yl]acetic acid Chemical compound C1CN(CC(O)=O)CCCN2CCN(CC(=O)O)CCCN1CC2 SYFGLWDDLZQFNI-UHFFFAOYSA-N 0.000 description 3
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 3
- 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 3
- 239000004475 Arginine Substances 0.000 description 3
- 206010005003 Bladder cancer Diseases 0.000 description 3
- COLNVLDHVKWLRT-MRVPVSSYSA-N D-phenylalanine Chemical compound OC(=O)[C@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-MRVPVSSYSA-N 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- 102000000802 Galectin 3 Human genes 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 239000007821 HATU Substances 0.000 description 3
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 3
- 235000010643 Leucaena leucocephala Nutrition 0.000 description 3
- 240000007472 Leucaena leucocephala Species 0.000 description 3
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- 208000007097 Urinary Bladder Neoplasms Diseases 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 239000000443 aerosol Substances 0.000 description 3
- 150000001412 amines Chemical class 0.000 description 3
- 239000012491 analyte Substances 0.000 description 3
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 3
- 229960003121 arginine Drugs 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 238000004364 calculation method Methods 0.000 description 3
- 239000002775 capsule Substances 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 125000003636 chemical group Chemical group 0.000 description 3
- 239000003086 colorant Substances 0.000 description 3
- 229940127089 cytotoxic agent Drugs 0.000 description 3
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 235000019800 disodium phosphate Nutrition 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 230000009977 dual effect Effects 0.000 description 3
- 239000000839 emulsion Substances 0.000 description 3
- 150000002148 esters Chemical class 0.000 description 3
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 210000004185 liver Anatomy 0.000 description 3
- 239000008176 lyophilized powder Substances 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 108010059074 monomethylauristatin F Proteins 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000002504 physiological saline solution Substances 0.000 description 3
- 239000006187 pill Substances 0.000 description 3
- 238000006722 reduction reaction Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 238000013341 scale-up Methods 0.000 description 3
- RMAQACBXLXPBSY-UHFFFAOYSA-N silicic acid Chemical compound O[Si](O)(O)O RMAQACBXLXPBSY-UHFFFAOYSA-N 0.000 description 3
- 239000006188 syrup Substances 0.000 description 3
- 235000020357 syrup Nutrition 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 230000002100 tumorsuppressive effect Effects 0.000 description 3
- 201000005112 urinary bladder cancer Diseases 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- PUPZLCDOIYMWBV-UHFFFAOYSA-N (+/-)-1,3-Butanediol Chemical compound CC(O)CCO PUPZLCDOIYMWBV-UHFFFAOYSA-N 0.000 description 2
- ZPGDWQNBZYOZTI-SFHVURJKSA-N (2s)-1-(9h-fluoren-9-ylmethoxycarbonyl)pyrrolidine-2-carboxylic acid Chemical compound OC(=O)[C@@H]1CCCN1C(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21 ZPGDWQNBZYOZTI-SFHVURJKSA-N 0.000 description 2
- CBPJQFCAFFNICX-IBGZPJMESA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-4-methylpentanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC(C)C)C(O)=O)C3=CC=CC=C3C2=C1 CBPJQFCAFFNICX-IBGZPJMESA-N 0.000 description 2
- BLUGYPPOFIHFJS-UUFHNPECSA-N (2s)-n-[(2s)-1-[[(3r,4s,5s)-3-methoxy-1-[(2s)-2-[(1r,2r)-1-methoxy-2-methyl-3-oxo-3-[[(1s)-2-phenyl-1-(1,3-thiazol-2-yl)ethyl]amino]propyl]pyrrolidin-1-yl]-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]-3-methyl-2-(methylamino)butanamid Chemical compound CN[C@@H](C(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 BLUGYPPOFIHFJS-UUFHNPECSA-N 0.000 description 2
- VBICKXHEKHSIBG-UHFFFAOYSA-N 1-monostearoylglycerol Chemical compound CCCCCCCCCCCCCCCCCC(=O)OCC(O)CO VBICKXHEKHSIBG-UHFFFAOYSA-N 0.000 description 2
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 2
- JHALWMSZGCVVEM-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CC1 JHALWMSZGCVVEM-UHFFFAOYSA-N 0.000 description 2
- CUZWESFCFFQUME-UHFFFAOYSA-N 2-[4-(carboxymethyl)-7-[(4-isothiocyanatophenyl)methyl]-1,4,7-triazonan-1-yl]acetic acid Chemical compound C1CN(CC(=O)O)CCN(CC(O)=O)CCN1CC1=CC=C(N=C=S)C=C1 CUZWESFCFFQUME-UHFFFAOYSA-N 0.000 description 2
- CKBDZRFNRUQSEI-UHFFFAOYSA-N 2-[4-[[4-[2-(2-aminoethylamino)-2-oxoethyl]phenyl]methyl]-7-(carboxymethyl)-1,4,7-triazonan-1-yl]acetic acid Chemical compound NCCNC(=O)CC1=CC=C(CN2CCN(CC(O)=O)CCN(CC(O)=O)CC2)C=C1 CKBDZRFNRUQSEI-UHFFFAOYSA-N 0.000 description 2
- XUSKJHCMMWAAHV-SANMLTNESA-N 220913-32-6 Chemical compound C1=C(O)C=C2C([Si](C)(C)C(C)(C)C)=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 XUSKJHCMMWAAHV-SANMLTNESA-N 0.000 description 2
- UQQQAKFVWNQYTP-UHFFFAOYSA-N 3,6,10,13,16,19-hexazabicyclo[6.6.6]icosane-1,8-diamine Chemical compound C1NCCNCC2(N)CNCCNCC1(N)CNCCNC2 UQQQAKFVWNQYTP-UHFFFAOYSA-N 0.000 description 2
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 2
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 2
- IIKWGTMGEYTNCQ-UHFFFAOYSA-N 5-(2-aminoethylamino)-5-oxo-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetrazacyclododec-1-yl]pentanoic acid Chemical compound NCCNC(CCC(C(=O)O)N1CCN(CCN(CCN(CC1)CC(=O)O)CC(=O)O)CC(=O)O)=O IIKWGTMGEYTNCQ-UHFFFAOYSA-N 0.000 description 2
- FUXVKZWTXQUGMW-FQEVSTJZSA-N 9-Aminocamptothecin Chemical compound C1=CC(N)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 FUXVKZWTXQUGMW-FQEVSTJZSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 2
- NOHFVHQBEMKKMB-UHFFFAOYSA-N C(=O)(O)C(CCC(=O)NCCN1C(C=CC1=O)=O)N1CCN(CCN(CCN(CC1)CC(=O)O)CC(=O)O)CC(=O)O Chemical compound C(=O)(O)C(CCC(=O)NCCN1C(C=CC1=O)=O)N1CCN(CCN(CCN(CC1)CC(=O)O)CC(=O)O)CC(=O)O NOHFVHQBEMKKMB-UHFFFAOYSA-N 0.000 description 2
- KSCCMOOFLZFMFP-UHFFFAOYSA-N C(C)(C)(C)OC(CN1CCN(CCN(CCN(CC1)CC(=O)OC(C)(C)C)CC(NCC#C)=O)CC(=O)OC(C)(C)C)=O Chemical compound C(C)(C)(C)OC(CN1CCN(CCN(CCN(CC1)CC(=O)OC(C)(C)C)CC(NCC#C)=O)CC(=O)OC(C)(C)C)=O KSCCMOOFLZFMFP-UHFFFAOYSA-N 0.000 description 2
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 2
- 102000004225 Cathepsin B Human genes 0.000 description 2
- 108090000712 Cathepsin B Proteins 0.000 description 2
- 206010008342 Cervix carcinoma Diseases 0.000 description 2
- ROSDSFDQCJNGOL-UHFFFAOYSA-N Dimethylamine Chemical compound CNC ROSDSFDQCJNGOL-UHFFFAOYSA-N 0.000 description 2
- 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 description 2
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- QUSNBJAOOMFDIB-UHFFFAOYSA-N Ethylamine Chemical compound CCN QUSNBJAOOMFDIB-UHFFFAOYSA-N 0.000 description 2
- 102100037362 Fibronectin Human genes 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 101001027128 Homo sapiens Fibronectin Proteins 0.000 description 2
- 101001027081 Homo sapiens Killer cell immunoglobulin-like receptor 2DL1 Proteins 0.000 description 2
- 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 2
- 102100037363 Killer cell immunoglobulin-like receptor 2DL1 Human genes 0.000 description 2
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 2
- 108010001831 LDL receptors Proteins 0.000 description 2
- 102100024640 Low-density lipoprotein receptor Human genes 0.000 description 2
- 241000124008 Mammalia Species 0.000 description 2
- BAVYZALUXZFZLV-UHFFFAOYSA-N Methylamine Chemical compound NC BAVYZALUXZFZLV-UHFFFAOYSA-N 0.000 description 2
- 206010048723 Multiple-drug resistance Diseases 0.000 description 2
- QPCDCPDFJACHGM-UHFFFAOYSA-N N,N-bis{2-[bis(carboxymethyl)amino]ethyl}glycine Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CC(O)=O QPCDCPDFJACHGM-UHFFFAOYSA-N 0.000 description 2
- DTPTWBYXQVMEMQ-UHFFFAOYSA-N NCCNC(=O)CCC(C(O)=O)N1CCN(CC(O)=O)CCN(CC(O)=O)CC1 Chemical compound NCCNC(=O)CCC(C(O)=O)N1CCN(CC(O)=O)CCN(CC(O)=O)CC1 DTPTWBYXQVMEMQ-UHFFFAOYSA-N 0.000 description 2
- 235000019483 Peanut oil Nutrition 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- GLUUGHFHXGJENI-UHFFFAOYSA-N Piperazine Chemical compound C1CNCCN1 GLUUGHFHXGJENI-UHFFFAOYSA-N 0.000 description 2
- 108010072866 Prostate-Specific Antigen Proteins 0.000 description 2
- 102100038358 Prostate-specific antigen Human genes 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 239000008156 Ringer's lactate solution Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 108010033576 Transferrin Receptors Proteins 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 108060008683 Tumor Necrosis Factor Receptor Proteins 0.000 description 2
- 208000006105 Uterine Cervical Neoplasms Diseases 0.000 description 2
- HPDRGGNSEBLDKL-NRFANRHFSA-N ac1l3zgz Chemical compound C1=CC=C2C(CO)=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 HPDRGGNSEBLDKL-NRFANRHFSA-N 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 239000002671 adjuvant Substances 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 125000000539 amino acid group Chemical group 0.000 description 2
- 150000008064 anhydrides Chemical class 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 230000001773 anti-convulsant effect Effects 0.000 description 2
- 239000000611 antibody drug conjugate Substances 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 239000001961 anticonvulsive agent Substances 0.000 description 2
- 229960003965 antiepileptics Drugs 0.000 description 2
- 229940041181 antineoplastic drug Drugs 0.000 description 2
- 239000003443 antiviral agent Substances 0.000 description 2
- LNHWXBUNXOXMRL-VWLOTQADSA-N belotecan Chemical compound C1=CC=C2C(CCNC(C)C)=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 LNHWXBUNXOXMRL-VWLOTQADSA-N 0.000 description 2
- 229950011276 belotecan Drugs 0.000 description 2
- SESFRYSPDFLNCH-UHFFFAOYSA-N benzyl benzoate Chemical compound C=1C=CC=CC=1C(=O)OCC1=CC=CC=C1 SESFRYSPDFLNCH-UHFFFAOYSA-N 0.000 description 2
- WGQKYBSKWIADBV-UHFFFAOYSA-N benzylamine Chemical compound NCC1=CC=CC=C1 WGQKYBSKWIADBV-UHFFFAOYSA-N 0.000 description 2
- 210000000941 bile Anatomy 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 229960000074 biopharmaceutical Drugs 0.000 description 2
- CJZGTCYPCWQAJB-UHFFFAOYSA-L calcium stearate Chemical class [Ca+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O CJZGTCYPCWQAJB-UHFFFAOYSA-L 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 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 2
- 229960000590 celecoxib Drugs 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 201000010881 cervical cancer Diseases 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 235000005687 corn oil Nutrition 0.000 description 2
- 239000002285 corn oil Substances 0.000 description 2
- POADTFBBIXOWFJ-VWLOTQADSA-N cositecan Chemical compound C1=CC=C2C(CC[Si](C)(C)C)=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 POADTFBBIXOWFJ-VWLOTQADSA-N 0.000 description 2
- 235000012343 cottonseed oil Nutrition 0.000 description 2
- 239000002385 cottonseed oil Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 108700013553 diamsar chelate Proteins 0.000 description 2
- LFQCJSBXBZRMTN-OAQYLSRUSA-N diflomotecan Chemical compound CC[C@@]1(O)CC(=O)OCC(C2=O)=C1C=C1N2CC2=CC3=CC(F)=C(F)C=C3N=C21 LFQCJSBXBZRMTN-OAQYLSRUSA-N 0.000 description 2
- 230000008030 elimination Effects 0.000 description 2
- 238000003379 elimination reaction Methods 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- DEFVIWRASFVYLL-UHFFFAOYSA-N ethylene glycol bis(2-aminoethyl)tetraacetic acid Chemical compound OC(=O)CN(CC(O)=O)CCOCCOCCN(CC(O)=O)CC(O)=O DEFVIWRASFVYLL-UHFFFAOYSA-N 0.000 description 2
- ZVYVPGLRVWUPMP-FYSMJZIKSA-N exatecan Chemical compound C1C[C@H](N)C2=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC3=CC(F)=C(C)C1=C32 ZVYVPGLRVWUPMP-FYSMJZIKSA-N 0.000 description 2
- 229950009429 exatecan Drugs 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 239000000945 filler Substances 0.000 description 2
- 239000012065 filter cake Substances 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 235000013355 food flavoring agent Nutrition 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- UIVFUQKYVFCEKJ-OPTOVBNMSA-N gimatecan Chemical compound C1=CC=C2C(\C=N\OC(C)(C)C)=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 UIVFUQKYVFCEKJ-OPTOVBNMSA-N 0.000 description 2
- 229950009073 gimatecan Drugs 0.000 description 2
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 2
- 239000008187 granular material Substances 0.000 description 2
- 231100000086 high toxicity Toxicity 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 239000003701 inert diluent Substances 0.000 description 2
- 230000008595 infiltration Effects 0.000 description 2
- 238000001764 infiltration Methods 0.000 description 2
- 102000006495 integrins Human genes 0.000 description 2
- 108010044426 integrins Proteins 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 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 description 2
- 229960004768 irinotecan Drugs 0.000 description 2
- 229960004592 isopropanol Drugs 0.000 description 2
- 239000000787 lecithin Substances 0.000 description 2
- 235000010445 lecithin Nutrition 0.000 description 2
- 229940067606 lecithin Drugs 0.000 description 2
- 239000008297 liquid dosage form Substances 0.000 description 2
- 231100000053 low toxicity Toxicity 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- RVFGKBWWUQOIOU-NDEPHWFRSA-N lurtotecan Chemical compound O=C([C@]1(O)CC)OCC(C(N2CC3=4)=O)=C1C=C2C3=NC1=CC=2OCCOC=2C=C1C=4CN1CCN(C)CC1 RVFGKBWWUQOIOU-NDEPHWFRSA-N 0.000 description 2
- 229950002654 lurtotecan Drugs 0.000 description 2
- ORIWMYRMOGIXGG-ZXVJYWQYSA-N lurtotecan dihydrochloride Chemical compound Cl.Cl.O=C([C@]1(O)CC)OCC(C(N2CC3=4)=O)=C1C=C2C3=NC1=CC=2OCCOC=2C=C1C=4CN1CCN(C)CC1 ORIWMYRMOGIXGG-ZXVJYWQYSA-N 0.000 description 2
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 239000012120 mounting media Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 231100000252 nontoxic Toxicity 0.000 description 2
- 230000003000 nontoxic effect Effects 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 150000007523 nucleic acids Chemical class 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000010603 pastilles Nutrition 0.000 description 2
- 239000000312 peanut oil Substances 0.000 description 2
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 2
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 2
- 229920000053 polysorbate 80 Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 239000003755 preservative agent Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 210000002307 prostate Anatomy 0.000 description 2
- 210000000064 prostate epithelial cell Anatomy 0.000 description 2
- 238000010791 quenching Methods 0.000 description 2
- 230000000171 quenching effect Effects 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- VHXNKPBCCMUMSW-FQEVSTJZSA-N rubitecan Chemical compound C1=CC([N+]([O-])=O)=C2C=C(CN3C4=CC5=C(C3=O)COC(=O)[C@]5(O)CC)C4=NC2=C1 VHXNKPBCCMUMSW-FQEVSTJZSA-N 0.000 description 2
- 229950009213 rubitecan Drugs 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000008159 sesame oil Substances 0.000 description 2
- 235000011803 sesame oil Nutrition 0.000 description 2
- 208000000587 small cell lung carcinoma Diseases 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- WGTYBPLFGIVFAS-UHFFFAOYSA-M tetramethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)C WGTYBPLFGIVFAS-UHFFFAOYSA-M 0.000 description 2
- 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 description 2
- 229960000303 topotecan Drugs 0.000 description 2
- 231100000331 toxic Toxicity 0.000 description 2
- 230000002588 toxic effect Effects 0.000 description 2
- 230000026683 transduction Effects 0.000 description 2
- 238000010361 transduction Methods 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 102000003298 tumor necrosis factor receptor Human genes 0.000 description 2
- 239000000080 wetting agent Substances 0.000 description 2
- JNYAEWCLZODPBN-JGWLITMVSA-N (2r,3r,4s)-2-[(1r)-1,2-dihydroxyethyl]oxolane-3,4-diol Chemical compound OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O JNYAEWCLZODPBN-JGWLITMVSA-N 0.000 description 1
- UGNIYGNGCNXHTR-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-methylbutanoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](C(C)C)C(O)=O)C3=CC=CC=C3C2=C1 UGNIYGNGCNXHTR-SFHVURJKSA-N 0.000 description 1
- SJVFAHZPLIXNDH-QFIPXVFZSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)-3-phenylpropanoic acid Chemical compound C([C@@H](C(=O)O)NC(=O)OCC1C2=CC=CC=C2C2=CC=CC=C21)C1=CC=CC=C1 SJVFAHZPLIXNDH-QFIPXVFZSA-N 0.000 description 1
- DJGMNCKHNMRKFM-SFHVURJKSA-N (2s)-2-(9h-fluoren-9-ylmethoxycarbonylamino)pent-4-ynoic acid Chemical compound C1=CC=C2C(COC(=O)N[C@@H](CC#C)C(=O)O)C3=CC=CC=C3C2=C1 DJGMNCKHNMRKFM-SFHVURJKSA-N 0.000 description 1
- XUNKPNYCNUKOAU-VXJRNSOOSA-N (2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-[[(2s)-2-amino-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]amino]-5-(diaminomethylideneamino)pentanoyl]a Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUNKPNYCNUKOAU-VXJRNSOOSA-N 0.000 description 1
- POGSZHUEECCEAP-ZETCQYMHSA-N (2s)-2-amino-3-(3-amino-4-hydroxyphenyl)propanoic acid Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C(N)=C1 POGSZHUEECCEAP-ZETCQYMHSA-N 0.000 description 1
- FPJGLSZLQLNZIW-VIFPVBQESA-N (2s)-2-amino-3-(4-methyl-1h-indol-3-yl)propanoic acid Chemical compound CC1=CC=CC2=C1C(C[C@H](N)C(O)=O)=CN2 FPJGLSZLQLNZIW-VIFPVBQESA-N 0.000 description 1
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 description 1
- 229940058015 1,3-butylene glycol Drugs 0.000 description 1
- QBPPRVHXOZRESW-UHFFFAOYSA-N 1,4,7,10-tetraazacyclododecane Chemical compound C1CNCCNCCNCCN1 QBPPRVHXOZRESW-UHFFFAOYSA-N 0.000 description 1
- WZYLHNBQPWGVDJ-UHFFFAOYSA-N 1,4,7,10-tetrazacyclotridec-5-ylmethanamine Chemical compound NCC1CNCCNCCCNCCN1 WZYLHNBQPWGVDJ-UHFFFAOYSA-N 0.000 description 1
- KIUIVKNVSSLOAG-UHFFFAOYSA-N 1,4,7,10-tetrazacyclotridecan-11-one Chemical compound O=C1CCNCCNCCNCCN1 KIUIVKNVSSLOAG-UHFFFAOYSA-N 0.000 description 1
- MDAXKAUIABOHTD-UHFFFAOYSA-N 1,4,8,11-tetraazacyclotetradecane Chemical compound C1CNCCNCCCNCCNC1 MDAXKAUIABOHTD-UHFFFAOYSA-N 0.000 description 1
- HHCPAKJHIFSCSD-UHFFFAOYSA-N 1,4,8,11-tetrazacyclotetradecan-5-one Chemical compound O=C1CCNCCNCCCNCCN1 HHCPAKJHIFSCSD-UHFFFAOYSA-N 0.000 description 1
- BGVLBVASHIQNIO-UHFFFAOYSA-N 1,4,8,11-tetrazacyclotetradecane-5,7-dione Chemical compound O=C1CC(=O)NCCNCCCNCCN1 BGVLBVASHIQNIO-UHFFFAOYSA-N 0.000 description 1
- PWJHXHMUGFXPSN-UHFFFAOYSA-N 1,7-dioxa-4,10-diazacyclododecane Chemical compound C1COCCNCCOCCN1 PWJHXHMUGFXPSN-UHFFFAOYSA-N 0.000 description 1
- BNLDMZVBFXARKJ-UHFFFAOYSA-N 1,8-dimethyl-1,4,8,11-tetrazacyclotetradecane Chemical compound CN1CCCNCCN(C)CCCNCC1 BNLDMZVBFXARKJ-UHFFFAOYSA-N 0.000 description 1
- LUTLAXLNPLZCOF-UHFFFAOYSA-N 1-Methylhistidine Natural products OC(=O)C(N)(C)CC1=NC=CN1 LUTLAXLNPLZCOF-UHFFFAOYSA-N 0.000 description 1
- FNJKRVYABOAQHB-UHFFFAOYSA-N 1-benzyl-1,4,7-triazonane Chemical compound C=1C=CC=CC=1CN1CCNCCNCC1 FNJKRVYABOAQHB-UHFFFAOYSA-N 0.000 description 1
- XLLFEZGASHWCGB-UHFFFAOYSA-N 1-benzyl-1,5,9-triazacyclododecane Chemical compound C=1C=CC=CC=1CN1CCCNCCCNCCC1 XLLFEZGASHWCGB-UHFFFAOYSA-N 0.000 description 1
- 101150029062 15 gene Proteins 0.000 description 1
- VILCJCGEZXAXTO-UHFFFAOYSA-N 2,2,2-tetramine Chemical compound NCCNCCNCCN VILCJCGEZXAXTO-UHFFFAOYSA-N 0.000 description 1
- LDGWQMRUWMSZIU-LQDDAWAPSA-M 2,3-bis[(z)-octadec-9-enoxy]propyl-trimethylazanium;chloride Chemical compound [Cl-].CCCCCCCC\C=C/CCCCCCCCOCC(C[N+](C)(C)C)OCCCCCCCC\C=C/CCCCCCCC LDGWQMRUWMSZIU-LQDDAWAPSA-M 0.000 description 1
- NTOIKDYVJIWVSU-UHFFFAOYSA-N 2,3-dihydroxy-2,3-bis(4-methylbenzoyl)butanedioic acid Chemical class C1=CC(C)=CC=C1C(=O)C(O)(C(O)=O)C(O)(C(O)=O)C(=O)C1=CC=C(C)C=C1 NTOIKDYVJIWVSU-UHFFFAOYSA-N 0.000 description 1
- VYHUGVOVCKRCKG-UHFFFAOYSA-N 2-(2-phenylethylsulfinylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)CCC1=CC=CC=C1 VYHUGVOVCKRCKG-UHFFFAOYSA-N 0.000 description 1
- BFAYIORIWKEONN-UHFFFAOYSA-N 2-(2-phenylethylsulfonylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)(=O)CCC1=CC=CC=C1 BFAYIORIWKEONN-UHFFFAOYSA-N 0.000 description 1
- JKMKVJHRNQTGPW-UHFFFAOYSA-N 2-(3-phenylpropylsulfinylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)CCCC1=CC=CC=C1 JKMKVJHRNQTGPW-UHFFFAOYSA-N 0.000 description 1
- OTZATDKDOPYOAI-UHFFFAOYSA-N 2-(3-phenylpropylsulfonylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)(=O)CCCC1=CC=CC=C1 OTZATDKDOPYOAI-UHFFFAOYSA-N 0.000 description 1
- RPNMGUBLKCLAEK-UHFFFAOYSA-N 2-(4-chlorophenyl)sulfanyl-n,n-diethylethanamine;hydrochloride Chemical compound [Cl-].CC[NH+](CC)CCSC1=CC=C(Cl)C=C1 RPNMGUBLKCLAEK-UHFFFAOYSA-N 0.000 description 1
- FTDAIZALXRHAIP-UHFFFAOYSA-N 2-(benzylsulfonylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)(=O)CC1=CC=CC=C1 FTDAIZALXRHAIP-UHFFFAOYSA-N 0.000 description 1
- UBPNBZGCZKGWIK-UHFFFAOYSA-N 2-(butylsulfinylmethyl)pentanedioic acid Chemical compound CCCCS(=O)CC(C(O)=O)CCC(O)=O UBPNBZGCZKGWIK-UHFFFAOYSA-N 0.000 description 1
- AQLGDVIEDOXUGY-UHFFFAOYSA-N 2-(butylsulfonylmethyl)pentanedioic acid Chemical compound CCCCS(=O)(=O)CC(C(O)=O)CCC(O)=O AQLGDVIEDOXUGY-UHFFFAOYSA-N 0.000 description 1
- NAWOQIXSDKDBEM-UHFFFAOYSA-N 2-(ethylsulfinylmethyl)pentanedioic acid Chemical compound CCS(=O)CC(C(O)=O)CCC(O)=O NAWOQIXSDKDBEM-UHFFFAOYSA-N 0.000 description 1
- SXSQDMPJGXEWGO-UHFFFAOYSA-N 2-(ethylsulfonylmethyl)pentanedioic acid Chemical compound CCS(=O)(=O)CC(C(O)=O)CCC(O)=O SXSQDMPJGXEWGO-UHFFFAOYSA-N 0.000 description 1
- BLDFSDCBQJUWFG-UHFFFAOYSA-N 2-(methylamino)-1,2-diphenylethanol Chemical compound C=1C=CC=CC=1C(NC)C(O)C1=CC=CC=C1 BLDFSDCBQJUWFG-UHFFFAOYSA-N 0.000 description 1
- YKJPKUXMGYHQKV-UHFFFAOYSA-N 2-(methylsulfinylmethyl)pentanedioic acid Chemical compound CS(=O)CC(C(O)=O)CCC(O)=O YKJPKUXMGYHQKV-UHFFFAOYSA-N 0.000 description 1
- GYGXSGJIYJXSFJ-UHFFFAOYSA-N 2-(propylsulfinylmethyl)pentanedioic acid Chemical compound CCCS(=O)CC(C(O)=O)CCC(O)=O GYGXSGJIYJXSFJ-UHFFFAOYSA-N 0.000 description 1
- CDALMBXQUMRRBP-UHFFFAOYSA-N 2-(pyridin-4-ylsulfinylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)C1=CC=NC=C1 CDALMBXQUMRRBP-UHFFFAOYSA-N 0.000 description 1
- ORVBOXYBVFKIAM-UHFFFAOYSA-N 2-(pyridin-4-ylsulfonylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CS(=O)(=O)C1=CC=NC=C1 ORVBOXYBVFKIAM-UHFFFAOYSA-N 0.000 description 1
- UTWIARBFTRTHPR-UHFFFAOYSA-N 2-(sulfinylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)C=S=O UTWIARBFTRTHPR-UHFFFAOYSA-N 0.000 description 1
- RHPCHKVYDYPCAJ-UHFFFAOYSA-N 2-(sulfonylmethyl)pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)C=S(=O)=O RHPCHKVYDYPCAJ-UHFFFAOYSA-N 0.000 description 1
- BYBCIVKIWIFVFD-UHFFFAOYSA-N 2-[4,10-bis(carboxymethyl)-7-(2,6-dioxooxan-3-yl)-1,4,7,10-tetrazacyclododec-1-yl]acetic acid Chemical compound C1CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CCN1C1C(=O)OC(=O)CC1 BYBCIVKIWIFVFD-UHFFFAOYSA-N 0.000 description 1
- XSVWFLQICKPQAA-UHFFFAOYSA-N 2-[4,10-bis(carboxymethyl)-7-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetic acid Chemical compound C1CN(CC(O)=O)CCN(CC(=O)O)CCN(CC(O)=O)CCN1CC(=O)ON1C(=O)CCC1=O XSVWFLQICKPQAA-UHFFFAOYSA-N 0.000 description 1
- FQIHLPGWBOBPSG-UHFFFAOYSA-N 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetamide Chemical compound NC(=O)CN1CCN(CC(N)=O)CCN(CC(N)=O)CCN(CC(N)=O)CC1 FQIHLPGWBOBPSG-UHFFFAOYSA-N 0.000 description 1
- DFPHZEYJGWLQJE-UHFFFAOYSA-N 2-[4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetic acid Chemical compound NC(=O)CN1CCN(CC(N)=O)CCN(CC(O)=O)CCN(CC(N)=O)CC1 DFPHZEYJGWLQJE-UHFFFAOYSA-N 0.000 description 1
- ZDJBZFMQLZGRRK-UHFFFAOYSA-N 2-[4,7-bis(2-amino-2-oxoethyl)-1,4,7,10-tetrazacyclododec-1-yl]acetamide Chemical compound NC(=O)CN1CCNCCN(CC(N)=O)CCN(CC(N)=O)CC1 ZDJBZFMQLZGRRK-UHFFFAOYSA-N 0.000 description 1
- ALRKEASEQOCKTJ-UHFFFAOYSA-N 2-[4,7-bis(2-amino-2-oxoethyl)-1,4,7-triazonan-1-yl]acetamide Chemical compound NC(=O)CN1CCN(CC(N)=O)CCN(CC(N)=O)CC1 ALRKEASEQOCKTJ-UHFFFAOYSA-N 0.000 description 1
- KHYQZCZUWQXKHB-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]-5-(2,5-dioxopyrrolidin-1-yl)oxy-5-oxopentanoic acid Chemical compound C1CN(CC(=O)O)CCN(CC(O)=O)CCN1C(C(O)=O)CCC(=O)ON1C(=O)CCC1=O KHYQZCZUWQXKHB-UHFFFAOYSA-N 0.000 description 1
- CFGSZUCRHWLSKD-UHFFFAOYSA-N 2-[4,7-bis(carboxymethyl)-1,4,7-triazonan-1-yl]-5-[(4-isothiocyanatophenyl)methylamino]-5-oxopentanoic acid Chemical compound C(=O)(O)C(CCC(=O)NCC1=CC=C(C=C1)N=C=S)N1CCN(CCN(CC1)CC(=O)O)CC(=O)O CFGSZUCRHWLSKD-UHFFFAOYSA-N 0.000 description 1
- JZFBXLDURQNNQU-UHFFFAOYSA-N 2-[4-(carboxymethyl)-7-[2-[2-(2,5-dioxopyrrol-1-yl)ethylamino]-2-oxoethyl]-1,4,7-triazonan-1-yl]acetic acid Chemical compound C1CN(CC(=O)O)CCN(CC(O)=O)CCN1CC(=O)NCCN1C(=O)C=CC1=O JZFBXLDURQNNQU-UHFFFAOYSA-N 0.000 description 1
- WRUJMBQKNNMUAY-UHFFFAOYSA-N 2-[7-(carboxymethyl)-4,10-bis[2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetic acid Chemical compound CC(C)(C)OC(=O)CN1CCN(CC(O)=O)CCN(CC(=O)OC(C)(C)C)CCN(CC(O)=O)CC1 WRUJMBQKNNMUAY-UHFFFAOYSA-N 0.000 description 1
- ZSUPTWOVEHNLSW-UHFFFAOYSA-N 2-[[aminomethyl(hydroxy)phosphoryl]methyl]pentanedioic acid Chemical compound NCP(O)(=O)CC(C(O)=O)CCC(O)=O ZSUPTWOVEHNLSW-UHFFFAOYSA-N 0.000 description 1
- NNCRKIKXRSDRFI-UHFFFAOYSA-N 2-[[benzyl(hydroxy)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)CC1=CC=CC=C1 NNCRKIKXRSDRFI-UHFFFAOYSA-N 0.000 description 1
- MIINQJNPWDYQGB-UHFFFAOYSA-N 2-[[butyl(hydroxy)phosphoryl]methyl]pentanedioic acid Chemical compound CCCCP(O)(=O)CC(C(O)=O)CCC(O)=O MIINQJNPWDYQGB-UHFFFAOYSA-N 0.000 description 1
- UNHSYVCKZOSDNN-UHFFFAOYSA-N 2-[[cyclohexyl(hydroxy)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)C1CCCCC1 UNHSYVCKZOSDNN-UHFFFAOYSA-N 0.000 description 1
- ZDECCUDWIMXEKV-UHFFFAOYSA-N 2-[[ethyl(hydroxy)phosphoryl]methyl]pentanedioic acid Chemical compound CCP(O)(=O)CC(C(O)=O)CCC(O)=O ZDECCUDWIMXEKV-UHFFFAOYSA-N 0.000 description 1
- YIXLYFSFJYJUBT-UHFFFAOYSA-N 2-[[hydroxy(3-phenylpentyl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)CCC(CC)C1=CC=CC=C1 YIXLYFSFJYJUBT-UHFFFAOYSA-N 0.000 description 1
- VAZDQVAVNHZARH-UHFFFAOYSA-N 2-[[hydroxy(3-phenylpropyl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)CCCC1=CC=CC=C1 VAZDQVAVNHZARH-UHFFFAOYSA-N 0.000 description 1
- ZLVSTOJKRGCGEB-UHFFFAOYSA-N 2-[[hydroxy(4-phenylbutyl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)CCCCC1=CC=CC=C1 ZLVSTOJKRGCGEB-UHFFFAOYSA-N 0.000 description 1
- WATYBUNAXQXJTC-UHFFFAOYSA-N 2-[[hydroxy(4-phenylpentyl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)CCCC(C)C1=CC=CC=C1 WATYBUNAXQXJTC-UHFFFAOYSA-N 0.000 description 1
- CAGAVCIHAJPIJR-UHFFFAOYSA-N 2-[[hydroxy(methyl)phosphoryl]methyl]pentanedioic acid Chemical compound CP(O)(=O)CC(C(O)=O)CCC(O)=O CAGAVCIHAJPIJR-UHFFFAOYSA-N 0.000 description 1
- VDCGGAXVFSYLTM-UHFFFAOYSA-N 2-[[hydroxy(oxan-2-yl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)C1CCCCO1 VDCGGAXVFSYLTM-UHFFFAOYSA-N 0.000 description 1
- UIVPEQQNMNDWDC-UHFFFAOYSA-N 2-[[hydroxy(phenyl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)C1=CC=CC=C1 UIVPEQQNMNDWDC-UHFFFAOYSA-N 0.000 description 1
- IKWYHDVSCUMYSM-UHFFFAOYSA-N 2-[[hydroxy(propyl)phosphoryl]methyl]pentanedioic acid Chemical compound CCCP(O)(=O)CC(C(O)=O)CCC(O)=O IKWYHDVSCUMYSM-UHFFFAOYSA-N 0.000 description 1
- WIJGRKWAABBWJZ-UHFFFAOYSA-N 2-[[hydroxy(pyridin-4-ylmethyl)phosphoryl]methyl]pentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)CP(O)(=O)CC1=CC=NC=C1 WIJGRKWAABBWJZ-UHFFFAOYSA-N 0.000 description 1
- OXJXOPBWKDCAHJ-UHFFFAOYSA-N 2-[benzyl(hydroxy)phosphoryl]oxypentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)OP(O)(=O)CC1=CC=CC=C1 OXJXOPBWKDCAHJ-UHFFFAOYSA-N 0.000 description 1
- MXFCNMASDUEDAA-UHFFFAOYSA-N 2-[butyl(hydroxy)phosphoryl]oxypentanedioic acid Chemical compound CCCCP(O)(=O)OC(C(O)=O)CCC(O)=O MXFCNMASDUEDAA-UHFFFAOYSA-N 0.000 description 1
- ZOQVILHRYCANIG-UHFFFAOYSA-N 2-[hydroxy(methyl)phosphoryl]oxypentanedioic acid Chemical compound CP(O)(=O)OC(C(O)=O)CCC(O)=O ZOQVILHRYCANIG-UHFFFAOYSA-N 0.000 description 1
- VZEGIOSPGNDJBZ-UHFFFAOYSA-N 2-[hydroxy(phenyl)phosphoryl]oxypentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)OP(O)(=O)C1=CC=CC=C1 VZEGIOSPGNDJBZ-UHFFFAOYSA-N 0.000 description 1
- WQMVADYYRIKMPQ-UHFFFAOYSA-N 2-[hydroxy(propyl)phosphoryl]oxypentanedioic acid Chemical compound CCCP(O)(=O)OC(C(O)=O)CCC(O)=O WQMVADYYRIKMPQ-UHFFFAOYSA-N 0.000 description 1
- QMGPVQRWSFZHJK-UHFFFAOYSA-N 2-[hydroxy(pyridin-2-ylmethyl)phosphoryl]oxypentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)OP(O)(=O)CC1=CC=CC=N1 QMGPVQRWSFZHJK-UHFFFAOYSA-N 0.000 description 1
- CPKYVOYKTIWOHJ-UHFFFAOYSA-N 2-[hydroxy(pyridin-4-ylmethyl)phosphoryl]oxypentanedioic acid Chemical compound OC(=O)CCC(C(O)=O)OP(O)(=O)CC1=CC=NC=C1 CPKYVOYKTIWOHJ-UHFFFAOYSA-N 0.000 description 1
- GOJUJUVQIVIZAV-UHFFFAOYSA-N 2-amino-4,6-dichloropyrimidine-5-carbaldehyde Chemical group NC1=NC(Cl)=C(C=O)C(Cl)=N1 GOJUJUVQIVIZAV-UHFFFAOYSA-N 0.000 description 1
- KZMAWJRXKGLWGS-UHFFFAOYSA-N 2-chloro-n-[4-(4-methoxyphenyl)-1,3-thiazol-2-yl]-n-(3-methoxypropyl)acetamide Chemical compound S1C(N(C(=O)CCl)CCCOC)=NC(C=2C=CC(OC)=CC=2)=C1 KZMAWJRXKGLWGS-UHFFFAOYSA-N 0.000 description 1
- PJISLFCKHOHLLP-UHFFFAOYSA-N 2-diethoxyphosphorylsulfanyl-n,n-diethylethanamine Chemical compound CCOP(=O)(OCC)SCCN(CC)CC PJISLFCKHOHLLP-UHFFFAOYSA-N 0.000 description 1
- ARSWQPLPYROOBG-ZETCQYMHSA-N 2-methylleucine Chemical compound CC(C)C[C@](C)(N)C(O)=O ARSWQPLPYROOBG-ZETCQYMHSA-N 0.000 description 1
- 125000000094 2-phenylethyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])* 0.000 description 1
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 description 1
- 125000006201 3-phenylpropyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 125000000339 4-pyridyl group Chemical group N1=C([H])C([H])=C([*])C([H])=C1[H] 0.000 description 1
- SUAUFMLRKFUOID-UHFFFAOYSA-N 5-[(2-methylpropan-2-yl)oxy]-5-oxo-4-[4,7,10-tris[2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]pentanoic acid Chemical compound CC(C)(C)OC(=O)CN1CCN(CC(=O)OC(C)(C)C)CCN(C(CCC(O)=O)C(=O)OC(C)(C)C)CCN(CC(=O)OC(C)(C)C)CC1 SUAUFMLRKFUOID-UHFFFAOYSA-N 0.000 description 1
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 1
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 1
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 description 1
- 229940124321 AIDS medicine Drugs 0.000 description 1
- DLFVBJFMPXGRIB-UHFFFAOYSA-N Acetamide Chemical compound CC(N)=O DLFVBJFMPXGRIB-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000005995 Aluminium silicate Substances 0.000 description 1
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 1
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- SJUXYJQNMNJVFX-UHFFFAOYSA-N C(=O)(O)C(CCC(=O)NCCN1C(C=CC1=O)=O)N1CCN(CCN(CC1)CC(=O)O)CC(=O)O Chemical compound C(=O)(O)C(CCC(=O)NCCN1C(C=CC1=O)=O)N1CCN(CCN(CC1)CC(=O)O)CC(=O)O SJUXYJQNMNJVFX-UHFFFAOYSA-N 0.000 description 1
- 101100243399 Caenorhabditis elegans pept-2 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 102000003900 Calpain-2 Human genes 0.000 description 1
- 108090000232 Calpain-2 Proteins 0.000 description 1
- 102000046744 Calpain-3 Human genes 0.000 description 1
- 108030001375 Calpain-3 Proteins 0.000 description 1
- 241000759905 Camptotheca acuminata Species 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 102000004172 Cathepsin L Human genes 0.000 description 1
- 108090000624 Cathepsin L Proteins 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 229910021589 Copper(I) bromide Inorganic materials 0.000 description 1
- 108010037462 Cyclooxygenase 2 Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- BWLUMTFWVZZZND-UHFFFAOYSA-N Dibenzylamine Chemical compound C=1C=CC=CC=1CNCC1=CC=CC=C1 BWLUMTFWVZZZND-UHFFFAOYSA-N 0.000 description 1
- 235000019739 Dicalciumphosphate Nutrition 0.000 description 1
- XBPCUCUWBYBCDP-UHFFFAOYSA-N Dicyclohexylamine Chemical compound C1CCCCC1NC1CCCCC1 XBPCUCUWBYBCDP-UHFFFAOYSA-N 0.000 description 1
- 108010013369 Enteropeptidase Proteins 0.000 description 1
- 102100029727 Enteropeptidase Human genes 0.000 description 1
- 241000283073 Equus caballus Species 0.000 description 1
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 description 1
- 206010015548 Euthanasia Diseases 0.000 description 1
- 108010054265 Factor VIIa Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 239000004606 Fillers/Extenders Substances 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101150110792 GNRHR gene Proteins 0.000 description 1
- 241000206672 Gelidium Species 0.000 description 1
- 101001133056 Homo sapiens Mucin-1 Proteins 0.000 description 1
- 101001051709 Homo sapiens Ribosomal protein S6 kinase-related protein Proteins 0.000 description 1
- 101500024338 Homo sapiens Somatostatin-14 Proteins 0.000 description 1
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 1
- 206010062904 Hormone-refractory prostate cancer Diseases 0.000 description 1
- 108010071893 Human Immunodeficiency Virus rev Gene Products Proteins 0.000 description 1
- CPELXLSAUQHCOX-UHFFFAOYSA-N Hydrogen bromide Chemical class Br CPELXLSAUQHCOX-UHFFFAOYSA-N 0.000 description 1
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 1
- 102100022339 Integrin alpha-L Human genes 0.000 description 1
- 208000022120 Jeavons syndrome Diseases 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 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
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 102100032114 Lumican Human genes 0.000 description 1
- 108010064548 Lymphocyte Function-Associated Antigen-1 Proteins 0.000 description 1
- 108010010995 MART-1 Antigen Proteins 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 description 1
- 240000003183 Manihot esculenta Species 0.000 description 1
- 235000016735 Manihot esculenta subsp esculenta Nutrition 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010076502 Matrix Metalloproteinase 11 Proteins 0.000 description 1
- 102100028389 Melanoma antigen recognized by T-cells 1 Human genes 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-M Methanesulfonate Chemical compound CS([O-])(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-M 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- BRMWTNUJHUMWMS-LURJTMIESA-N N(tele)-methyl-L-histidine Chemical compound CN1C=NC(C[C@H](N)C(O)=O)=C1 BRMWTNUJHUMWMS-LURJTMIESA-N 0.000 description 1
- HTLZVHNRZJPSMI-UHFFFAOYSA-N N-ethylpiperidine Chemical compound CCN1CCCCC1 HTLZVHNRZJPSMI-UHFFFAOYSA-N 0.000 description 1
- MBBZMMPHUWSWHV-BDVNFPICSA-N N-methylglucamine Chemical compound CNC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO MBBZMMPHUWSWHV-BDVNFPICSA-N 0.000 description 1
- 101710138657 Neurotoxin Proteins 0.000 description 1
- 102100030411 Neutrophil collagenase Human genes 0.000 description 1
- 101710118230 Neutrophil collagenase Proteins 0.000 description 1
- 241000209018 Nyssaceae Species 0.000 description 1
- 239000005642 Oleic acid Substances 0.000 description 1
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 description 1
- 108091034117 Oligonucleotide Proteins 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- HCBIBCJNVBAKAB-UHFFFAOYSA-N Procaine hydrochloride Chemical compound Cl.CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 HCBIBCJNVBAKAB-UHFFFAOYSA-N 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-N Propionic acid Chemical class CCC(O)=O XBDQKXXYIPTUBI-UHFFFAOYSA-N 0.000 description 1
- 108010022052 Proprotein Convertase 5 Proteins 0.000 description 1
- 102000012210 Proprotein Convertase 5 Human genes 0.000 description 1
- 102100038280 Prostaglandin G/H synthase 2 Human genes 0.000 description 1
- 206010036909 Prostate cancer metastatic Diseases 0.000 description 1
- 101800004937 Protein C Proteins 0.000 description 1
- 101710149951 Protein Tat Proteins 0.000 description 1
- 238000003559 RNA-seq method Methods 0.000 description 1
- 102100024914 Ribosomal protein S6 kinase-related protein Human genes 0.000 description 1
- 101100539927 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) NAN1 gene Proteins 0.000 description 1
- 102400000827 Saposin-D Human genes 0.000 description 1
- 101800001700 Saposin-D Proteins 0.000 description 1
- 108020004682 Single-Stranded DNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N Sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 235000002595 Solanum tuberosum Nutrition 0.000 description 1
- 244000061456 Solanum tuberosum Species 0.000 description 1
- 102400000820 Somatostatin-14 Human genes 0.000 description 1
- 101150038447 Sstr2 gene Proteins 0.000 description 1
- 102100028847 Stromelysin-3 Human genes 0.000 description 1
- 241000282898 Sus scrofa Species 0.000 description 1
- GUGOEEXESWIERI-UHFFFAOYSA-N Terfenadine Chemical compound C1=CC(C(C)(C)C)=CC=C1C(O)CCCN1CCC(C(O)(C=2C=CC=CC=2)C=2C=CC=CC=2)CC1 GUGOEEXESWIERI-UHFFFAOYSA-N 0.000 description 1
- WDLRUFUQRNWCPK-UHFFFAOYSA-N Tetraxetan Chemical compound OC(=O)CN1CCN(CC(O)=O)CCN(CC(O)=O)CCN(CC(O)=O)CC1 WDLRUFUQRNWCPK-UHFFFAOYSA-N 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 101000980463 Treponema pallidum (strain Nichols) Chaperonin GroEL Proteins 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000251539 Vertebrata <Metazoa> Species 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- JVVXZOOGOGPDRZ-SLFFLAALSA-N [(1R,4aS,10aR)-1,4a-dimethyl-7-propan-2-yl-2,3,4,9,10,10a-hexahydrophenanthren-1-yl]methanamine Chemical compound NC[C@]1(C)CCC[C@]2(C)C3=CC=C(C(C)C)C=C3CC[C@H]21 JVVXZOOGOGPDRZ-SLFFLAALSA-N 0.000 description 1
- 239000001089 [(2R)-oxolan-2-yl]methanol Substances 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000003655 absorption accelerator Substances 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 238000005917 acylation reaction Methods 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 235000010419 agar Nutrition 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 1
- WNROFYMDJYEPJX-UHFFFAOYSA-K aluminium hydroxide Chemical compound [OH-].[OH-].[OH-].[Al+3] WNROFYMDJYEPJX-UHFFFAOYSA-K 0.000 description 1
- 235000012211 aluminium silicate Nutrition 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 239000001099 ammonium carbonate Substances 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 229940035674 anesthetics Drugs 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000001772 anti-angiogenic effect Effects 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000001430 anti-depressive effect Effects 0.000 description 1
- 230000003474 anti-emetic effect Effects 0.000 description 1
- 230000001387 anti-histamine Effects 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 230000000648 anti-parkinson Effects 0.000 description 1
- 230000000561 anti-psychotic effect Effects 0.000 description 1
- 230000001754 anti-pyretic effect Effects 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000000935 antidepressant agent Substances 0.000 description 1
- 229940005513 antidepressants Drugs 0.000 description 1
- 239000002111 antiemetic agent Substances 0.000 description 1
- 239000003429 antifungal agent Substances 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000000739 antihistaminic agent Substances 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 239000004599 antimicrobial Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000003096 antiparasitic agent Substances 0.000 description 1
- 239000000939 antiparkinson agent Substances 0.000 description 1
- 239000003904 antiprotozoal agent Substances 0.000 description 1
- 239000002221 antipyretic Substances 0.000 description 1
- 108010038288 antisecretory factor Proteins 0.000 description 1
- 239000000074 antisense oligonucleotide Substances 0.000 description 1
- 238000012230 antisense oligonucleotides Methods 0.000 description 1
- 229960004676 antithrombotic agent Drugs 0.000 description 1
- RHJPBGWFGOAEID-UHFFFAOYSA-N aplysiatoxin Natural products O1C2(OC(O)(CC(=O)OC(CC(=O)O3)C(C)O)C(C)CC2(C)C)CC3C(C)C1C(C)CCC(OC)C1=CC(O)=CC=C1Br RHJPBGWFGOAEID-UHFFFAOYSA-N 0.000 description 1
- 239000007900 aqueous suspension Substances 0.000 description 1
- 239000008135 aqueous vehicle Substances 0.000 description 1
- 108010072041 arginyl-glycyl-aspartic acid Proteins 0.000 description 1
- 125000003289 ascorbyl group Chemical class [H]O[C@@]([H])(C([H])([H])O*)[C@@]1([H])OC(=O)C(O*)=C1O* 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- UPABQMWFWCMOFV-UHFFFAOYSA-N benethamine Chemical compound C=1C=CC=CC=1CNCCC1=CC=CC=C1 UPABQMWFWCMOFV-UHFFFAOYSA-N 0.000 description 1
- 239000000440 bentonite Substances 0.000 description 1
- 229910000278 bentonite Inorganic materials 0.000 description 1
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 1
- JUHORIMYRDESRB-UHFFFAOYSA-N benzathine Chemical compound C=1C=CC=CC=1CNCCNCC1=CC=CC=C1 JUHORIMYRDESRB-UHFFFAOYSA-N 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- 229940050390 benzoate Drugs 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 229960002903 benzyl benzoate Drugs 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N biotin Natural products N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-M bisulphate group Chemical group S([O-])(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-M 0.000 description 1
- 150000001642 boronic acid derivatives Chemical class 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 150000005693 branched-chain amino acids Chemical class 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 235000019437 butane-1,3-diol Nutrition 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 235000010216 calcium carbonate Nutrition 0.000 description 1
- AXCZMVOFGPJBDE-UHFFFAOYSA-L calcium dihydroxide Chemical compound [OH-].[OH-].[Ca+2] AXCZMVOFGPJBDE-UHFFFAOYSA-L 0.000 description 1
- 239000000920 calcium hydroxide Substances 0.000 description 1
- 229910001861 calcium hydroxide Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 239000008116 calcium stearate Substances 0.000 description 1
- 235000013539 calcium stearate Nutrition 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- BMLSTPRTEKLIPM-UHFFFAOYSA-I calcium;potassium;disodium;hydrogen carbonate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].OC([O-])=O BMLSTPRTEKLIPM-UHFFFAOYSA-I 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 238000012754 cardiac puncture Methods 0.000 description 1
- 239000002327 cardiovascular agent Substances 0.000 description 1
- 229940125692 cardiovascular agent Drugs 0.000 description 1
- 239000004359 castor oil Substances 0.000 description 1
- 235000019438 castor oil Nutrition 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- RHJPBGWFGOAEID-BEDNPZBZSA-N chembl1256416 Chemical compound C1([C@H](CC[C@H](C)[C@@H]2[C@H]([C@@H]3C[C@@]4(O[C@@](O)(CC(=O)O[C@H](CC(=O)O3)[C@@H](C)O)[C@H](C)CC4(C)C)O2)C)OC)=CC(O)=CC=C1Br RHJPBGWFGOAEID-BEDNPZBZSA-N 0.000 description 1
- 230000002925 chemical effect Effects 0.000 description 1
- 229940044683 chemotherapy drug Drugs 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- VDANGULDQQJODZ-UHFFFAOYSA-N chloroprocaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1Cl VDANGULDQQJODZ-UHFFFAOYSA-N 0.000 description 1
- 229960002023 chloroprocaine Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- YSPZOYMEWUTYDA-UHFFFAOYSA-N cis-glyoxal-cyclen Chemical compound C1CN2CCN(CC3)C2C2N3CCN21 YSPZOYMEWUTYDA-UHFFFAOYSA-N 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 239000004927 clay Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 230000000536 complexating effect Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- HCAJEUSONLESMK-UHFFFAOYSA-N cyclohexylsulfamic acid Chemical class OS(=O)(=O)NC1CCCCC1 HCAJEUSONLESMK-UHFFFAOYSA-N 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 229940124448 dermatologic drug Drugs 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008355 dextrose injection Substances 0.000 description 1
- NEFBYIFKOOEVPA-UHFFFAOYSA-K dicalcium phosphate Chemical compound [Ca+2].[Ca+2].[O-]P([O-])([O-])=O NEFBYIFKOOEVPA-UHFFFAOYSA-K 0.000 description 1
- 229940038472 dicalcium phosphate Drugs 0.000 description 1
- 229910000390 dicalcium phosphate Inorganic materials 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- ZBCBWPMODOFKDW-UHFFFAOYSA-N diethanolamine Chemical compound OCCNCCO ZBCBWPMODOFKDW-UHFFFAOYSA-N 0.000 description 1
- 229940043237 diethanolamine Drugs 0.000 description 1
- HPNMFZURTQLUMO-UHFFFAOYSA-N diethylamine Chemical compound CCNCC HPNMFZURTQLUMO-UHFFFAOYSA-N 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 description 1
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical class CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 1
- BJAJDJDODCWPNS-UHFFFAOYSA-N dotp Chemical compound O=C1N2CCOC2=NC2=C1SC=C2 BJAJDJDODCWPNS-UHFFFAOYSA-N 0.000 description 1
- 229960004679 doxorubicin Drugs 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 210000001163 endosome Anatomy 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 239000002532 enzyme inhibitor Substances 0.000 description 1
- 229940125532 enzyme inhibitor Drugs 0.000 description 1
- 210000000918 epididymis Anatomy 0.000 description 1
- 201000010063 epididymitis Diseases 0.000 description 1
- 210000003238 esophagus Anatomy 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-M ethanesulfonate Chemical compound CCS([O-])(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-M 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 229940093499 ethyl acetate Drugs 0.000 description 1
- MNJVRJDLRVPLFE-UHFFFAOYSA-N etoricoxib Chemical compound C1=NC(C)=CC=C1C1=NC=C(Cl)C=C1C1=CC=C(S(C)(=O)=O)C=C1 MNJVRJDLRVPLFE-UHFFFAOYSA-N 0.000 description 1
- 229960004945 etoricoxib Drugs 0.000 description 1
- 210000000887 face Anatomy 0.000 description 1
- 229940012414 factor viia Drugs 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 238000000684 flow cytometry Methods 0.000 description 1
- 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 description 1
- 238000002073 fluorescence micrograph Methods 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-L fumarate(2-) Chemical class [O-]C(=O)\C=C\C([O-])=O VZCYOOQTPOCHFL-OWOJBTEDSA-L 0.000 description 1
- 230000006870 function Effects 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 239000003193 general anesthetic agent Substances 0.000 description 1
- 125000005612 glucoheptonate group Chemical group 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000002310 glutaric acid derivatives Chemical class 0.000 description 1
- YQEMORVAKMFKLG-UHFFFAOYSA-N glycerine monostearate Natural products CCCCCCCCCCCCCCCCCC(=O)OC(CO)CO YQEMORVAKMFKLG-UHFFFAOYSA-N 0.000 description 1
- SVUQHVRAGMNPLW-UHFFFAOYSA-N glycerol monostearate Natural products CCCCCCCCCCCCCCCCC(=O)OCC(O)CO SVUQHVRAGMNPLW-UHFFFAOYSA-N 0.000 description 1
- ZEMPKEQAKRGZGQ-XOQCFJPHSA-N glycerol triricinoleate Natural products CCCCCC[C@@H](O)CC=CCCCCCCCC(=O)OC[C@@H](COC(=O)CCCCCCCC=CC[C@@H](O)CCCCCC)OC(=O)CCCCCCCC=CC[C@H](O)CCCCCC ZEMPKEQAKRGZGQ-XOQCFJPHSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 229960002885 histidine Drugs 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 150000003840 hydrochlorides Chemical class 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 230000000147 hypnotic effect Effects 0.000 description 1
- 239000012216 imaging agent Substances 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 229960003444 immunosuppressant agent Drugs 0.000 description 1
- 230000001861 immunosuppressant effect Effects 0.000 description 1
- 239000003018 immunosuppressive agent Substances 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 description 1
- 239000000644 isotonic solution Substances 0.000 description 1
- 239000007951 isotonicity adjuster Substances 0.000 description 1
- 238000001948 isotopic labelling Methods 0.000 description 1
- NLYAJNPCOHFWQQ-UHFFFAOYSA-N kaolin Chemical compound O.O.O=[Al]O[Si](=O)O[Si](=O)O[Al]=O NLYAJNPCOHFWQQ-UHFFFAOYSA-N 0.000 description 1
- 230000002147 killing effect Effects 0.000 description 1
- 150000003893 lactate salts Chemical class 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 210000002429 large intestine Anatomy 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 244000144972 livestock Species 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000003589 local anesthetic agent Substances 0.000 description 1
- 239000007937 lozenge Substances 0.000 description 1
- 210000003712 lysosome Anatomy 0.000 description 1
- 230000001868 lysosomic effect Effects 0.000 description 1
- VWHRYODZTDMVSS-QMMMGPOBSA-N m-fluoro-L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC(F)=C1 VWHRYODZTDMVSS-QMMMGPOBSA-N 0.000 description 1
- 229910052749 magnesium Inorganic materials 0.000 description 1
- 239000011777 magnesium Substances 0.000 description 1
- VTHJTEIRLNZDEV-UHFFFAOYSA-L magnesium dihydroxide Chemical compound [OH-].[OH-].[Mg+2] VTHJTEIRLNZDEV-UHFFFAOYSA-L 0.000 description 1
- 239000000347 magnesium hydroxide Substances 0.000 description 1
- 229910001862 magnesium hydroxide Inorganic materials 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- 150000002690 malonic acid derivatives Chemical class 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 208000010658 metastatic prostate carcinoma Diseases 0.000 description 1
- MYWUZJCMWCOHBA-VIFPVBQESA-N methamphetamine Chemical compound CN[C@@H](C)CC1=CC=CC=C1 MYWUZJCMWCOHBA-VIFPVBQESA-N 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000004170 methylsulfonyl group Chemical group [H]C([H])([H])S(*)(=O)=O 0.000 description 1
- 239000004530 micro-emulsion Substances 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 230000002438 mitochondrial effect Effects 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- CQDGTJPVBWZJAZ-UHFFFAOYSA-N monoethyl carbonate Chemical compound CCOC(O)=O CQDGTJPVBWZJAZ-UHFFFAOYSA-N 0.000 description 1
- 239000002324 mouth wash Substances 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 239000003158 myorelaxant agent Substances 0.000 description 1
- JKQNZLKTODZGSY-UHFFFAOYSA-N n,n-diethyl-2-[4,8,11-tris[2-(diethylamino)-2-oxoethyl]-1,4,8,11-tetrazacyclotetradec-1-yl]acetamide Chemical compound CCN(CC)C(=O)CN1CCCN(CC(=O)N(CC)CC)CCN(CC(=O)N(CC)CC)CCCN(CC(=O)N(CC)CC)CC1 JKQNZLKTODZGSY-UHFFFAOYSA-N 0.000 description 1
- CMWYAOXYQATXSI-UHFFFAOYSA-N n,n-dimethylformamide;piperidine Chemical compound CN(C)C=O.C1CCNCC1 CMWYAOXYQATXSI-UHFFFAOYSA-N 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 125000005487 naphthalate group Chemical group 0.000 description 1
- 239000007922 nasal spray Substances 0.000 description 1
- 229940097496 nasal spray Drugs 0.000 description 1
- 229940000041 nervous system drug Drugs 0.000 description 1
- 239000002581 neurotoxin Substances 0.000 description 1
- 231100000618 neurotoxin Toxicity 0.000 description 1
- 150000002823 nitrates Chemical class 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 description 1
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 239000000346 nonvolatile oil Substances 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 229960002969 oleic acid Drugs 0.000 description 1
- 235000021313 oleic acid Nutrition 0.000 description 1
- 239000004006 olive oil Substances 0.000 description 1
- 235000008390 olive oil Nutrition 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 150000003891 oxalate salts Chemical class 0.000 description 1
- 150000002942 palmitic acid derivatives Chemical class 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229960004662 parecoxib Drugs 0.000 description 1
- TZRHLKRLEZJVIJ-UHFFFAOYSA-N parecoxib Chemical compound C1=CC(S(=O)(=O)NC(=O)CC)=CC=C1C1=C(C)ON=C1C1=CC=CC=C1 TZRHLKRLEZJVIJ-UHFFFAOYSA-N 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000002304 perfume Substances 0.000 description 1
- 229940021222 peritoneal dialysis isotonic solution Drugs 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 125000003170 phenylsulfonyl group Chemical group C1(=CC=CC=C1)S(=O)(=O)* 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229940127126 plasminogen activator Drugs 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 239000000244 polyoxyethylene sorbitan monooleate Substances 0.000 description 1
- 239000003910 polypeptide antibiotic agent Substances 0.000 description 1
- 229920000136 polysorbate Polymers 0.000 description 1
- 229940068968 polysorbate 80 Drugs 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 159000000001 potassium salts Chemical class 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 229960001309 procaine hydrochloride Drugs 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000003180 prostaglandins Chemical class 0.000 description 1
- 229960000856 protein c Drugs 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 150000003856 quaternary ammonium compounds Chemical class 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 230000002285 radioactive effect Effects 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 229940000044 respiratory system drug Drugs 0.000 description 1
- 239000003340 retarding agent Substances 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- RZJQGNCSTQAWON-UHFFFAOYSA-N rofecoxib Chemical compound C1=CC(S(=O)(=O)C)=CC=C1C1=C(C=2C=CC=CC=2)C(=O)OC1 RZJQGNCSTQAWON-UHFFFAOYSA-N 0.000 description 1
- 229960000371 rofecoxib Drugs 0.000 description 1
- 150000003873 salicylate salts Chemical class 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 150000004760 silicates Chemical class 0.000 description 1
- 235000012239 silicon dioxide Nutrition 0.000 description 1
- 210000002027 skeletal muscle Anatomy 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- WBHQBSYUUJJSRZ-UHFFFAOYSA-M sodium bisulfate Chemical compound [Na+].OS([O-])(=O)=O WBHQBSYUUJJSRZ-UHFFFAOYSA-M 0.000 description 1
- 229910000342 sodium bisulfate Inorganic materials 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000012312 sodium hydride Substances 0.000 description 1
- 229910000104 sodium hydride Inorganic materials 0.000 description 1
- 235000019333 sodium laurylsulphate Nutrition 0.000 description 1
- 239000007909 solid dosage form Substances 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 210000000952 spleen Anatomy 0.000 description 1
- 108010088201 squamous cell carcinoma-related antigen Proteins 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 230000003637 steroidlike Effects 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 150000003890 succinate salts Chemical class 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- IIACRCGMVDHOTQ-UHFFFAOYSA-N sulfamic acid Chemical class NS(O)(=O)=O IIACRCGMVDHOTQ-UHFFFAOYSA-N 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 150000003892 tartrate salts Chemical class 0.000 description 1
- TVUPYCMMXREXSS-UHFFFAOYSA-N tert-butyl 2-[4-[2-(2-aminoethylamino)-2-oxoethyl]-7,10-bis[2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetate Chemical compound CC(C)(C)OC(=O)CN1CCN(CC(=O)NCCN)CCN(CC(=O)OC(C)(C)C)CCN(CC(=O)OC(C)(C)C)CC1 TVUPYCMMXREXSS-UHFFFAOYSA-N 0.000 description 1
- XIICJKXKEDCZEE-UHFFFAOYSA-N tert-butyl 2-[7-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxoethyl]-4,10-bis[2-[(2-methylpropan-2-yl)oxy]-2-oxoethyl]-1,4,7,10-tetrazacyclododec-1-yl]acetate Chemical compound C1CN(CC(=O)OC(C)(C)C)CCN(CC(=O)OC(C)(C)C)CCN(CC(=O)OC(C)(C)C)CCN1CC(=O)ON1C(=O)CCC1=O XIICJKXKEDCZEE-UHFFFAOYSA-N 0.000 description 1
- 230000002381 testicular Effects 0.000 description 1
- 208000022345 tetraamelia syndrome Diseases 0.000 description 1
- CBXCPBUEXACCNR-UHFFFAOYSA-N tetraethylammonium Chemical compound CC[N+](CC)(CC)CC CBXCPBUEXACCNR-UHFFFAOYSA-N 0.000 description 1
- BSYVTEYKTMYBMK-UHFFFAOYSA-N tetrahydrofurfuryl alcohol Chemical compound OCC1CCCO1 BSYVTEYKTMYBMK-UHFFFAOYSA-N 0.000 description 1
- QEMXHQIAXOOASZ-UHFFFAOYSA-N tetramethylammonium Chemical compound C[N+](C)(C)C QEMXHQIAXOOASZ-UHFFFAOYSA-N 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical class CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 235000010487 tragacanth Nutrition 0.000 description 1
- 239000000196 tragacanth Substances 0.000 description 1
- 229940116362 tragacanth Drugs 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 230000032895 transmembrane transport Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000000439 tumor marker Substances 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- LNPDTQAFDNKSHK-UHFFFAOYSA-N valdecoxib Chemical compound CC=1ON=C(C=2C=CC=CC=2)C=1C1=CC=C(S(N)(=O)=O)C=C1 LNPDTQAFDNKSHK-UHFFFAOYSA-N 0.000 description 1
- 229960002004 valdecoxib Drugs 0.000 description 1
- NQPDZGIKBAWPEJ-UHFFFAOYSA-M valerate Chemical class CCCCC([O-])=O NQPDZGIKBAWPEJ-UHFFFAOYSA-M 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 229940124549 vasodilator Drugs 0.000 description 1
- 239000003071 vasodilator agent Substances 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 150000003722 vitamin derivatives Chemical class 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- UGZADUVQMDAIAO-UHFFFAOYSA-L zinc hydroxide Chemical compound [OH-].[OH-].[Zn+2] UGZADUVQMDAIAO-UHFFFAOYSA-L 0.000 description 1
- 229940007718 zinc hydroxide Drugs 0.000 description 1
- 229910021511 zinc hydroxide Inorganic materials 0.000 description 1
Abstract
A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules. Also involved is a pharmaceutical composition, comprising the conjugate compound or the pharmaceutically acceptable salt thereof. In addition, involved are a method for delivering a payload to an object in need, and a method for treating a disease, wherein the methods both comprise administering to the object a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
Description
Description BI-LIGAND DRUG CONJUGATE AND USE THEREOF
Technical Field The present application relates to the field of biomedical chemistry. More particularly, the present application relates to a bi-ligand drug conjugate, a pharmaceutical composition comprising the bi-ligand drug conjugate, a method for delivering a payload to a subject in need thereof by using the bi-ligand drug 3.13 conjugate, and a method for treating a disease with the bi-ligand drug conjugate.
Background Art In general, diseased cells and nottnal cells are significantly different in both pathological and physiological characteristics, and one of the differences lies in that diseased cells have specific or overexpressed materials (such as antigens, chemical signals and receptors) on their surfaces, whereas these materials are absent or lowly expressed in nottnal cells. On this basis, an antibody-drug conjugate (ADC) and a polypeptide-drug conjugate (PDC) are developed for the treatment of diseases.
At present, some ADC- and PDC-based drugs have been marketed or have entered clinical researches; however, due to their design rationale, ADC- and PDC-based drugs have a great limitation in clinical application.
Due to the complexity and relatively large molecular weight of ADCs, the development thereof faces many difficulties, including lack of suitable targets, difficulty in production and poor drug stability. Currently, ADCs are mainly used in the field of tumor treatment. In some instances, the affinity of a targeting antibody to a cancer cell surface antigen can reach 10-9-10-12 (Kd, mole/liter).
Therefore, when having a high specificity to target cells, ADCs also have a high specificity to normal cells with the same targeting receptor as the target cells. Moreover, it may take a long time (1 to 3 weeks) to metabolize ADCs in vivo, during which ADCs continuously kill nottnal cells, leading to significantly increased toxic and side effects. Therefore, ADCs are more applicable to diseases characterized by a very Date Recue/Date Received 2021-07-27 large difference in the amount of cell surface antigens between tumor cells and normal cells. However, very few diseases known in the art can meet such strict requirement.
PDCs have been used to treat a variety of diseases in clinical or pre-clinical researches, but in such case chemotherapeutics are simply connected to polypeptides, or polypeptides are added to nanoparticles or polymer materials embedded with chemotherapeutic drugs, which will make most of the polypeptides unable to enter cells due to their large molecular weights and bearing charges.
Therefore, most of the PDCs are currently only suitable for an extracellular treatment and the application scopes and efficacy of PDCs are severely limited.
A drug conjugate compound can also be a ligand-drug conjugate (LDC), wherein the ligand may be a peptide or a small molecule. However, in terms of bioavailability, stability, efficacy, toxicity, etc., there are various problems in the application of LDCs. For example, due to large molecular weights, lipophilicity or other properties, many ligands are incapable of entering cells, which limits their therapeutic applications. In addition, ligands usually have low efficacy when conjugated with conventional chemotherapeutics (such as doxorubicin and paclitaxel), and when conjugated with highly effective drug molecules (such as MMAE and DM1), ligands may produce a high toxicity, thereby poisoning and even killing animals before a therapeutically effective amount for treating a tumor is achieved.
Therefore, there is also an urgent need in the art to obtain an improved LDC, which can act on highly-expressed receptors widely existing on the surface of diseased cells, broaden a targeting range and a treatment window, enhance drug efficacy and avoid drug side effects.
Summary of the Invention In one aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively.
Technical Field The present application relates to the field of biomedical chemistry. More particularly, the present application relates to a bi-ligand drug conjugate, a pharmaceutical composition comprising the bi-ligand drug conjugate, a method for delivering a payload to a subject in need thereof by using the bi-ligand drug 3.13 conjugate, and a method for treating a disease with the bi-ligand drug conjugate.
Background Art In general, diseased cells and nottnal cells are significantly different in both pathological and physiological characteristics, and one of the differences lies in that diseased cells have specific or overexpressed materials (such as antigens, chemical signals and receptors) on their surfaces, whereas these materials are absent or lowly expressed in nottnal cells. On this basis, an antibody-drug conjugate (ADC) and a polypeptide-drug conjugate (PDC) are developed for the treatment of diseases.
At present, some ADC- and PDC-based drugs have been marketed or have entered clinical researches; however, due to their design rationale, ADC- and PDC-based drugs have a great limitation in clinical application.
Due to the complexity and relatively large molecular weight of ADCs, the development thereof faces many difficulties, including lack of suitable targets, difficulty in production and poor drug stability. Currently, ADCs are mainly used in the field of tumor treatment. In some instances, the affinity of a targeting antibody to a cancer cell surface antigen can reach 10-9-10-12 (Kd, mole/liter).
Therefore, when having a high specificity to target cells, ADCs also have a high specificity to normal cells with the same targeting receptor as the target cells. Moreover, it may take a long time (1 to 3 weeks) to metabolize ADCs in vivo, during which ADCs continuously kill nottnal cells, leading to significantly increased toxic and side effects. Therefore, ADCs are more applicable to diseases characterized by a very Date Recue/Date Received 2021-07-27 large difference in the amount of cell surface antigens between tumor cells and normal cells. However, very few diseases known in the art can meet such strict requirement.
PDCs have been used to treat a variety of diseases in clinical or pre-clinical researches, but in such case chemotherapeutics are simply connected to polypeptides, or polypeptides are added to nanoparticles or polymer materials embedded with chemotherapeutic drugs, which will make most of the polypeptides unable to enter cells due to their large molecular weights and bearing charges.
Therefore, most of the PDCs are currently only suitable for an extracellular treatment and the application scopes and efficacy of PDCs are severely limited.
A drug conjugate compound can also be a ligand-drug conjugate (LDC), wherein the ligand may be a peptide or a small molecule. However, in terms of bioavailability, stability, efficacy, toxicity, etc., there are various problems in the application of LDCs. For example, due to large molecular weights, lipophilicity or other properties, many ligands are incapable of entering cells, which limits their therapeutic applications. In addition, ligands usually have low efficacy when conjugated with conventional chemotherapeutics (such as doxorubicin and paclitaxel), and when conjugated with highly effective drug molecules (such as MMAE and DM1), ligands may produce a high toxicity, thereby poisoning and even killing animals before a therapeutically effective amount for treating a tumor is achieved.
Therefore, there is also an urgent need in the art to obtain an improved LDC, which can act on highly-expressed receptors widely existing on the surface of diseased cells, broaden a targeting range and a treatment window, enhance drug efficacy and avoid drug side effects.
Summary of the Invention In one aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively.
2 Date Recue/Date Received 2021-07-27 In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a ligand moiety represented by formula (I), respectively:
(D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A-/
(I).
In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
H 0 (s) N AN
H H H
0 .
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO (s) NANNAN
H H H H
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
(D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A-/
(I).
In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
H 0 (s) N AN
H H H
0 .
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO (s) NANNAN
H H H H
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
3 Date Recue/Date Received 2021-07-27 HO 0 0õOH
y j 0 --- 0 HO
(s) N N (s) 0 H H H H
0 j(i\j,&ti N (s) H
0 \
HO .
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
_ HO (s) NANNAN 0 1.4 0 H H H H
0 j(N,(sA
H H
0 \
In some embodiments, the two targeting molecules comprised in the conjugate compound or the pharmaceutically acceptable salt thereof are different.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof is a cell-interacting molecule.
In some embodiments, the two targeting molecules comprised in the conjugate compound or the pharmaceutically acceptable salt thereof are cell-interacting molecules that interact with different cell molecules.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof is an endocytosis molecule capable of mediating endocytosis.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40,
y j 0 --- 0 HO
(s) N N (s) 0 H H H H
0 j(i\j,&ti N (s) H
0 \
HO .
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
_ HO (s) NANNAN 0 1.4 0 H H H H
0 j(N,(sA
H H
0 \
In some embodiments, the two targeting molecules comprised in the conjugate compound or the pharmaceutically acceptable salt thereof are different.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof is a cell-interacting molecule.
In some embodiments, the two targeting molecules comprised in the conjugate compound or the pharmaceutically acceptable salt thereof are cell-interacting molecules that interact with different cell molecules.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof is an endocytosis molecule capable of mediating endocytosis.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40,
4 Date Recue/Date Received 2021-07-27 CD4OL, 0X40, OX4OL, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4, SIGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3 and CXCR4.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand moiety and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises a ligand moiety represented by formula (I) and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.
In yet some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule moiety, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof is a folate or an analog thereof In some embodiments, the analog of folate is selected from the group consisting of 5-methyltetrahydrofolate, 5-foitnyltetrahydrofolate, methotrexate, and
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises a prostate-specific membrane antigen ligand moiety and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises a ligand moiety represented by formula (I) and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.
In yet some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises P10 and a synergistic molecule moiety, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
In some embodiments, the synergistic molecule comprised in the conjugate compound or the pharmaceutically acceptable salt thereof is a folate or an analog thereof In some embodiments, the analog of folate is selected from the group consisting of 5-methyltetrahydrofolate, 5-foitnyltetrahydrofolate, methotrexate, and
5,10-methylenetetrahydrofolate.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises one, two, three, four or more payloads. In some embodiments, the payload is selected from the group consisting of a small molecule compound, a nucleotide, a peptide and a protein. In some embodiments, the payload is a small molecule compound. In some embodiments, the small molecule compound is selected from the group consisting of camptothecin and any derivative Date Recue/Date Received 2021-07-27 thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, a radionuclide complex, a cyclooxygenase-2 inhibitor, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin or any derivative thereof, auristatin or any derivative thereof, maytansine or any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor.
In some embodiments, the payload comprised in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is linked to at least one targeting molecule via a linker.
In some embodiments, the linker comprised in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is a peptide linker, a disulfide linker, a pH-dependent linker or a combination of the above-mentioned linkers.
In some embodiments, the peptide linker is cleavable under a certain physiological environment by protease cleavage or reduction. In some embodiments, the peptide linker is selected from the group consisting of cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino acids is amidated.
In some embodiments, the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM, and NDMDS.
In some embodiments, the pH-dependent linker is a cis-aconitic anhydride.
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application has the following structure:
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof comprises one, two, three, four or more payloads. In some embodiments, the payload is selected from the group consisting of a small molecule compound, a nucleotide, a peptide and a protein. In some embodiments, the payload is a small molecule compound. In some embodiments, the small molecule compound is selected from the group consisting of camptothecin and any derivative Date Recue/Date Received 2021-07-27 thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, a radionuclide complex, a cyclooxygenase-2 inhibitor, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin or any derivative thereof, auristatin or any derivative thereof, maytansine or any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor.
In some embodiments, the payload comprised in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is linked to at least one targeting molecule via a linker.
In some embodiments, the linker comprised in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is a peptide linker, a disulfide linker, a pH-dependent linker or a combination of the above-mentioned linkers.
In some embodiments, the peptide linker is cleavable under a certain physiological environment by protease cleavage or reduction. In some embodiments, the peptide linker is selected from the group consisting of cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino acids is amidated.
In some embodiments, the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM, and NDMDS.
In some embodiments, the pH-dependent linker is a cis-aconitic anhydride.
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application has the following structure:
6 Date Recue/Date Received 2021-07-27 NH
,(SA
0 HN (S) 0 *
0 , NH
N.(sA
N N (S) 0 0 , NH
0 Ho [N1 N N
0 0 y\
s' NH
N N (s) N
= H
(R) 0 0 *
H N0 0 0 1\
,(SA
0 HN (S) 0 *
0 , NH
N.(sA
N N (S) 0 0 , NH
0 Ho [N1 N N
0 0 y\
s' NH
N N (s) N
= H
(R) 0 0 *
H N0 0 0 1\
7 Date Recue/Date Received 2021-07-27 NH
N
N (S) (R) 0 61-=!),r0 NH
N rE1 N N (s) H
(R) 1/4.= 0 ? 0 OY\J NH2 HO
H
= H H
Nizz.N
Iir0 0 0 o NIrorN 00j )y31 $ 0 0
N
N (S) (R) 0 61-=!),r0 NH
N rE1 N N (s) H
(R) 1/4.= 0 ? 0 OY\J NH2 HO
H
= H H
Nizz.N
Iir0 0 0 o NIrorN 00j )y31 $ 0 0
8 Date Recue/Date Received 2021-07-27 /1\1_-:N
3&) 0 0 N 1\11rOf N
HN
/NH
ArCNO
\jLO 110 0 0) N NlrorN
HN
/NH N.õ..N
(s) \)L0 0O 0) N (s) N IrOThrN0(:)) HN
H (s) HN
or the linker is a combination of the above-mentioned structures and a peptide linker.
3&) 0 0 N 1\11rOf N
HN
/NH
ArCNO
\jLO 110 0 0) N NlrorN
HN
/NH N.õ..N
(s) \)L0 0O 0) N (s) N IrOThrN0(:)) HN
H (s) HN
or the linker is a combination of the above-mentioned structures and a peptide linker.
9 Date Recue/Date Received 2021-07-27 In some embodiments, the two targeting molecules comprised in the conjugate compound or the phannaceutically acceptable salt thereof of the present application are linked to each other via a spacer. In some embodiments, the spacer described in the present application comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg and Pro-Leu-Gly.
In some embodiments, the conjugate compound of the present application is CB-20B which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
)=I21 HN Fl(s) N N OH
HN
HN
HNO
(s) u<OH
NH HN
(=-======,(OH
0 (S) HOy..
0 s 0-1,1 0 )NH..= (s) 0(S) N'ILNH 2 NH
0, HmN) .0 0 (R) (R) NH
(R) (8) OH
In some embodiments, the conjugate compound of the present application is CB-20BK which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
(s) H2N (s) )=N OH
HNO
N N
HN
HN HN
(s) OH HN
(s) OH
Hoy NH NH
(s) 0 (R
).. NEclo NH
P
(D
HI)_<
(s) (s) NET
ER
(sN) 0 (R) (R) NH
(R) (S) OH
In some embodiments, the conjugate compound of the present application is CB-60S which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
(s) )=N
N)¨ N (s) OH
HN
HN
HN
6s) OH
NH HN
(s) OH
NH
HOy.. (s) (s) 0 (s) ro,N_NõN
NH
(s) HN
OH
0 Nz 0 ¨
\ N
0 (s) In some embodiments, the conjugate compound of the present application is CB-60SK which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
(s) H2N (s) =
)=N OH
HN tO
N
HN
HN HN
(s) OH HN
(s) __ yOH
NH NH
HO((s) = HN
0 (s) NH2 (s) 0 (s) ro,N,NoN
'o NH
C) (s) HN
OH
O N z O ¨
\ N
0 (s) In some embodiments, the conjugate compound of the present application is CB-20C which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
(s) H2N (s) )=N OH
HN
N N
HN
HN HN
=
(s) OH HN
(s) OH
NH NH
HOy, (s) 0 (s) NH2 (S) 0 (R) N
Oi NH
)1..
of\I)1N1H2 NH
P
(D
NH
:S
0' N¨N
\
In some embodiments, the conjugate compound of the present application is CB-1020 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH p ...,,))õ,,N,ri,NI-12 1/....(OH (s) (S) N
HN 0 o(1=..,---1, Ht.( 0 0 HN
(s) ===.<
OH HO (s) o (R) ....( HN HN
HN H2N (s) NH
o (s) ...,,OH
HN
(s) o HN N HN--""
01..õ)/N
HOy.,..= 0 0 (s) ,N H2 01 0 0 ..., NH HN
HO,ir,- 0-NI HN
tO
HN-..õ....õ..,,....
\O
S 0) 0 0-) .L0 j ) ii\IH
o2 H
NH
P
>... si.s7 FII\..<
(s) \etl¨
i .H0 \
(isµl) (R) (R) NH
(R) In some embodiments, the conjugate compound of the present application is CB-1320 which has the following structural foi tnula:
Date Recue/Date Received 2021-07-27 F1 ....
OH
(s) H6s) ....80 I, \OH
HN
HN
HNa HN HN
0H N¨' (s) 0 NH 0 I NH (s) HOy,. HN N
0 01 ¶ 01-1 1 NH
0 (,$)0 NH
L H* (S) NH HoN¨c.,_<
O
H HN
H N
N-... js ..,..õ.,õ.,..- 0 0 (0 is 0 C-0 ¨) \_ JO
N
Oi >. /NH
4Ths..,""N)I'NH2 NH
P
o )... ,(Ns¨) o HN
o s) N-- 0 .0 \
o N\ (s) Q(R) (R-NH
(R) (S) OH
In some embodiments, the conjugate compound of the present application is CB-1820 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
1....OH
H
HN . /
FINC))NHO , HN (s) NH 0 HN HN (RiN H , H041,õ3), (R
O
(c... ;
HN Hd 0 4-Thr(S OH HN 0 HO(0 HN
HN
0 NH OH HO,r,.... 04 HN--_ _______ 0.--1-:\I
Oj HN I
NH
oP \ ....sisT
/ o Ni...<
o x_to \
\ (is) o (R) (R) NH
((R) 8) OH
In some embodiments, the conjugate compound of the present application is CR19428 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
(s) H2N (s) =
)=N OH
HN
N N
HN
HN HN
(s) OH HN
(s) OH
NH NH
HO( = (s) HN
(s) NH2 = (S) 0 (R) N
NH
HN
NH
(s) HN
OR
NH
(o s V \
0 ==
'OH
In some embodiments, the conjugate compound of the present application is 20R-SMO9 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 HO (s) N AN isN )-LN 0 0 H H 0 H H 0 0 .$) H 0 (s) ?
N (S) 'LN (s) NH2 H 0 = H 0 FIC::
00, _OH HN
0 rCOOH
H
(s) 0 7----0 NfrN
N -Th 0 N N¨\
N)'NN
1 H \N) COON
H
HOOCH
In some embodiments, the conjugate compound of the present application is CB-20R which has the following structural foimula:
HO 0 0, _OH
HO (s) N)-LN N AN 0 (1::i 0 j.rH 0 H H H H
0 N ka,[1, H j-1, N (s) N (s) NH2 H , H
HO
0 õOH HN
0 rCOOH
N f-ENI (s) o0 /---_ HN- ( "Th N H )cNN 0 1 H c___.._) COON
H
HOOCH
wherein M is a radionuclide.
In some embodiments, the conjugate compound of the present application is CB-18G which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
_________________________________________ HN
(s) (W(s) *
HN (1'NH
HO
0 (/µµ
0 HN (R) O
NH
H2N = (S) )=N HO 0 HN)_t0 HN 0 HN
HO _,_0 N(S) 0 (s) = 4DH
NO) NH 0 R) oi NH
NH
C) N¨
I(S) HN
(s) Xs) tFT
(SN
0 (R) (R) NH
(R) (S) OH
Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound of the present application is CR19426 which has the following structural foimula:
OH
H 2 N 0 = H 0 (s) ___________________________________________ HN
(s) Ws) NH
HN (17-NH
HO Vs) (11' NH
)=N HO 0 HN
HO HN
(s) 0 O
(s) OH
HN õIR) 2\IH 0 \ 0 co HN
OR
NH
ioHN
NH
( 0 \ N
s V \
o In some embodiments, the conjugate compound of the present application is CB-10S which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
(s) H2 HN NH
(s) (s) HO (s) NH
(R) ( HN
H2 N (s) NH
(s) HN
OH
)N (s) HNON
0 (s) N N
HN
HN (s) (s) =.,, HN
(s) H N NH
NH
ro NH
NH
(s 0 0 , / \
N z OH
In some embodiments, the conjugate compound of the present application is CR19425 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
o (s) HN NH
o (s) (s) HN
HO (s) NH
(R) ( HN
H2N (s) NH
(s) HN
)=N (s) HNI)_t0 ON (s) N N
HN
HN (s) (s) (s) HN NH
\O
NH
HN
NH
(s) HN
NH
(o 0 \ N
s V \
'OH
In some embodiments, the conjugate compound of the present application is CB-50S which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
0 (s) FN1 y NH2 HN NH
O
(s) (s) HN
HO (s) N H
(R) .. ( HN
H2 N (s) N H
(s) HN
)=N (s) )¨ N N 0(s) HN
H N (s) = 0 N H
(s) (s) " OH .. HO
H N N H
NH
NH
=
/
N z OH
In another aspect, the present application discloses a pharmaceutical composition comprising the conjugate compound or the pharmaceutically Date Recue/Date Received 2021-07-27 acceptable salt thereof of the present application, and a pharmaceutically acceptable carrier.
In some embodiments, the composition is administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly.
In another aspect, the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application.
In another aspect, the present application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application.
In some embodiments, the method for treating a disease in a subject of the present application further comprises administering one or more therapeutic agents in combination with the conjugate compound or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
In yet another aspect, the present application discloses use of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application in the preparation of a drug for treating a disease in a subject.
In yet another aspect, the present application discloses the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application for treating a disease in a subject.
In some embodiments, the disease is selected from the group consisting of a cancer, an immunological disease, a cardiovascular disease, a metabolic disease, and a neurological disease.
In some embodiments, the cancer is selected from the group consisting of prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian cancer, Date Recue/Date Received 2021-07-27 gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.
In some embodiments, the immunological disease is an autoimmune disease.
In some embodiments, the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
In some embodiments, the cardiovascular disease is selected from the group consisting of angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital heart disease.
In some embodiments, the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
In some embodiments, the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
Brief Description of the Drawings Fig. 1 shows chemical structural foiniulas of conjugate compounds CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S.
Fig. 2A shows a curve of Cy5-pep-20BK binding and endocytosis by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells) over time. Fig. 2B shows a curve of Cy5-pep-20AK binding and endocytosis by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells) over time.
Fig. 3 shows fluorescence images of Cy5-FA binding and endocytosis by different cells over time, wherein the fluorescence shown as a complete circle is the fluorescence of cell nuclei, and the fluorescence distributed in a spotty pattern is the fluorescence of Cy5-FA.
Date Recue/Date Received 2021-07-27 Fig. 4A shows an inhibitory activity of conjugate compound CB-20BK on the amplification of tumor cells as shown. Fig. 4B shows an inhibitory activity of conjugate compound CB-20B on the amplification of tumor cells as shown. Fig.
shows an inhibitory activity of conjugate compound CB-10S on the amplification of tumor cells as shown. Fig. 4D shows an inhibitory activity of conjugate compound CB-605 on the amplification of tumor cells as shown. Fig. 4E shows an inhibitory activity of conjugate compound CB-605K on the amplification of tumor cells as shown. Fig. 4F shows an inhibitory activity of conjugate compound CB-18G on the amplification of tumor cells as shown. Fig. 4G shows an inhibitory activity of conjugate compound CB-505 on the amplification of tumor cells as shown.
Figs. 5A-5E show tumor suppressive effects of conjugate compound CB-20BK
in mice.
Figs. 6A-6C show tumor suppressive effects of conjugate compound CB-20B
in mice.
Figs. 7A-7E show tumor suppressive effects of conjugate compound CB-18G
in mice.
Figs. 8A-8B show effects of CBP-1018 for injection on tumor volumes of LU2505 lung cancer model and LU1206 lung cancer model.
Detailed Description of Embodiments While various aspects and embodiments will be disclosed in the present application, it is apparent that a person skilled in the art may make various equivalent changes and modifications to the aspects and embodiments without deviating from the subject spirit and scope of the present application. The various aspects and embodiments disclosed in the present application are only for the purposes of illustration and are not intended to be limiting, with the true scope being indicated by the appended claims. All publications, patents or patent applications cited in the present application are incorporated by reference in their entirety.
Unless defined otherwise, the technical and scientific teinis as used herein have the same meanings as commonly understood by a person skilled in the art to which the present application belongs.
Date Recue/Date Received 2021-07-27 As used herein and in the appended claims, the singular foinis "a", "an", and "the" include plural reference unless the context clearly dictates otherwise.
The Wails "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the teinis "comprise/comprising", "include/including", and "have/having" can be used interchangeably.
As used herein and in the appended claims, the teini "analog" includes a structural analog and a functional analog. The structural analog refers to a class of compounds with similar chemical structures, which may comprise one or more different atoms or one or more different functional groups. The functional analog refers to a class of compounds that have the same or similar chemical, biological or pharmacological effects. For example, the analogs of folate include 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methyl enetetrahydro folate .
As used herein and in the appended claims, the teini "derivative" refers to a relatively complex compound derived from a parent compound molecule in which one or more atoms or atomic groups are substituted with other atoms or atomic groups. For example, camptothecin derivatives include irinotecan, SN-38, Dxd, topotecan, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 10-hydroxyc amptothe c in, (20 S)-c amptothec in, 9-nitrocamptothecin, gimatecan, karenitecin, silatecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625 In one aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively.
In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a ligand moiety represented by foimula (I), respectively:
Date Recue/Date Received 2021-07-27 (D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A¨
/
(I).
In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof The tem" "payload" as used herein refers to a molecule or material to be delivered to a target cell or tissue. Without limitation, the payload may be any molecule or material that is intended for use in the diagnosis, treatment, or prevention of a disease in a subject. In some embodiments, the payload has a molecular weight of less than or equal to about 5 kDa. In some embodiments, the payload has a molecular weight of less than or equal to about 1.5 kDa. In some embodiments, the payload is a drug or a diagnostic reagent that has been deemed safe and effective for use by appropriate drug approval and registration agencies (such as FDA, EMEA, or NMPA).
In some embodiments, the payload of the present application is a small molecule compound, a nucleotide (such as a DNA, a plasmid DNA, an RNA, an siRNA, an antisense oligonucleotide and an aptamer), a peptide, or a protein (for example, an enzyme). In some embodiments, the payload is a small molecule compound.
In some embodiments, the payloads of the present application include, but are not limited to: an anticancer drug, a radioactive substance, a vitamin, an anti-AIDS
drug, an antibiotic, an immunosuppressant, an antiviral drug, an enzyme inhibitor, a neurotoxin, an opioid, a regulator of cell-extracellular matrix interaction, a vasodilator, an antihypertensive drug, a hypnotic, an antihistamine, an anticonvulsant, a muscle relaxant, an anti-Parkinson substance, an anticonvulsant and a drug for inhibiting muscle contraction, an antiparasitic drug and/or an anti-antiprotozoal drug, an analgesic drug, an antipyretic, a steroidal or Date Recue/Date Received 2021-07-27 non-steroidal anti-inflammatory drug, an anti-angiogenic factor, an antisecretory factor, an anticoagulant and/or an antithrombotic agent, a local anesthetic, a prostaglandin, an antidepressant, an antipsychotic, an antiemetic, or an imaging agent.
In some embodiments, the payload of the present application has a free amino or carboxyl group before being conjugated with the conjugate compound of the present application, and the payload is conjugated with the conjugate compound through an acylation reaction between the above-mentioned amino or carboxyl group and the corresponding part (for example, a linker) of the conjugate compound.
In some embodiments, a modification on the above-mentioned free amino or carboxyl group (for example, by conjugating with the conjugate compound of the present application) may significantly reduce the activity of the payload (for example, by at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%).
The "small molecule compound" as used herein refers to a compound having a molecular weight of less than or equal to about 2 kDa. In some embodiments, the small molecule compound has a molecular weight of less than or equal to about 1.5 kDa. In some preferred embodiments, the small molecule compound has a molecular weight of less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da. In some embodiments, the small molecule compound of the present application is selected from the group consisting of camptothecin and any derivative thereof (for example, SN38 or Dxd), auristatin and any derivative thereof (for example, MMAE and MMAF), maytansine and any derivative thereof, a cyclooxygenase-2 inhibitor (for example, celecoxib), a radionuclide complex, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin or any derivative thereof, auristatin or any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor. In some embodiments, the small molecule compound described in the present application is a drug for relieving or treating a cancer. In some embodiments, the Date Recue/Date Received 2021-07-27 small molecule compound described in the present application is a drug for relieving or treating an autoimmune disease.
The ten," "camptothecin" as used herein refers to a cytotoxic alkaloid, mainly derived from Camptotheca acuminata (Nyssaceae) and showing a strong anti-tumor activity. The camptothecin and derivative thereof of the present application include a camptothecin and a derivative thereof that are already in existence or will be produced later. The camptothecin and derivative thereof of the present application include, but are not limited to: camptothecin, irinotecan, SN-38, Dxd, topotecan, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 10-hydroxyc amptothe c in, (20 S)-c amptothec in, 9-nitrocamptothecin, gimatecan, karenitecin, silatecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625.
The tettn "auristatin and any derivative thereof/auristatin or any derivative thereof' as used herein refers to a natural anti-tumor product, aplysiatoxin
In some embodiments, the conjugate compound of the present application is CB-20B which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
)=I21 HN Fl(s) N N OH
HN
HN
HNO
(s) u<OH
NH HN
(=-======,(OH
0 (S) HOy..
0 s 0-1,1 0 )NH..= (s) 0(S) N'ILNH 2 NH
0, HmN) .0 0 (R) (R) NH
(R) (8) OH
In some embodiments, the conjugate compound of the present application is CB-20BK which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
(s) H2N (s) )=N OH
HNO
N N
HN
HN HN
(s) OH HN
(s) OH
Hoy NH NH
(s) 0 (R
).. NEclo NH
P
(D
HI)_<
(s) (s) NET
ER
(sN) 0 (R) (R) NH
(R) (S) OH
In some embodiments, the conjugate compound of the present application is CB-60S which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
(s) )=N
N)¨ N (s) OH
HN
HN
HN
6s) OH
NH HN
(s) OH
NH
HOy.. (s) (s) 0 (s) ro,N_NõN
NH
(s) HN
OH
0 Nz 0 ¨
\ N
0 (s) In some embodiments, the conjugate compound of the present application is CB-60SK which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
(s) H2N (s) =
)=N OH
HN tO
N
HN
HN HN
(s) OH HN
(s) __ yOH
NH NH
HO((s) = HN
0 (s) NH2 (s) 0 (s) ro,N,NoN
'o NH
C) (s) HN
OH
O N z O ¨
\ N
0 (s) In some embodiments, the conjugate compound of the present application is CB-20C which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
(s) H2N (s) )=N OH
HN
N N
HN
HN HN
=
(s) OH HN
(s) OH
NH NH
HOy, (s) 0 (s) NH2 (S) 0 (R) N
Oi NH
)1..
of\I)1N1H2 NH
P
(D
NH
:S
0' N¨N
\
In some embodiments, the conjugate compound of the present application is CB-1020 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH p ...,,))õ,,N,ri,NI-12 1/....(OH (s) (S) N
HN 0 o(1=..,---1, Ht.( 0 0 HN
(s) ===.<
OH HO (s) o (R) ....( HN HN
HN H2N (s) NH
o (s) ...,,OH
HN
(s) o HN N HN--""
01..õ)/N
HOy.,..= 0 0 (s) ,N H2 01 0 0 ..., NH HN
HO,ir,- 0-NI HN
tO
HN-..õ....õ..,,....
\O
S 0) 0 0-) .L0 j ) ii\IH
o2 H
NH
P
>... si.s7 FII\..<
(s) \etl¨
i .H0 \
(isµl) (R) (R) NH
(R) In some embodiments, the conjugate compound of the present application is CB-1320 which has the following structural foi tnula:
Date Recue/Date Received 2021-07-27 F1 ....
OH
(s) H6s) ....80 I, \OH
HN
HN
HNa HN HN
0H N¨' (s) 0 NH 0 I NH (s) HOy,. HN N
0 01 ¶ 01-1 1 NH
0 (,$)0 NH
L H* (S) NH HoN¨c.,_<
O
H HN
H N
N-... js ..,..õ.,õ.,..- 0 0 (0 is 0 C-0 ¨) \_ JO
N
Oi >. /NH
4Ths..,""N)I'NH2 NH
P
o )... ,(Ns¨) o HN
o s) N-- 0 .0 \
o N\ (s) Q(R) (R-NH
(R) (S) OH
In some embodiments, the conjugate compound of the present application is CB-1820 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
1....OH
H
HN . /
FINC))NHO , HN (s) NH 0 HN HN (RiN H , H041,õ3), (R
O
(c... ;
HN Hd 0 4-Thr(S OH HN 0 HO(0 HN
HN
0 NH OH HO,r,.... 04 HN--_ _______ 0.--1-:\I
Oj HN I
NH
oP \ ....sisT
/ o Ni...<
o x_to \
\ (is) o (R) (R) NH
((R) 8) OH
In some embodiments, the conjugate compound of the present application is CR19428 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
OH
(s) H2N (s) =
)=N OH
HN
N N
HN
HN HN
(s) OH HN
(s) OH
NH NH
HO( = (s) HN
(s) NH2 = (S) 0 (R) N
NH
HN
NH
(s) HN
OR
NH
(o s V \
0 ==
'OH
In some embodiments, the conjugate compound of the present application is 20R-SMO9 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 HO (s) N AN isN )-LN 0 0 H H 0 H H 0 0 .$) H 0 (s) ?
N (S) 'LN (s) NH2 H 0 = H 0 FIC::
00, _OH HN
0 rCOOH
H
(s) 0 7----0 NfrN
N -Th 0 N N¨\
N)'NN
1 H \N) COON
H
HOOCH
In some embodiments, the conjugate compound of the present application is CB-20R which has the following structural foimula:
HO 0 0, _OH
HO (s) N)-LN N AN 0 (1::i 0 j.rH 0 H H H H
0 N ka,[1, H j-1, N (s) N (s) NH2 H , H
HO
0 õOH HN
0 rCOOH
N f-ENI (s) o0 /---_ HN- ( "Th N H )cNN 0 1 H c___.._) COON
H
HOOCH
wherein M is a radionuclide.
In some embodiments, the conjugate compound of the present application is CB-18G which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
_________________________________________ HN
(s) (W(s) *
HN (1'NH
HO
0 (/µµ
0 HN (R) O
NH
H2N = (S) )=N HO 0 HN)_t0 HN 0 HN
HO _,_0 N(S) 0 (s) = 4DH
NO) NH 0 R) oi NH
NH
C) N¨
I(S) HN
(s) Xs) tFT
(SN
0 (R) (R) NH
(R) (S) OH
Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound of the present application is CR19426 which has the following structural foimula:
OH
H 2 N 0 = H 0 (s) ___________________________________________ HN
(s) Ws) NH
HN (17-NH
HO Vs) (11' NH
)=N HO 0 HN
HO HN
(s) 0 O
(s) OH
HN õIR) 2\IH 0 \ 0 co HN
OR
NH
ioHN
NH
( 0 \ N
s V \
o In some embodiments, the conjugate compound of the present application is CB-10S which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
(s) H2 HN NH
(s) (s) HO (s) NH
(R) ( HN
H2 N (s) NH
(s) HN
OH
)N (s) HNON
0 (s) N N
HN
HN (s) (s) =.,, HN
(s) H N NH
NH
ro NH
NH
(s 0 0 , / \
N z OH
In some embodiments, the conjugate compound of the present application is CR19425 which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
o (s) HN NH
o (s) (s) HN
HO (s) NH
(R) ( HN
H2N (s) NH
(s) HN
)=N (s) HNI)_t0 ON (s) N N
HN
HN (s) (s) (s) HN NH
\O
NH
HN
NH
(s) HN
NH
(o 0 \ N
s V \
'OH
In some embodiments, the conjugate compound of the present application is CB-50S which has the following structural foimula:
Date Recue/Date Received 2021-07-27 OH
0 (s) FN1 y NH2 HN NH
O
(s) (s) HN
HO (s) N H
(R) .. ( HN
H2 N (s) N H
(s) HN
)=N (s) )¨ N N 0(s) HN
H N (s) = 0 N H
(s) (s) " OH .. HO
H N N H
NH
NH
=
/
N z OH
In another aspect, the present application discloses a pharmaceutical composition comprising the conjugate compound or the pharmaceutically Date Recue/Date Received 2021-07-27 acceptable salt thereof of the present application, and a pharmaceutically acceptable carrier.
In some embodiments, the composition is administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly.
In another aspect, the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application.
In another aspect, the present application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application.
In some embodiments, the method for treating a disease in a subject of the present application further comprises administering one or more therapeutic agents in combination with the conjugate compound or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition.
In yet another aspect, the present application discloses use of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application in the preparation of a drug for treating a disease in a subject.
In yet another aspect, the present application discloses the conjugate compound or the pharmaceutically acceptable salt thereof of the present application, or the pharmaceutical composition of the present application for treating a disease in a subject.
In some embodiments, the disease is selected from the group consisting of a cancer, an immunological disease, a cardiovascular disease, a metabolic disease, and a neurological disease.
In some embodiments, the cancer is selected from the group consisting of prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian cancer, Date Recue/Date Received 2021-07-27 gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.
In some embodiments, the immunological disease is an autoimmune disease.
In some embodiments, the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
In some embodiments, the cardiovascular disease is selected from the group consisting of angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital heart disease.
In some embodiments, the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
In some embodiments, the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
Brief Description of the Drawings Fig. 1 shows chemical structural foiniulas of conjugate compounds CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S.
Fig. 2A shows a curve of Cy5-pep-20BK binding and endocytosis by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells) over time. Fig. 2B shows a curve of Cy5-pep-20AK binding and endocytosis by different cells (from top to bottom: LNCaP cells, SKOV3 cells, DU145 cells and NCI-H460 cells) over time.
Fig. 3 shows fluorescence images of Cy5-FA binding and endocytosis by different cells over time, wherein the fluorescence shown as a complete circle is the fluorescence of cell nuclei, and the fluorescence distributed in a spotty pattern is the fluorescence of Cy5-FA.
Date Recue/Date Received 2021-07-27 Fig. 4A shows an inhibitory activity of conjugate compound CB-20BK on the amplification of tumor cells as shown. Fig. 4B shows an inhibitory activity of conjugate compound CB-20B on the amplification of tumor cells as shown. Fig.
shows an inhibitory activity of conjugate compound CB-10S on the amplification of tumor cells as shown. Fig. 4D shows an inhibitory activity of conjugate compound CB-605 on the amplification of tumor cells as shown. Fig. 4E shows an inhibitory activity of conjugate compound CB-605K on the amplification of tumor cells as shown. Fig. 4F shows an inhibitory activity of conjugate compound CB-18G on the amplification of tumor cells as shown. Fig. 4G shows an inhibitory activity of conjugate compound CB-505 on the amplification of tumor cells as shown.
Figs. 5A-5E show tumor suppressive effects of conjugate compound CB-20BK
in mice.
Figs. 6A-6C show tumor suppressive effects of conjugate compound CB-20B
in mice.
Figs. 7A-7E show tumor suppressive effects of conjugate compound CB-18G
in mice.
Figs. 8A-8B show effects of CBP-1018 for injection on tumor volumes of LU2505 lung cancer model and LU1206 lung cancer model.
Detailed Description of Embodiments While various aspects and embodiments will be disclosed in the present application, it is apparent that a person skilled in the art may make various equivalent changes and modifications to the aspects and embodiments without deviating from the subject spirit and scope of the present application. The various aspects and embodiments disclosed in the present application are only for the purposes of illustration and are not intended to be limiting, with the true scope being indicated by the appended claims. All publications, patents or patent applications cited in the present application are incorporated by reference in their entirety.
Unless defined otherwise, the technical and scientific teinis as used herein have the same meanings as commonly understood by a person skilled in the art to which the present application belongs.
Date Recue/Date Received 2021-07-27 As used herein and in the appended claims, the singular foinis "a", "an", and "the" include plural reference unless the context clearly dictates otherwise.
The Wails "a" (or "an"), "one or more" and "at least one" can be used interchangeably herein. It is also to be noted that the teinis "comprise/comprising", "include/including", and "have/having" can be used interchangeably.
As used herein and in the appended claims, the teini "analog" includes a structural analog and a functional analog. The structural analog refers to a class of compounds with similar chemical structures, which may comprise one or more different atoms or one or more different functional groups. The functional analog refers to a class of compounds that have the same or similar chemical, biological or pharmacological effects. For example, the analogs of folate include 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methyl enetetrahydro folate .
As used herein and in the appended claims, the teini "derivative" refers to a relatively complex compound derived from a parent compound molecule in which one or more atoms or atomic groups are substituted with other atoms or atomic groups. For example, camptothecin derivatives include irinotecan, SN-38, Dxd, topotecan, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 10-hydroxyc amptothe c in, (20 S)-c amptothec in, 9-nitrocamptothecin, gimatecan, karenitecin, silatecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625 In one aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively.
In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a ligand moiety represented by foimula (I), respectively:
Date Recue/Date Received 2021-07-27 (D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A¨
/
(I).
In another aspect, the present application discloses a conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof The tem" "payload" as used herein refers to a molecule or material to be delivered to a target cell or tissue. Without limitation, the payload may be any molecule or material that is intended for use in the diagnosis, treatment, or prevention of a disease in a subject. In some embodiments, the payload has a molecular weight of less than or equal to about 5 kDa. In some embodiments, the payload has a molecular weight of less than or equal to about 1.5 kDa. In some embodiments, the payload is a drug or a diagnostic reagent that has been deemed safe and effective for use by appropriate drug approval and registration agencies (such as FDA, EMEA, or NMPA).
In some embodiments, the payload of the present application is a small molecule compound, a nucleotide (such as a DNA, a plasmid DNA, an RNA, an siRNA, an antisense oligonucleotide and an aptamer), a peptide, or a protein (for example, an enzyme). In some embodiments, the payload is a small molecule compound.
In some embodiments, the payloads of the present application include, but are not limited to: an anticancer drug, a radioactive substance, a vitamin, an anti-AIDS
drug, an antibiotic, an immunosuppressant, an antiviral drug, an enzyme inhibitor, a neurotoxin, an opioid, a regulator of cell-extracellular matrix interaction, a vasodilator, an antihypertensive drug, a hypnotic, an antihistamine, an anticonvulsant, a muscle relaxant, an anti-Parkinson substance, an anticonvulsant and a drug for inhibiting muscle contraction, an antiparasitic drug and/or an anti-antiprotozoal drug, an analgesic drug, an antipyretic, a steroidal or Date Recue/Date Received 2021-07-27 non-steroidal anti-inflammatory drug, an anti-angiogenic factor, an antisecretory factor, an anticoagulant and/or an antithrombotic agent, a local anesthetic, a prostaglandin, an antidepressant, an antipsychotic, an antiemetic, or an imaging agent.
In some embodiments, the payload of the present application has a free amino or carboxyl group before being conjugated with the conjugate compound of the present application, and the payload is conjugated with the conjugate compound through an acylation reaction between the above-mentioned amino or carboxyl group and the corresponding part (for example, a linker) of the conjugate compound.
In some embodiments, a modification on the above-mentioned free amino or carboxyl group (for example, by conjugating with the conjugate compound of the present application) may significantly reduce the activity of the payload (for example, by at least 50%, 60%, 70%, 80%, 90%, 95%, 98% or 99%).
The "small molecule compound" as used herein refers to a compound having a molecular weight of less than or equal to about 2 kDa. In some embodiments, the small molecule compound has a molecular weight of less than or equal to about 1.5 kDa. In some preferred embodiments, the small molecule compound has a molecular weight of less than or equal to about 1 kDa, 800 Da, 700 Da, 600 Da, or 500 Da. In some embodiments, the small molecule compound of the present application is selected from the group consisting of camptothecin and any derivative thereof (for example, SN38 or Dxd), auristatin and any derivative thereof (for example, MMAE and MMAF), maytansine and any derivative thereof, a cyclooxygenase-2 inhibitor (for example, celecoxib), a radionuclide complex, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C. In some embodiments, the small molecule compound is camptothecin or any derivative thereof, auristatin or any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor. In some embodiments, the small molecule compound described in the present application is a drug for relieving or treating a cancer. In some embodiments, the Date Recue/Date Received 2021-07-27 small molecule compound described in the present application is a drug for relieving or treating an autoimmune disease.
The ten," "camptothecin" as used herein refers to a cytotoxic alkaloid, mainly derived from Camptotheca acuminata (Nyssaceae) and showing a strong anti-tumor activity. The camptothecin and derivative thereof of the present application include a camptothecin and a derivative thereof that are already in existence or will be produced later. The camptothecin and derivative thereof of the present application include, but are not limited to: camptothecin, irinotecan, SN-38, Dxd, topotecan, GI-147211C, 9-aminocamptothecin, 7-hydroxymethyl camptothecin, 7-aminomethyl camptothecin, 10-hydroxyc amptothe c in, (20 S)-c amptothec in, 9-nitrocamptothecin, gimatecan, karenitecin, silatecan, exatecan, diflomotecan, belotecan, lurtotecan and S39625.
The tettn "auristatin and any derivative thereof/auristatin or any derivative thereof' as used herein refers to a natural anti-tumor product, aplysiatoxin
10, and a series of derivatives thereof, wherein such compounds interfere with microscopic self-assembly to arrest cells in a mitotic phase, which has a strong lethality to the cells. The auristatin and any derivative thereof of the present application include auristatin and any derivative thereof that are already in existence or will be produced later. The auristatin and derivative thereof of the present application include, but are not limited to auristatin, monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), monomethyl auristatin D (MMAD), AFP and AFHPA.
The tettn "cyclooxygenase-2 inhibitor" as used herein is a class of specific cyclooxygenase-2 inhibitors. Cyclooxygenase-2 participates in the development and infiltration of malignant tumors through a variety of mechanisms.
Cyclooxygenase-2 inhibitors can inhibit tumor cell migration and adhesion and intravascular infiltration, thereby inhibiting the occurrence and development of malignant tumors. The cyclooxygenase-2 inhibitors of the present application include cyclooxygenase-2 inhibitors that are already in existence or will be produced later. Cyclooxygenase-2 inhibitors include, but are not limited to celecoxib, rofecoxib, parecoxib, valdecoxib and etoricoxib.
Date Recue/Date Received 2021-07-27 The tem" "radionuclide complex" as used herein refers to a special class of complexes containing radionuclides, wherein the chelating agent in the complex can be chelated with the radionuclide and provide a linking part that more stably binds to a target substance. The tem" "radionuclide" as used herein refers to an element that can spontaneously emit radiation (such as a-rays, I3-rays, or y-rays).
The radionuclides of the present application include all radionuclides for treatment and diagnosis that are already in existence or will be produced later. The radionuclides of the present application include, but are not limited to 67cu, 64cu., 90y, 109pd, 111Ag, 149pm, 153sm, 165H0, 166H0, 177Lu, 186-e K, 188Re, 99mTC, 67Ga, 68Ga, "In, 90y, 177Lu, 186Re, 188Re, 197AU, 198AU, 'Au, io5Rh, 161Tb, 149pm, 44se, 47se, 70As, 71As, 72As, 73AS, 74AS, 76AS, 77As, 212pb, 212Bi, 213Bi, 225Ac, 117msb, 67Ga, 201T1, 1231, 1311, 160Gd, 148Nd, 89Sr and 211At. In some embodiments, the chelating agent is a macrocyclic chelating agent. The chelating agents of the present application include, but are not limited to H2dedpa, H4octapa, H2azapa, DTPA, CHX-A"-DTPA, DTPA-bis anhydride, Maleimide-DTPA, DTPA(tBu)4, DiamSar CB-TE2A, Cyclam, DO2A, DOTA, OTA-GA(tBu)4, Maleimide-DOTA-GA, p-NCS-Bz-DOTA-GA, NH2-DOTA-GA, DOTA-GA anhydride, DOTA-tris(tBu) ester, Propargyl-DOTA-tris(tBu) ester, DO3AM-acetic acid, DO3AM-N-(2-aminoethyl)ethanamide, DO3AtBu-N-(2-aminoethyl)ethanamide, DOTA-di(tBu)ester, DOTA-tris(tBu)ester NHS ester, DOTA-NHS ester, Propargyl-DOTA-tris(tBu) ester, DOTADOTA-GA anhydride, DOTA-GA(tBu)4, p-NCS-Bz-DOTA-GA, NH2-DOTA-GA, Maleimide-DOTA-GA, AGuIX, Gado-H, CYCLEN, DO2AtBu, DO3AtBu, DO3AEt, DO3AM, DOTAEt, DOTPrEt, cis-Glyoxal-Cyclen, Mono-N-Benzyl-Cyclen, trans-N-Dibenyl-Cyclen, TriB0C-Cyclen, Mono-N-Benzyl-TACN, DiB0C-TACN, Cross-bridge-Cyclam (CB-Cyclam), (1 3)aneN4, TACN, TACN=3HC1, TACD, Mono-N-benzyl-TACD, DiB0C-TACD, 1,7-Dioxa-4,10-diazacyclododecane, C-Methyl-Ester-Cyclam, C-Carboxylic-Acid-Cyclam, trans-N-Dimethyl-Cyclam, TETRAM, TETAEt, TETAMEt2, TETAMMe2, TETAM, CPTA, CB-Cyclam derivatives, CB-TE2A, Methylamino-(13)aneN4, Bis-(13)aneN4, Oxo-(1 3)aneN4, Mono-N-Benzyl-(13)aneN4, TriB0C-(1 3)aneN4, TRITRAM, TRI3AEt, TRI3AtBu, Date Recue/Date Received 2021-07-27 TRITAM, TRITA, Mono-N-Benzyl-Cyclam, Foi _______________ inaldehyde-Cyc lam, c is -Glyoxal-Cyc lam, Dioxocyclam, Oxocyc lam, trans-N-D ibenzyl-Cyc lam, TriB0C-Cyclam, DOTP, DOTMA, TETA, DOTAM, DiAmSar, CB-Cyclam, CB-TE2A, NOTA, NOTAM, NH2-NODA-GA, Iodo-NODA-GA, NCS-MP-NODA, NH2-MPAA-NODA, NODA-GA(tBu)3, NODA-GA-NHS ester, Maleimide-NODA-GA, NO TA -NH S ester, Maleimide-NOTA, Propargyl-NOTA(tBu)2, p-NCS-benzyl-NODA-GA, NOTA(tBu)2, NCS-MP-NODA, NH2-MPAA-NODA, NH2-NODA-GA, Iodo-NODA-GA and TACN.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises one payload. In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises two or more payloads. For example, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more payloads. In a conjugate compound containing multiple payloads, each of the payloads may be identical to or different from each other. In some embodiments, at least two of the payloads are different from each other.
The term "targeting molecule" as used herein refers to any molecule or moiety capable of targeting the conjugate compound of the present application to a target site, a target tissue, a target organ, a target cell, or a target intracellular region. In some embodiments, the targeting molecule allows the conjugate compound of the present application to be distributed more in a target site, a target tissue, a target organ, a target cell or a target intracellular region compared with a non-target site, a non-target tissue, a non-target organ, a non-target cell or a non-target intracellular region, for example, at least 10% more, 20% more, 50% more, 80% more, 100%
more, 150% more, 200% more, 300% more, 400% more, 500% more, etc. In some embodiments, the targeting molecule allows a conjugate compound containing a targeting molecule, compared with a conjugate compound containing no targeting molecule, to be distributed more in a target site, a target tissue, a target organ, a target cell or a target intracellular region, for example, at least 10% more, 20% more, 50% more, 80% more, 100% more, 150% more, 200% more, 300% more, 400%
Date Recue/Date Received 2021-07-27 more, 500% more, etc. In some embodiments, the targeting molecule can trigger or promote a conjugate compound containing such targeting molecules to specifically bind to a target molecule, trigger or promote endocytosis of the conjugate compound by a target cell, and trigger or promote the conjugate compound to be enriched around a target cell and/or enter a target cell.
In some embodiments, the conjugate compound of the present application comprises at least two targeting molecules. In some embodiments, the two or more targeting molecules comprised in the conjugate compound of the present application are identical or different. In some embodiments, at least two targeting molecules of the two or more targeting molecules comprised in the conjugate compound of the present application are different. In some embodiments, the two or more targeting molecules comprised in the conjugate compound of the present application are all different from each other. In some embodiments, at least two targeting molecules of the two or more targeting molecules comprised in the conjugate compound of the present application can specifically bind to different cell surface proteins or markers.
In some embodiments, the two or more targeting molecules comprised in the conjugate compound of the present application can specifically bind to different cell surface proteins or markers.
In some embodiments, the conjugate compound of the present application comprises at least two targeting molecules, at least one of which is a synergistic molecule.
The teini "synergistic molecule" as used herein refers to any molecule or moiety that is capable of working synergistically with other targeting molecules comprised in the conjugate compound of the present application to better trigger or promote the conjugate compound to specifically bind to a target molecule, trigger or promote the endocytosis of the conjugate compound by a target cell, trigger or promote the conjugate compound to be enriched around a target cell and/or enter a target cell, and/or cause the conjugate compound, in a different manner, to specifically bind to a target cell and be maintained. In some embodiments, the synergistic molecule allows the conjugate compound of the present application to be distributed more in a target site, a target tissue, a target organ, a target cell or a Date Recue/Date Received 2021-07-27 target intracellular region compared with a non-target site, a non-target tissue, a non-target organ, a non-target cell or a non-target intracellular region, for example, at least 10% more, 20% more, 50% more, 80% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500% more, etc. In some embodiments, the synergistic molecule allows a conjugate compound containing a synergistic molecule, compared with a conjugate compound containing no synergistic molecule, to be distributed more in a target site, a target tissue, a target organ, a target cell or a target intracellular region, for example, at least 10% more, 20% more, 50%
more, 80% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500%
more, etc. In some embodiments, the synergistic molecule allows a conjugate compound containing a synergistic molecule, compared with a conjugate compound containing no synergistic molecule, to have a higher activity on a target cell, for example, at least 10% higher, 20% higher, 50% higher, 80% higher, 100% higher, 150% higher, 200% higher, 300% higher, 400% higher, 500% higher, etc.
In some embodiments, the synergistic molecule of the present application is a cell-interacting molecule.
The teini "cell-interacting molecule" as used herein refers to a molecule that is capable of interacting with a cell surface material of a target cell to trigger or promote a conjugate compound containing such cell-interacting molecules to specifically bind to a cell, to trigger or promote endocytosis of the conjugate compound by a target cell, and/or to trigger or promote the conjugate compound to be enriched around a target cell and/or enter a target cell.
The cell-interacting molecule may be a small chemical molecule or a large biomolecule. In some embodiments, the cell-interacting molecule is a small molecule compound or a polypeptide. In some embodiments, the cell-interacting molecule is a small molecule compound, or a polypeptide comprising 2-50, 2-40, 2-30, 2-25, 2-22, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 4-50, 5-50, 5-40, 5-30, 5-25, 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 6, 7, 8, or 9 amino acids.
In some embodiments, the targeting molecule is a ligand capable of binding to a cell surface receptor or other molecules. In some embodiments, at least one of the Date Recue/Date Received 2021-07-27 targeting molecules is a ligand capable of binding to a cell surface receptor or other molecules.
The ligands of the present application can include a variety of chemical or biological molecules, which can have a specific binding affinity to a selected target, wherein the selected target can be, for example, a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the ligand can specifically bind to a protein or a marker expressed on the surface of a target cell. In some embodiments, the ligand of the present application binds to a cell surface protein or marker with an affinity of 1 0-6-10-11M (Ka value). In some embodiments, 113 the ligand of the present application binds to a cell surface protein or marker with an affinity of at least 10-7, at least 10-8 and at least 10-9 M (Ka value). In some embodiments, the ligand of the present application binds to a cell surface protein or marker with an affinity of less than 10-6, less than 10-7 and less than 10-8 M
(Ka value). In some embodiments, the ligand of the present application binds to a cell surface protein or marker with a certain affinity, wherein the certain affinity refers to the affinity of the ligand to a target cell surface protein or marker which is at least two, three, four, five, six, eight, ten, twenty, fifty, one hundred or more times higher than that to a non-target cell surface protein or marker. In some embodiments, the expression of the cell surface protein or marker of the present application in target cells (e.g. cancer cells) is significantly higher than that in nottnal cells.
The tettn "significantly" as used herein refers to statistically significant differences, or significant differences that can be recognized by a person skilled in the art.
In some embodiments, the expression level of the cell surface protein or marker of the present application in target cells (e.g. cancer cells) are 2 to 1,000,000 times higher than that in normal cells; for example, the expression level in target cells (e.g. cancer cells) are 2 to 10, 2 to 100, 2 to 1,000, 2 to 10,000, 2 to 100,000 or 2 to 1,000,000 (which can be equal to any value within the above numerical range, and the end values of this range included) times higher than that in nottnal cells. In some embodiments, the expression level of the cell surface receptor in target cells (e.g. cancer cells) is at least 10 times higher, or 100 times higher, or 1,000 times higher, or 10,000 times higher, or 100,000 times higher than that in nottnal cells. In Date Recue/Date Received 2021-07-27 some embodiments, compared with the level of the cell surface protein or marker on target cells (e.g. cancer cells), the level of the cell surface receptor on nonnal cells is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the cell surface protein or marker described in the present application is undetectable in nonnal cells.
In some embodiments, the cell surface protein or marker of the present application is a cell surface receptor.
In some embodiments, the cell surface receptor of the present application is selected from the group consisting of a transferrin receptor (TFR), a low-density lipoprotein receptor (LDLR), a folate receptor (FR), a growth hoinione-inhibiting hormone receptor, a uric acid kinase receptor, a tumor necrosis factor receptor (TNFR), an integrin receptor (LFA-1), an SST-14 receptor (SSTR2), a GNRH
receptor (GNRHR), a TRPV6 and an integrin a receptor.
In some embodiments, the cell surface protein or marker of the present application is a cell surface antigen.
In some embodiments, the cell surface antigen of the present application is selected from the group consisting of a prostate-specific membrane antigen, a MUC1 mucin, an acute lymphoblast common antigen, a Thy-1 cell surface antigen, a Melan-A protein, a squamous cell carcinoma antigen, a galectin 3 and a human leukocyte antigen.
In some embodiments, the cell-interacting molecule of the present application can bind to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR
family, CD40, CD4OL, 0X40, OX4OL, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4, 5IGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3 and CXCR4.
Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises a prostate-specific membrane antigen ligand moiety and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises a ligand moiety represented by formula (I) and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.
In yet some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises P10 and a synergistic molecule moiety, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
In some embodiments, one of the synergistic molecules in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is an endocytosis molecule moiety capable of mediating endocytosis. The term "endocytosis" as used herein means that the conjugate compound or the pharmaceutically acceptable salt thereof interacts with a target cell and then is capable of mediating its own endocytosis, internalization or uptake by the target cell.
The teini "endocytosis molecule" as used herein refers to a molecule that interacts with a target cell and then is capable of mediating the endocytosis, internalization, or uptake of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application by the target cell.
In some embodiments, the endocytosis molecule is selected from the group consisting of a folate and an analog thereof, a peptide capable of mediating endocytosis, and a cell-penetrating peptide.
In some embodiments, the endocytosis molecule of the present application is a folate or an analog thereof Folate is beneficial for forming a chemical bond with other groups due to its small molecule weight, non-immunogenicity, and good stability. Folate can be Date Recue/Date Received 2021-07-27 associated with a folate receptor expressed on a cell surface with a high affinity to mediate a cellular uptake of the folate. Although expressed at a very low level in most normal cells, a folate receptor is expressed at a high level in numerous cancer cells to meet the high folate demand of rapidly dividing cells under a low folate condition (see Kelemen LE, Int J Cancer, 2006; 119:243-50; Kane MA, et al., J
Clin Invest. 1988; 81: 1398-406; Matsue H, et al., Proc Natl Acad Sci USA.
1992;
89: 6006-9; Zhao R, et al., Annu Rev Nutr. 2011; 31: 177-201). Folate is capable of specifically binding to a folate receptor on a cell surface, and is also capable of mediating endocytosis of a conjugate compound or a pharmaceutically acceptable salt thereof into target cells.
In some embodiments, the analog of folate is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methyl enetetrahydro folate .
In some embodiments, the endocytosis molecule is a peptide capable of mediating endocytosis.
In some embodiments, the peptide capable of mediating endocytosis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18 and Arg-Gly-Asp (named as RGD), and homologous peptides having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence homology to any of SEQ ID NOs:
16-18, wherein the homologous peptides are functional equivalents of the peptides as shown in SEQ ID NOs: 16-18, respectively.
In some embodiments, the peptide capable of mediating endocytosis as described in the present application has a conservative substitution of an amino acid at only one amino acid site compared to the sequences of SEQ ID NOs: 16-20 and RGD. In some embodiments, the peptide capable of mediating endocytosis as described in the present application has a conservative substitution of an amino acid at 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sites compared to the sequences of SEQ ID
NOs: 16-20.
Date Recue/Date Received 2021-07-27 Under the premise of not affecting its biological activity, the peptide capable of mediating endocytosis as described in the present application may also contain non-naturally occurring amino acids, including, for example, I3-fluoroalanine, 1-methyl-histidine, y-methylene-glutamic acid, a-methyl-leucine, 4,5 -dehydro-lysine, hydroxypro line, 3 -fluoro-phenylalanine, 3-amino-tyrosine, 4-methyl-tryptophan, and the like.
The percentage of homology can be determined by various well-known methods in the art. For example, the comparison of sequences can be achieved by the following publically available tools: BLASTp software (available from the website of National Center for Biotechnology Infoitnation (NCBI):
http://blast.ncbi.nlm.nih.gov/Blast.cgi; also see: Altschul S.F. et al., J.
Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the website of European Bioinfounatics Institute:
http://www.ebi.ac.uk/Tools/msa/clustalw2/; also see: Higgins D. G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and Tcoffee (available from the website of Sweden Bioinformatics Institute; also see: Poirot 0. et al., Nucleic Acids Res., 31(13): 3503-6 (2003); Notredame C. et al., J. Mol. Boil., 302(1): 205-17 (2000)). If the alignment of sequences is performed using a software, the default parameters available in the software may be used, or otherwise the parameters may be customized to suit the alignment purpose. All of these are within the scope of knowledge of a person skilled in the art.
The term "functional equivalent" as used herein refers to a derivative peptide that retains a biological activity that is substantially similar to that of the original peptide sequence that the derivative peptide derives from. The functional equivalent may be a natural derivative or is prepared synthetically. Exemplary functional equivalents include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of a peptide is maintained. The amino acid used for substitution desirably has chemico-physical properties similar to the amino acid to be substituted.
Desirable Date Recue/Date Received 2021-07-27 similar chemico-physical properties include, similarities in charges, bulkiness, hydrophobicity, hydrophilicity, and the like.
In some embodiments, the functional equivalents include a conservative substitution of an amino acid residue. The conservative substitution of an amino acid residue refers to a substitution between amino acids with similar properties, for example, a substitution between polar amino acids (such as a substitution between glutamine and asparagine), a substitution between hydrophobic amino acids (such as a substitution among leucine, isoleucine, methionine and valine), a substitution between amino acids with identical charges (such as a substitution among arginine, lysine and histidine, or a substitution between glutamic acid and aspartic acid), etc.
In some embodiments, the endocytosis molecule is a cell-penetrating peptide.
Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), are short peptides (generally less than 40 amino acids), with the ability to gain access to the interior of cells in a receptor-independent manner. The cell-penetrating peptides, when conjugated with payloads, are capable of mediating the transmembrane transport of the payloads and have a protein transduction activity.
In some embodiments, the cell-penetrating peptide described in the present application is selected from the group consisting of a tumor-homing peptide, a mitochondrial penetrating peptide, an activatable cell-penetrating peptide, and an antibacterial peptide. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (RRRRRRRRR, named as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which is a Tat peptide, i.e. a cell-penetrating peptide of the HIV transactivator of transcription protein).
In some embodiments, one targeting molecule in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is a prostate-specific membrane antigen ligand moiety.
The term "prostate-specific membrane antigen" as used herein refers to a type II transmembrane glycoprotein that exists in the membrane of prostate epithelial cells and consists of 750 amino acids, comprising 19 amino acids in the intracellular region, 24 amino acids in the transmembrane region and 707 amino acids in the Date Recue/Date Received 2021-07-27 extracellular region. The prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but is expressed at a much higher level in prostate cancer cells. Compared with the traditional prostate-specific antigen used for clinical detection, the prostate-specific membrane antigen is a more sensitive and specific prostate cancer tumor marker, and especially, it is highly expressed in both hormone-refractory prostate cancer and prostate cancer metastatic lesions, and has a high sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors. Moreover, in a variety of nonprostate solid tumors (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer and colorectal cancer), the prostate-specific membrane antigen is also highly and specifically expressed on tumor vascular endothelial cells.
The tem" "prostate-specific membrane antigen ligand" as used herein refers to an antibody, an aptamer and a small molecule that is capable of specifically recognizing and binding to a prostate-specific membrane antigen. The prostate-specific membrane antigen ligands of the present application include the prostate-specific membrane antigen ligands that are already in existence or will be produced later, as well as fragments of the aforementioned ligands, as long as these fragments still retain the ability to bind to a prostate-specific membrane antigen.
Antibody ligands are the most common prostate-specific membrane antigen ligands, which include, but are not limited to monoclonal antibodies J591, J533, J415 and E99 (for example, see Liu H, Rajasekaran AK, Moy P et al. Constitutive and antibody-induced internalization of prostate-specific memberane antigen [J].
Cancer Res, 1998, 58 (18): 4055-4060). The aptamer is a single-stranded DNA or RNA that is obtained through technical screening by an exponential enrichment ligand system and can bind to prostate-specific membrane antigens with a high affinity and a high specificity. Such prostate-specific membrane antigen ligands include, but are not limited to an xPSM-A10 aptamer and a derivative thereof and an xPSM-A9 aptamer and a derivative thereof (for example, see Lupoid SE et al., Identification and Characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen, Cncer Res, 2002, 62(14):4029-4033). Compared with antibody ligands and aptamer ligands, the Date Recue/Date Received 2021-07-27 prostate-specific membrane antigen small molecule ligands have the advantages of small molecular weight, high pettneability, low immunogenicity, and ease of synthesis, and include but are not limited to glutamine urea small molecule ligands and phosphoramidate small molecule ligands.
In some embodiments, the prostate-specific membrane antigen small mole cule ligands of the present application can be selected from the group consist ing of 2-[[methylhydroxyphosphinyl]methyl]glutaric acid; 2-[[ethylhydroxyphos phinyl]methyl]glutaric acid; 2-[[propylhydroxyphosphinyl]methyl]glutaric acid;
2-[[butylhydroxyphosphinyl]methyl]glutaric acid; 2-[[cyclohexylhydroxyphosphin yl]methyl] glutaric acid; 2- [[phenylhydroxyphosphinyl]methyl] glutaric acid;
2- [ [2 -(tetrahydrofuranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(2-tetrahydropyra nyl)hydroxyphosphinyl]methyl] glutaric acid; 2 - [ [((4-pyridyl)methyl)hydroxyphos phinyl]methyl] glutaric acid; 2 - [ [((2-pyridyl)methyl)hydroxyphosphinyl]methyl] gl utaric acid; 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-p henylethyl)methyl)hydroxyphosphinyl]methyl] glutaric acid; 2- [ [((3 -phenylpropyl) methyl)hydroxyphosphinyl]methyl] glutaric acid; 2- [ [((3 -phenylbutyl)methyl)hydr oxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphin yl]methyl] glutaric acid; 2- [ [(4-phenylbutyl)hydroxypho sphinyl] methyl]
glutaric ac id; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[methyl h ydroxyphosphinyl] oxy] glutaric acid; 2- [ [ethyl hydroxyphosphinyl] oxy]
glutaric a cid; 2-[[propyl hydroxyphosphinyl]oxy]glutaric acid; 2-[[butyl hydroxyphosphin yl] oxy] glutaric acid; 2- [ [phenyl hydroxypho sphinyl] oxy] glutaric acid; 2-[ [((4-py ridyl)methyl)hydroxyphosphinyl] oxy] glutaric acid; 2- [ [((2-pyridyl)methyl)hydrox yphosphinyl] oxy] glutaric acid; 2- [ [(phenylmethyl)hydroxyphosphinyl] oxy]
glutaric acid; and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[(n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-methyl)carbamo yl]methyl]glutaric acid; 2-[[(n-butyl-n-hydroxyl)carbamoyl]methyl]glutaric acid;
2-[[(n-benzyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-phen yl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-2-phenylethyl)carbamoyl]m ethyl]glutaric acid; 2-[[(n-ethyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[ [(n-hydroxyl-n-propyl)carbamoyl]methyl] glutaric acid; 2- [ [(n-hydroxyl-n-3 -phen Date Recue/Date Received 2021-07-27 ylpropyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-4-pyridyl)carbamoyl]
methyl]glutaric acid; 2-[[(n-hydroxyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl (methyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(benzyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(phenyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(2-pheny lethyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(ethyl)amide]methyl]glutaric aci d; 2-[[n-hydroxyl(propyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(3-phenylpro pyl)amide]methyl]glutaric acid; and 2-[[n-hydroxyl(4-pyridyl)amide]methyl]gluta ric acid; 2-[(thionyl)methyl]glutaric acid; 2-[(methylthionyl)methyl]glutaric acid;
2-[(ethylthionyl)methyl]glutaric acid; 2-[(propylthionyl)methyl]glutaric acid;
[(butylthionyl)methyl]glutaric acid; 2-[(phenylthionyl]methyl]glutaric acid; 2-[[(2-phenylethyl)thionyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)thionyl]methyl]
glutaric acid; 2-[[(4-pyridyl)thionyl]methyl]glutaric acid; 2-[(benzylthionyl)meth yl]glutaric acid; 2-[(sulfonyl)methyl]glutaric acid; 2-[(methylsulfonyl)methyl]glu taric acid; 2-[(ethylsulfonyl)methyl]glutaric acid; 2-[(propylsulfonyl)methyl]gluta ric acid; 2-[(butylsulfonyl)methyl]glutaric acid; 2-[(phenylsulfonyl]methyl]glutar ic acid; 2-[[(2-phenylethyl)sulfonyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)sul fonyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfonyl]methyl]glutaric acid; 2-[(benz ylsulfonyl)methyl]glutaric acid; 2-[(sulfoximinyl)methyl]glutaric acid; 2-[(methy lsulfoximinyl)methyl]glutaric acid; 2-[(ethylsulfoximinyl)methyl]glutaric acid; 2-[(propylsulfoximinyl)methyl]glutaric acid; 2-[(butylsulfoximinyl)methyl]glutaric acid; 2-[(phenylsulfoximinyl]methyl]glutaric acid; 2-[[(2-phenylethyl)sulfoximiny l]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfoximinyl]methyl]glutaric acid;
[[(4-pyridyl)sulfoximinyl]methyl]glutaric acid; and 2-[(benzylsulfoximinyl)methy l]glutaric acid; n- [methyl hydroxyphosphinyl]glutamic acid; n- [ethyl hydroxyph osphinyl]glutamic acid; n-[propyl hydroxyphosphinyl]glutamic acid; n4butyl hy droxyphosphinyl]glutamic acid; n- [phenyl hydroxyphosphinyl]glutamic acid; n-[(phenylmethyl)hydroxyphosphinyl]glutamic acid; n-[((2-phenylethyl)methyl)hydr oxyphosphinyl]glutamic acid; and N-methyl-N4phenylhydroxyphosphinyl]gluta mic acid. The prostate-specific membrane antigen ligands of the present appli cation also include all prostate-specific membrane antigen small molecule liga nds disclosed in PCT applications WO 2010/108125 and WO 2006/093991, t Date Recue/Date Received 2021-07-27 he above-mentioned two patent applications are incorporated herein in their e ntirety.
In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application is a glutaric acid derivative. In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application is an aminocarbonyl derivative of glutaric acid.
In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application has the following structure:
H0( (s) NAN1/4 H H
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO
(s) NANiNC., H H H
0 .
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO 00 0, _OH
HO (S) NANNAN 0 H H H H
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO 0 0 , -OH
H 0 N AN : NA N 0 (s) (S) 0 H H H H
N (S) H
0 \
H 0 .
Date Recue/Date Received 2021-07-27 In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
_ HO
(S) NANNAN 0 i_i 0 H H H H
0 j(isj,(sA
H i H
0 \
HO
In some embodiments, one targeting molecule in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is a ligand moiety represented by formula (I):
> _____________________ (D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A¨
/
(I), or a ligand moiety having at least 70%, at least 80%, at least 85% or at least 90% amino acid sequence homology thereto or having at most 3, 2 or 1 amino acid substitutions (for example, conservative substitutions) therewith.
In some embodiments, the one targeting molecule in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is P10 or a ligand moiety having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% or at least 93% amino acid sequence homology thereto or having at most 3, 2 or 1 amino acid substitutions (for example, conservative substitutions) therewith.
The term "P10" as used herein refers to a peptide having an amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.
In some embodiments, the conjugate compound of the present application has targeting molecules selected from the group consisting of (1) a folate ligand and a prostate-specific membrane antigen ligand; (2) a TRPV6 ligand and a Date Recue/Date Received 2021-07-27 prostate-specific membrane antigen ligand; (3) a GNRHR ligand and a prostate-specific membrane antigen ligand; (4) an SSTR2 ligand and a prostate-specific membrane antigen ligand; (5) a folate ligand and an SSTR2 ligand;
or (6) a TRPV6 ligand and a folate ligand.
In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a folate or an analog thereof and a prostate-specific membrane antigen ligand moiety, respectively. Without wishing to be limited by the theory, a specific folate or an analog thereof and a prostate-specific membrane antigen ligand moiety that have a better stability than the ligand combinations in the prior art are selected.
In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a synergistic molecule moiety and a ligand moiety represented by formula (I), respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a folate or an analog thereof and a ligand moiety represented by formula (I), respectively.
In some embodiments, the two targeting molecules of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a synergistic molecule moiety and P10, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a folate or an analog thereof and P10, respectively.
Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound provided in the present application only comprises a single payload conjugated with the two targeting molecules. In some embodiments, the conjugate compound provided in the present application comprises multiple payloads conjugated with the two targeting molecules.
The teini "conjugated" as used herein refer to the linking through a covalent bond of two chemical groups, either directly founing a covalent bond between the two chemical groups, or indirectly linking the two chemical groups via a linker.
In some embodiments, the conjugate compound or the phannaceutically acceptable salt thereof comprises a payload(s) (for example, 1 payload) and two targeting molecules, wherein the payload is directly covalently linked to at least one of the targeting molecules. In some embodiments, the payload is directly covalently linked to the two targeting molecules.
In some embodiments, the conjugate compound or the phannaceutically acceptable salt thereof comprises a payload(s) (for example, 1 payload) and two targeting molecules, wherein the payload is covalently linked to at least one of the targeting molecules via a linker. In some embodiments, the payload is covalently linked to the two targeting molecules via a linker.
The teini "linker" as used herein refers to a molecule or moiety that covalently links a payload to a targeting molecule. The linkers include a functional group for linking a payload to at least one targeting molecule. In some embodiments, the functional group may comprise two reactive moieties, one for linking to a payload and the other for linking to a targeting molecule. In some embodiments, the functional groups are different from each other. In some embodiments, the functional groups include a group containing a thiol-reacting moiety and an amine-reacting moiety. In some embodiments, the functional groups are identical to each other. In some embodiments, the functional groups are maleimide groups.
In some embodiments, the linker contains an amino acid. In some embodiments, the carboxylic acid in the amino acid contained in the linker is amidated. In some embodiments, the linker contains a short chain polyethylene glycol (for example, comprising 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeating units).
Date Recue/Date Received 2021-07-27 In some embodiments, the linker of the present application is a multivalent linker capable of binding to at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and at least one targeting molecule. The payloads bound to the multivalent linker may be identical or different, and the targeting molecules bound to the multivalent linker may be identical or different.
In one aspect, the linker shall be sufficiently stable to avoid from unintended release of payloads during a blood circulation to increase an effective amount of payloads delivered to target cells or tissues and avoid toxicity. In another aspect, the linker shall be capable of releasing the payloads around or within target cells to efficiently kill target cells or block functions of target cells. In some embodiments, the linker comprises at least one cleavable functional group. Preferably, the cleavable functional group is sufficiently stable outside a target cell, but upon entry into the target cell, is cleaved to release a payload(s). In some embodiments, the cleavable functional group is cleaved at least 10, 20, 30, 50, 100 times or more efficiently in target cells than in the blood or serum.
The cleavable linker may be cleaved by a hydrolysis, an enzymatic reaction, or a reduction reaction, or by a pH change. In some embodiments, the linker is cleavable under a certain physiological environment (for example, under an appropriate pH environment). In some embodiments, the linker is cleavable in an acidic environment with a pH of about 6.5 or lower, or by reagents such as enzymes.
In some embodiments, the linker is susceptible to cleavage agents, for example, pH, redox potential or the presence of degradative molecules.
In some embodiments, the linker is non-cleavable. Non-cleavable linkers as used herein refer to linkers which remain basically intact during intracellular metabolism.
In some embodiments, the linker is a peptide linker consisting of a straight or branched chain amino acids linked by peptide bonds. In some embodiments, the peptide linker is cleavable by a protease that is highly or specifically expressed around or in target cells, for example, cathepsin B in the lysosome or endosome.
The peptide linker as used herein can be of varying lengths. Typically, the peptide linker of the present application is from 1 to 50 amino acids in length. In some Date Recue/Date Received 2021-07-27 embodiments, the peptide liker is from 1 to 45, from 1 to 40, from 1 to 35, from 1 to 30, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2 or 1 amino acids in length. In some embodiments, the peptide liker is from 2 to 45, from 2 to 40, from 2 to 35, from 2 to 30, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3 or 2 amino acids in length. The number of amino acids of the peptide linker as described in the present application can be equal to any integer value within the above numerical range, including the end values of this range. In some embodiments, the peptide linker is preferred to be 1, 2, 3, 4 or 5 amino acids in length. In some embodiments, the peptide linker is cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino acids is amidated.
In some embodiments, the linker is a disulfide linker containing a disulfide bond. The disulfide bond may be cleaved under an intracellular reductive environment, while remains stable in a circular system. The disulfide linker of the present application may be DSDM, DMDS, MDS, or NDMDS. The structures of these disulfide linkers are shown in Table 1 below.
Table 1: Structures of DSDM, DMDS, MDS and NDMDS
Name Structure DSDM NcS,s0.1\j I
DMDS N S -S7).L ,11-Date Recue/Date Received 2021-07-27 MDS I
,)-L _II?
I H
NS'70-11\i'l\r el 0 0.---.................---).(0, In some embodiments, the linker is a pH-dependent linker. The pH-dependent linker as described in the present application is cleavable under a certain pH
environment. In some embodiments, the pH-dependent linker may be stable under an alkaline condition, while cleavable under an acidic condition, for example, under a pH value of 6.5 or lower. In some embodiments, the pH-dependent linker is a cis-aconitic anhydride.
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof is NH
H H
N,(sA N 4 40 H
0 0 Oy\
0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
Date Recue/Date Received 2021-07-27 NH
\N(R) s 0 NtLz HN (S) 0 N 110 Oy\
0 0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
(S) N
- (R) 0 H 0 =
01r\
(S) or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
J,r1\1 N (S) = H
0 0 Oy\
HOs, Date Recue/Date Received 2021-07-27 or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
N-11\j'LSAN (S) N
= H
110 Olf\
HO
(s) 0 or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
N N (s) N
- H
(S) 0 a 0 N H2 0 (S) HN N,CSAN
= H H
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
Date Recue/Date Received 2021-07-27 In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
4N -rN 1,c!
0 (:) ) H H
NI-r-rNi 0()) le 10 0 0 0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
--2r0 (30 ) H H
N 10r N 023) 0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
Date Recue/Date Received 2021-07-27 s I
) 3.L0 110 0 0 H H
HN
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
/NH N,N
Ari\Aõ..N 0 ) H
N ) Ny,,cy...---)i,N 0C).õõ=-i H
HN
H2N , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
Date Recue/Date Received 2021-07-27 /NH N,N
0 H2N 00 0) 5,?,,H 0 (S) N
HN
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
N(S = " \/\ Nss (s) H N
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the present application may comprise any one of or a combination of the linkers as described above.
In some embodiments, the payload is conjugated with a first targeting molecule directly or indirectly, and the first targeting molecule is conjugated with a second targeting molecule directly or indirectly. In some embodiments, the payload is conjugated with each of the first and the second targeting molecule directly. In some embodiments, the payload is conjugated with each of the first and the second targeting molecule indirectly. In some embodiments, the payload is conjugated with the first targeting molecule indirectly, e.g. via a linker, and the first targeting molecule is conjugated with the second targeting molecule directly or indirectly. In some embodiments, the payload is conjugated with the first targeting molecule via a Date Recue/Date Received 2021-07-27 first linker, and the payload is conjugated with the second targeting molecule via a second linker. In some embodiments, the linker is a multivalent linker that binds to at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and two targeting molecules.
In some embodiments, the two targeting molecules are linked to each other via a spacer. In some embodiments, the spacer is cleavable by a protease that is specifically expressed in target cells or to be expressed in target cells.
Such proteases include, for example, the proteases as listed in Table 2 below. In some embodiments, the spacer comprises the amino acid sequence selected from any one of the amino acid sequences as listed in Table 2 below.
Table 2: List of Enzymatically Cleavable Sequences Amino acid sequence of Protease SEQ ID NO.
recognition site Cathepsin B RR -Legumain ASN -Matripase KSRAEDE SEQ ID NO: 1 MMP-2 PLGLAG SEQ ID NO: 2 Prostate-specific antigen SSLY SEQ ID NO: 3 Stromelysin-3 AAA -TMPRSS2 LLRSLIG SEQ ID NO: 4 Urokinase-type SSR -plasminogen activator Activated protein C LVKR SEQ ID NO: 5 Factor Ixa LVVR SEQ ID NO: 6 Factor VIIa QLTR SEQ ID NO: 7 Factor Xa LEGR SEQ ID NO: 8 Thrombin PR -Calpain-a PLFAEP SEQ ID NO: 9 Calpain-2 GLGSEP SEQ ID NO: 10 Enteropeptidase DDDDK SEQ ID NO: 11 MMP-8 GPSG SEQ ID NO: 12 Cathepsin L PLG -Date Recue/Date Received 2021-07-27 Proprotein convertase 5 RSKR SEQ ID
NO: 13 Calpain-3 VGVF SEQ ID
NO: 14 The Wails "cleavable" or "cleaved" as used herein refer to a metabolic process or reaction process on the conjugate compound provided in the present application, whereby a linker between a payload and a targeting molecule, or a spacer between targeting molecules are broken to release free payload or targeting molecule.
The linker and spacer is either cleaved by a protease or cleaved under a certain physiological environment, e.g. a pH environment.
In some embodiments, the conjugate compound has a structure of foimula I, II, III, or IV shown as follows, wherein n, m, p and q are independently 0 or 1, which represent that the linker and spacer are present or absent independently. The "molecule" in the following formula is an abbreviation for "targeting molecule".
Payload Linker )0 __ Molecule 14.J ( Spacer )03 ¨.Molecule 2 ( Formula i) ,N10 ec ule 1 4.0v Payload (Li nicer " Molecule Formula II) Date Recue/Date Received 2021-07-27 Molecule 1 4-Payload I, Multivalent linker Pa-y-1-o- ad 2 Molecule 2 ( Formula 0) Molecule I +I
.09**
Multivalent Payload linker Molecule 2 ( Formula M
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload as provided in the present application, two targeting molecules as provided in the present application and optionally a linker or spacer as provided in the present application. In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises one payload as provided in the present application, one ligand that specifically binds to a cell surface protein or marker as provided in the present application, one synergistic molecule as provided in the present application, and a linker or spacer as provided in the present application.
In some embodiments, the conjugate compound has a structure of formula V, VI, VII, or VIII shown as follows, wherein n, m, p, q and s are independently 0 or 1, which represent that the linker, multivalent linker and spacer are present or absent independently.
Date Recue/Date Received 2021-07-27 Payload _______________ ( Linker )n Ligand __ ( Spacer )m Synergistic molecule ( Formula V) Synergistic Payload ( Linker ) ¨ _________________ molecule ( Spacer )mn Ligand ( Formula VI) _________________________________________________ Ligand Payload .ver 2.4 _______________________________________ Synergistic molecule ( Formula VII) Ligand C
Payload __________________________________ Multivalent linker ¨^ Synergistic molecule Formula VIII) In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively, for example, CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SMO9 and CB-20R.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises one or more payloads and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a ligand moiety represented by formula (I), respectively, for example, CB-18G, CB-1820 and CR19426.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises one payload Date Recue/Date Received 2021-07-27 and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof, such as CB-10S, CR19425 and CB-50S.
In some embodiments, the conjugate compound of the present application is selected from the group consisting of the following compounds: CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S (the specific structure of each conjugate compound is shown in Fig. 1). In some embodiments, the conjugate compound of the present application is fottned by linking a linker-drug moiety to a ligand moiety via a covalent bond. The linker-drug moiety of the present application comprises a payload and a linker, and the ligand moiety of the present application comprises two targeting molecules and an optional spacer or linker, wherein the two moieties foal" the conjugate compound of the present application by reacting and fottning a covalent bond, and the covalent bond can be fottned between the linker in the linker-drug moiety and the ligand molecule in the ligand moiety, or can be formed between the linker in the linker-drug moiety and the spacer or linker in the ligand moiety.
The conjugate compound of the present application, CB-20B, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety 20B-SMO9 via a covalent bond. The conjugate compound of the present application, CB-20BK, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety 20BK-SMO9 via a covalent bond. The conjugate compound of the present application, CB-60S, is fottned by linking a linker-drug moiety LT2000C to a ligand moiety 60S-SM09 via a covalent bond. The conjugate compound of the present application, CB-60SK, is fottned by linking a linker-drug moiety LT2000C to a ligand moiety 60SK-SM09 via a covalent bond. The conjugate compound of the present application, CB-20C, is fottned by linking a linker-drug moiety LD1001 to a ligand moiety 20BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-1020, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety 1020BK-SMO9 via a covalent bond. The conjugate compound of the present application, CB-1320, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety Date Recue/Date Received 2021-07-27 1320BK-SMO9 via a covalent bond. The conjugate compound of the present application, CB-1820, is fonned by linking a linker-drug moiety LT1002 to a ligand moiety 1820BK-SMO9 via a covalent bond. The conjugate compound of the present application, CR19428, is fonned by linking a linker-drug moiety CR19423 to a ligand moiety 20BK-SMO9 via a covalent bond. The conjugate compound of the present application. CB-20R. is fonned by complexing 20R-SMO9 with a radionuclide ion M. The conjugate compound of the present application, CB-18G, is fonned by linking a linker-drug moiety LT1002 to a ligand moiety 18G-SMO9 via a covalent bond. The conjugate compound of the present application, CR19426, is fonned by linking a linker-drug moiety CR19423 to a ligand moiety 18G-SMO9 via a covalent bond. The conjugate compound of the present application, CB-10S, is fonned by linking a linker-drug moiety LT1000 to a ligand moiety CBSMO9 via a covalent bond. The conjugate compound of the present application, CR19425, is fonned by linking a linker-drug moiety CR19423 to a ligand moiety CBSMO9 via a covalent bond. The conjugate compound of the present application, CB-50S, is fonned by linking a linker-drug moiety LT1000N3 to a ligand moiety 50S-SMO9 via a covalent bond. Each structure is shown in Table 3 below.
Table 3: Structures of Linker-drug Moiety and Ligand Moiety Abbreviation Structure NH
LD1001 N¨
H
N
I N
OH
Hµ!`i \
0,0 N
(s) 0 Date Recue/Date Received 2021-07-27 Abbreviation Structure OH
H H
cN ,ro.iN
NI \
() 0 y N
(s) 0 ¨) 0 0) \ / 0 0 o NH
LT1002 H ii N Ali ? I V ? H
' H
R OH
0 y (e) N (S) (R) (s) 010 0 õA., 0 ,0 0 HO
\ /
I
/
A (s) LT2000C H H ii N, 0 iNlrOr - N
H
(K--) 0 0¨? NH2 \ /
HN
HO Hr_N 9 H \- 0 = H H
s) Ni,,,,,, s)N
. s) N .
(s) N lifiRN (s) N yrsl'N IfISI'N
Nlr'NH
H H H H H H H
(R) 0 ao N NH
N)?r N 'N 0 _, I
H
Date Recue/Date Received 2021-07-27 Abbreviation Structure 18G-SMO9 o Ho it 0 FIN 0 J\
HN (Rs 0,$) 'S
0, ,NH2 HN H 0 0 H r-o-,,..$) (R) ki (si s) ki 0 0) NH -HN60 0 õ H H
---7" 0 HN (s) H) ;--- 0 0 N
,..) .(R) HO HO (S) /
(R) õ SH
OOH
/ /
NH
N"..----"Thr H
N)-,NN 0 H2N Nle H
20B-SMO9 HO 0 (:),OH
Vli N 0 H 0 0 N,@-L
N (s) NH
H
HO
(R) SH
OOH
H
N NH
0 N(-r H
NJ,NN 0 0=,N H2 H
Date Recue/Date Received 2021-07-27 Abbreviation Structure HO 0 0, _OH
HO (s) N)N(N)-1\1 r I
H i H
HO
(R) SH
0 0, _OH
r /H
NiNNH
1\1)NN
H2N NIµl H
D 0 0 0,0H 0 HO
(s) N N N N
H H H H 0 ,$) CI?
(s) 'LNH2 H : H
0 - 0 -) \O
HO
COOH
,--4 0 /----..---\
0 la [\ilfil HN-Q - ) 1\1)?rN 0 N N-\
I H c-NJ b 0H
H
Hooc) NH HO X
HN N0 0 ¨( 0 , H \ 0 ---q ro o HrH, HolomAvli,i,,N)sN-1.0N S) NINA(c.)s)NN s)N Ir r., (s) N N'NH
0 H 0 H 0 H 0 H 0 H 0 0 \, 0, ,OH /
NH
0 el 11 ic) r-N
N N
1 __ H
Date Recue/Date Received 2021-07-27 Abbreviation Structure 60S-SMO9 HO 0 0, _OH
HO (S) N -LINI(.N)-NI 0 H
H H H H
N (s) . NH
H 0 \c) HO
(s) 0,0H
H
Nriµl-,QSNIH
H
N, NN 0 H2N NIµl H
yEiof 0 (:),OH HO
HO .
(s)NA NIN1j-LN 0 9 H H H H
0 N.(sP kil N (s) H = H
0 -\ 0 HO
(s) \
H
N _iN,a NH
H
NJ-,NN 0 H
H
HN N
HO H
0 ---( 0 H 0 H ,0 0H2OH,0 HONNN NN.k....--..,o, 01 1 H HH (:) NH2 HNC) (S) HO 0 0 0 ,OH HO
HO A
(s)N N NA N 0 LSHH 0 H H H H
N (s) N (s) H
0 -\ 0 HO
Date Recue/Date Received 2021-07-27 Abbreviation Structure H
HN
0 0 y H2NN s) N11.,4 .õ
,--,IH
OH H
, 0 i 0 HN N 0 O(R), s) Q H
The tettn "cyclooxygenase-2 inhibitor" as used herein is a class of specific cyclooxygenase-2 inhibitors. Cyclooxygenase-2 participates in the development and infiltration of malignant tumors through a variety of mechanisms.
Cyclooxygenase-2 inhibitors can inhibit tumor cell migration and adhesion and intravascular infiltration, thereby inhibiting the occurrence and development of malignant tumors. The cyclooxygenase-2 inhibitors of the present application include cyclooxygenase-2 inhibitors that are already in existence or will be produced later. Cyclooxygenase-2 inhibitors include, but are not limited to celecoxib, rofecoxib, parecoxib, valdecoxib and etoricoxib.
Date Recue/Date Received 2021-07-27 The tem" "radionuclide complex" as used herein refers to a special class of complexes containing radionuclides, wherein the chelating agent in the complex can be chelated with the radionuclide and provide a linking part that more stably binds to a target substance. The tem" "radionuclide" as used herein refers to an element that can spontaneously emit radiation (such as a-rays, I3-rays, or y-rays).
The radionuclides of the present application include all radionuclides for treatment and diagnosis that are already in existence or will be produced later. The radionuclides of the present application include, but are not limited to 67cu, 64cu., 90y, 109pd, 111Ag, 149pm, 153sm, 165H0, 166H0, 177Lu, 186-e K, 188Re, 99mTC, 67Ga, 68Ga, "In, 90y, 177Lu, 186Re, 188Re, 197AU, 198AU, 'Au, io5Rh, 161Tb, 149pm, 44se, 47se, 70As, 71As, 72As, 73AS, 74AS, 76AS, 77As, 212pb, 212Bi, 213Bi, 225Ac, 117msb, 67Ga, 201T1, 1231, 1311, 160Gd, 148Nd, 89Sr and 211At. In some embodiments, the chelating agent is a macrocyclic chelating agent. The chelating agents of the present application include, but are not limited to H2dedpa, H4octapa, H2azapa, DTPA, CHX-A"-DTPA, DTPA-bis anhydride, Maleimide-DTPA, DTPA(tBu)4, DiamSar CB-TE2A, Cyclam, DO2A, DOTA, OTA-GA(tBu)4, Maleimide-DOTA-GA, p-NCS-Bz-DOTA-GA, NH2-DOTA-GA, DOTA-GA anhydride, DOTA-tris(tBu) ester, Propargyl-DOTA-tris(tBu) ester, DO3AM-acetic acid, DO3AM-N-(2-aminoethyl)ethanamide, DO3AtBu-N-(2-aminoethyl)ethanamide, DOTA-di(tBu)ester, DOTA-tris(tBu)ester NHS ester, DOTA-NHS ester, Propargyl-DOTA-tris(tBu) ester, DOTADOTA-GA anhydride, DOTA-GA(tBu)4, p-NCS-Bz-DOTA-GA, NH2-DOTA-GA, Maleimide-DOTA-GA, AGuIX, Gado-H, CYCLEN, DO2AtBu, DO3AtBu, DO3AEt, DO3AM, DOTAEt, DOTPrEt, cis-Glyoxal-Cyclen, Mono-N-Benzyl-Cyclen, trans-N-Dibenyl-Cyclen, TriB0C-Cyclen, Mono-N-Benzyl-TACN, DiB0C-TACN, Cross-bridge-Cyclam (CB-Cyclam), (1 3)aneN4, TACN, TACN=3HC1, TACD, Mono-N-benzyl-TACD, DiB0C-TACD, 1,7-Dioxa-4,10-diazacyclododecane, C-Methyl-Ester-Cyclam, C-Carboxylic-Acid-Cyclam, trans-N-Dimethyl-Cyclam, TETRAM, TETAEt, TETAMEt2, TETAMMe2, TETAM, CPTA, CB-Cyclam derivatives, CB-TE2A, Methylamino-(13)aneN4, Bis-(13)aneN4, Oxo-(1 3)aneN4, Mono-N-Benzyl-(13)aneN4, TriB0C-(1 3)aneN4, TRITRAM, TRI3AEt, TRI3AtBu, Date Recue/Date Received 2021-07-27 TRITAM, TRITA, Mono-N-Benzyl-Cyclam, Foi _______________ inaldehyde-Cyc lam, c is -Glyoxal-Cyc lam, Dioxocyclam, Oxocyc lam, trans-N-D ibenzyl-Cyc lam, TriB0C-Cyclam, DOTP, DOTMA, TETA, DOTAM, DiAmSar, CB-Cyclam, CB-TE2A, NOTA, NOTAM, NH2-NODA-GA, Iodo-NODA-GA, NCS-MP-NODA, NH2-MPAA-NODA, NODA-GA(tBu)3, NODA-GA-NHS ester, Maleimide-NODA-GA, NO TA -NH S ester, Maleimide-NOTA, Propargyl-NOTA(tBu)2, p-NCS-benzyl-NODA-GA, NOTA(tBu)2, NCS-MP-NODA, NH2-MPAA-NODA, NH2-NODA-GA, Iodo-NODA-GA and TACN.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises one payload. In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises two or more payloads. For example, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more payloads. In a conjugate compound containing multiple payloads, each of the payloads may be identical to or different from each other. In some embodiments, at least two of the payloads are different from each other.
The term "targeting molecule" as used herein refers to any molecule or moiety capable of targeting the conjugate compound of the present application to a target site, a target tissue, a target organ, a target cell, or a target intracellular region. In some embodiments, the targeting molecule allows the conjugate compound of the present application to be distributed more in a target site, a target tissue, a target organ, a target cell or a target intracellular region compared with a non-target site, a non-target tissue, a non-target organ, a non-target cell or a non-target intracellular region, for example, at least 10% more, 20% more, 50% more, 80% more, 100%
more, 150% more, 200% more, 300% more, 400% more, 500% more, etc. In some embodiments, the targeting molecule allows a conjugate compound containing a targeting molecule, compared with a conjugate compound containing no targeting molecule, to be distributed more in a target site, a target tissue, a target organ, a target cell or a target intracellular region, for example, at least 10% more, 20% more, 50% more, 80% more, 100% more, 150% more, 200% more, 300% more, 400%
Date Recue/Date Received 2021-07-27 more, 500% more, etc. In some embodiments, the targeting molecule can trigger or promote a conjugate compound containing such targeting molecules to specifically bind to a target molecule, trigger or promote endocytosis of the conjugate compound by a target cell, and trigger or promote the conjugate compound to be enriched around a target cell and/or enter a target cell.
In some embodiments, the conjugate compound of the present application comprises at least two targeting molecules. In some embodiments, the two or more targeting molecules comprised in the conjugate compound of the present application are identical or different. In some embodiments, at least two targeting molecules of the two or more targeting molecules comprised in the conjugate compound of the present application are different. In some embodiments, the two or more targeting molecules comprised in the conjugate compound of the present application are all different from each other. In some embodiments, at least two targeting molecules of the two or more targeting molecules comprised in the conjugate compound of the present application can specifically bind to different cell surface proteins or markers.
In some embodiments, the two or more targeting molecules comprised in the conjugate compound of the present application can specifically bind to different cell surface proteins or markers.
In some embodiments, the conjugate compound of the present application comprises at least two targeting molecules, at least one of which is a synergistic molecule.
The teini "synergistic molecule" as used herein refers to any molecule or moiety that is capable of working synergistically with other targeting molecules comprised in the conjugate compound of the present application to better trigger or promote the conjugate compound to specifically bind to a target molecule, trigger or promote the endocytosis of the conjugate compound by a target cell, trigger or promote the conjugate compound to be enriched around a target cell and/or enter a target cell, and/or cause the conjugate compound, in a different manner, to specifically bind to a target cell and be maintained. In some embodiments, the synergistic molecule allows the conjugate compound of the present application to be distributed more in a target site, a target tissue, a target organ, a target cell or a Date Recue/Date Received 2021-07-27 target intracellular region compared with a non-target site, a non-target tissue, a non-target organ, a non-target cell or a non-target intracellular region, for example, at least 10% more, 20% more, 50% more, 80% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500% more, etc. In some embodiments, the synergistic molecule allows a conjugate compound containing a synergistic molecule, compared with a conjugate compound containing no synergistic molecule, to be distributed more in a target site, a target tissue, a target organ, a target cell or a target intracellular region, for example, at least 10% more, 20% more, 50%
more, 80% more, 100% more, 150% more, 200% more, 300% more, 400% more, 500%
more, etc. In some embodiments, the synergistic molecule allows a conjugate compound containing a synergistic molecule, compared with a conjugate compound containing no synergistic molecule, to have a higher activity on a target cell, for example, at least 10% higher, 20% higher, 50% higher, 80% higher, 100% higher, 150% higher, 200% higher, 300% higher, 400% higher, 500% higher, etc.
In some embodiments, the synergistic molecule of the present application is a cell-interacting molecule.
The teini "cell-interacting molecule" as used herein refers to a molecule that is capable of interacting with a cell surface material of a target cell to trigger or promote a conjugate compound containing such cell-interacting molecules to specifically bind to a cell, to trigger or promote endocytosis of the conjugate compound by a target cell, and/or to trigger or promote the conjugate compound to be enriched around a target cell and/or enter a target cell.
The cell-interacting molecule may be a small chemical molecule or a large biomolecule. In some embodiments, the cell-interacting molecule is a small molecule compound or a polypeptide. In some embodiments, the cell-interacting molecule is a small molecule compound, or a polypeptide comprising 2-50, 2-40, 2-30, 2-25, 2-22, 2-20, 2-18, 2-15, 2-12, 2-10, 2-8, 4-50, 5-50, 5-40, 5-30, 5-25, 5-22, 5-20, 5-18, 5-15, 5-12, 5-10, 6, 7, 8, or 9 amino acids.
In some embodiments, the targeting molecule is a ligand capable of binding to a cell surface receptor or other molecules. In some embodiments, at least one of the Date Recue/Date Received 2021-07-27 targeting molecules is a ligand capable of binding to a cell surface receptor or other molecules.
The ligands of the present application can include a variety of chemical or biological molecules, which can have a specific binding affinity to a selected target, wherein the selected target can be, for example, a cell surface receptor, a cell surface antigen, a cell, a tissue, an organ, etc. In some embodiments, the ligand can specifically bind to a protein or a marker expressed on the surface of a target cell. In some embodiments, the ligand of the present application binds to a cell surface protein or marker with an affinity of 1 0-6-10-11M (Ka value). In some embodiments, 113 the ligand of the present application binds to a cell surface protein or marker with an affinity of at least 10-7, at least 10-8 and at least 10-9 M (Ka value). In some embodiments, the ligand of the present application binds to a cell surface protein or marker with an affinity of less than 10-6, less than 10-7 and less than 10-8 M
(Ka value). In some embodiments, the ligand of the present application binds to a cell surface protein or marker with a certain affinity, wherein the certain affinity refers to the affinity of the ligand to a target cell surface protein or marker which is at least two, three, four, five, six, eight, ten, twenty, fifty, one hundred or more times higher than that to a non-target cell surface protein or marker. In some embodiments, the expression of the cell surface protein or marker of the present application in target cells (e.g. cancer cells) is significantly higher than that in nottnal cells.
The tettn "significantly" as used herein refers to statistically significant differences, or significant differences that can be recognized by a person skilled in the art.
In some embodiments, the expression level of the cell surface protein or marker of the present application in target cells (e.g. cancer cells) are 2 to 1,000,000 times higher than that in normal cells; for example, the expression level in target cells (e.g. cancer cells) are 2 to 10, 2 to 100, 2 to 1,000, 2 to 10,000, 2 to 100,000 or 2 to 1,000,000 (which can be equal to any value within the above numerical range, and the end values of this range included) times higher than that in nottnal cells. In some embodiments, the expression level of the cell surface receptor in target cells (e.g. cancer cells) is at least 10 times higher, or 100 times higher, or 1,000 times higher, or 10,000 times higher, or 100,000 times higher than that in nottnal cells. In Date Recue/Date Received 2021-07-27 some embodiments, compared with the level of the cell surface protein or marker on target cells (e.g. cancer cells), the level of the cell surface receptor on nonnal cells is reduced by at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. In some embodiments, the cell surface protein or marker described in the present application is undetectable in nonnal cells.
In some embodiments, the cell surface protein or marker of the present application is a cell surface receptor.
In some embodiments, the cell surface receptor of the present application is selected from the group consisting of a transferrin receptor (TFR), a low-density lipoprotein receptor (LDLR), a folate receptor (FR), a growth hoinione-inhibiting hormone receptor, a uric acid kinase receptor, a tumor necrosis factor receptor (TNFR), an integrin receptor (LFA-1), an SST-14 receptor (SSTR2), a GNRH
receptor (GNRHR), a TRPV6 and an integrin a receptor.
In some embodiments, the cell surface protein or marker of the present application is a cell surface antigen.
In some embodiments, the cell surface antigen of the present application is selected from the group consisting of a prostate-specific membrane antigen, a MUC1 mucin, an acute lymphoblast common antigen, a Thy-1 cell surface antigen, a Melan-A protein, a squamous cell carcinoma antigen, a galectin 3 and a human leukocyte antigen.
In some embodiments, the cell-interacting molecule of the present application can bind to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR
family, CD40, CD4OL, 0X40, OX4OL, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4, 5IGLEC15, NKG2A, NKG2D, SLAMF7, KIR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3 and CXCR4.
Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises a prostate-specific membrane antigen ligand moiety and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises a ligand moiety represented by formula (I) and a synergistic molecule moiety, wherein the synergistic molecule moiety binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.
In yet some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application comprises P10 and a synergistic molecule moiety, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
In some embodiments, one of the synergistic molecules in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is an endocytosis molecule moiety capable of mediating endocytosis. The term "endocytosis" as used herein means that the conjugate compound or the pharmaceutically acceptable salt thereof interacts with a target cell and then is capable of mediating its own endocytosis, internalization or uptake by the target cell.
The teini "endocytosis molecule" as used herein refers to a molecule that interacts with a target cell and then is capable of mediating the endocytosis, internalization, or uptake of the conjugate compound or the pharmaceutically acceptable salt thereof of the present application by the target cell.
In some embodiments, the endocytosis molecule is selected from the group consisting of a folate and an analog thereof, a peptide capable of mediating endocytosis, and a cell-penetrating peptide.
In some embodiments, the endocytosis molecule of the present application is a folate or an analog thereof Folate is beneficial for forming a chemical bond with other groups due to its small molecule weight, non-immunogenicity, and good stability. Folate can be Date Recue/Date Received 2021-07-27 associated with a folate receptor expressed on a cell surface with a high affinity to mediate a cellular uptake of the folate. Although expressed at a very low level in most normal cells, a folate receptor is expressed at a high level in numerous cancer cells to meet the high folate demand of rapidly dividing cells under a low folate condition (see Kelemen LE, Int J Cancer, 2006; 119:243-50; Kane MA, et al., J
Clin Invest. 1988; 81: 1398-406; Matsue H, et al., Proc Natl Acad Sci USA.
1992;
89: 6006-9; Zhao R, et al., Annu Rev Nutr. 2011; 31: 177-201). Folate is capable of specifically binding to a folate receptor on a cell surface, and is also capable of mediating endocytosis of a conjugate compound or a pharmaceutically acceptable salt thereof into target cells.
In some embodiments, the analog of folate is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methyl enetetrahydro folate .
In some embodiments, the endocytosis molecule is a peptide capable of mediating endocytosis.
In some embodiments, the peptide capable of mediating endocytosis comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 16, SEQ
ID NO: 17, SEQ ID NO: 18 and Arg-Gly-Asp (named as RGD), and homologous peptides having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% amino acid sequence homology to any of SEQ ID NOs:
16-18, wherein the homologous peptides are functional equivalents of the peptides as shown in SEQ ID NOs: 16-18, respectively.
In some embodiments, the peptide capable of mediating endocytosis as described in the present application has a conservative substitution of an amino acid at only one amino acid site compared to the sequences of SEQ ID NOs: 16-20 and RGD. In some embodiments, the peptide capable of mediating endocytosis as described in the present application has a conservative substitution of an amino acid at 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid sites compared to the sequences of SEQ ID
NOs: 16-20.
Date Recue/Date Received 2021-07-27 Under the premise of not affecting its biological activity, the peptide capable of mediating endocytosis as described in the present application may also contain non-naturally occurring amino acids, including, for example, I3-fluoroalanine, 1-methyl-histidine, y-methylene-glutamic acid, a-methyl-leucine, 4,5 -dehydro-lysine, hydroxypro line, 3 -fluoro-phenylalanine, 3-amino-tyrosine, 4-methyl-tryptophan, and the like.
The percentage of homology can be determined by various well-known methods in the art. For example, the comparison of sequences can be achieved by the following publically available tools: BLASTp software (available from the website of National Center for Biotechnology Infoitnation (NCBI):
http://blast.ncbi.nlm.nih.gov/Blast.cgi; also see: Altschul S.F. et al., J.
Mol. Biol., 215:403-410 (1990); Stephen F. et al, Nucleic Acids Res., 25:3389-3402 (1997)), ClustalW2 (available from the website of European Bioinfounatics Institute:
http://www.ebi.ac.uk/Tools/msa/clustalw2/; also see: Higgins D. G. et al., Methods in Enzymology, 266:383-402 (1996); Larkin M.A. et al., Bioinformatics (Oxford, England), 23(21): 2947-8 (2007)), and Tcoffee (available from the website of Sweden Bioinformatics Institute; also see: Poirot 0. et al., Nucleic Acids Res., 31(13): 3503-6 (2003); Notredame C. et al., J. Mol. Boil., 302(1): 205-17 (2000)). If the alignment of sequences is performed using a software, the default parameters available in the software may be used, or otherwise the parameters may be customized to suit the alignment purpose. All of these are within the scope of knowledge of a person skilled in the art.
The term "functional equivalent" as used herein refers to a derivative peptide that retains a biological activity that is substantially similar to that of the original peptide sequence that the derivative peptide derives from. The functional equivalent may be a natural derivative or is prepared synthetically. Exemplary functional equivalents include amino acid sequences having substitutions, deletions, or additions of one or more amino acids, provided that the biological activity of a peptide is maintained. The amino acid used for substitution desirably has chemico-physical properties similar to the amino acid to be substituted.
Desirable Date Recue/Date Received 2021-07-27 similar chemico-physical properties include, similarities in charges, bulkiness, hydrophobicity, hydrophilicity, and the like.
In some embodiments, the functional equivalents include a conservative substitution of an amino acid residue. The conservative substitution of an amino acid residue refers to a substitution between amino acids with similar properties, for example, a substitution between polar amino acids (such as a substitution between glutamine and asparagine), a substitution between hydrophobic amino acids (such as a substitution among leucine, isoleucine, methionine and valine), a substitution between amino acids with identical charges (such as a substitution among arginine, lysine and histidine, or a substitution between glutamic acid and aspartic acid), etc.
In some embodiments, the endocytosis molecule is a cell-penetrating peptide.
Cell-penetrating peptides (CPPs), also known as protein transduction domains (PTDs), are short peptides (generally less than 40 amino acids), with the ability to gain access to the interior of cells in a receptor-independent manner. The cell-penetrating peptides, when conjugated with payloads, are capable of mediating the transmembrane transport of the payloads and have a protein transduction activity.
In some embodiments, the cell-penetrating peptide described in the present application is selected from the group consisting of a tumor-homing peptide, a mitochondrial penetrating peptide, an activatable cell-penetrating peptide, and an antibacterial peptide. In some embodiments, the cell-penetrating peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 19 (RRRRRRRRR, named as R9) and SEQ ID NO: 20 (GRKKRRQRRRPPQ, which is a Tat peptide, i.e. a cell-penetrating peptide of the HIV transactivator of transcription protein).
In some embodiments, one targeting molecule in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is a prostate-specific membrane antigen ligand moiety.
The term "prostate-specific membrane antigen" as used herein refers to a type II transmembrane glycoprotein that exists in the membrane of prostate epithelial cells and consists of 750 amino acids, comprising 19 amino acids in the intracellular region, 24 amino acids in the transmembrane region and 707 amino acids in the Date Recue/Date Received 2021-07-27 extracellular region. The prostate-specific membrane antigen is expressed in normal prostate epithelial cells, but is expressed at a much higher level in prostate cancer cells. Compared with the traditional prostate-specific antigen used for clinical detection, the prostate-specific membrane antigen is a more sensitive and specific prostate cancer tumor marker, and especially, it is highly expressed in both hormone-refractory prostate cancer and prostate cancer metastatic lesions, and has a high sensitivity and specificity in distinguishing prostate cancer from other types of malignant tumors. Moreover, in a variety of nonprostate solid tumors (such as lung cancer, bladder cancer, gastric cancer, pancreatic cancer, kidney cancer and colorectal cancer), the prostate-specific membrane antigen is also highly and specifically expressed on tumor vascular endothelial cells.
The tem" "prostate-specific membrane antigen ligand" as used herein refers to an antibody, an aptamer and a small molecule that is capable of specifically recognizing and binding to a prostate-specific membrane antigen. The prostate-specific membrane antigen ligands of the present application include the prostate-specific membrane antigen ligands that are already in existence or will be produced later, as well as fragments of the aforementioned ligands, as long as these fragments still retain the ability to bind to a prostate-specific membrane antigen.
Antibody ligands are the most common prostate-specific membrane antigen ligands, which include, but are not limited to monoclonal antibodies J591, J533, J415 and E99 (for example, see Liu H, Rajasekaran AK, Moy P et al. Constitutive and antibody-induced internalization of prostate-specific memberane antigen [J].
Cancer Res, 1998, 58 (18): 4055-4060). The aptamer is a single-stranded DNA or RNA that is obtained through technical screening by an exponential enrichment ligand system and can bind to prostate-specific membrane antigens with a high affinity and a high specificity. Such prostate-specific membrane antigen ligands include, but are not limited to an xPSM-A10 aptamer and a derivative thereof and an xPSM-A9 aptamer and a derivative thereof (for example, see Lupoid SE et al., Identification and Characterization of nuclease-stabilized RNA molecules that bind human prostate cancer cells via the prostate-specific membrane antigen, Cncer Res, 2002, 62(14):4029-4033). Compared with antibody ligands and aptamer ligands, the Date Recue/Date Received 2021-07-27 prostate-specific membrane antigen small molecule ligands have the advantages of small molecular weight, high pettneability, low immunogenicity, and ease of synthesis, and include but are not limited to glutamine urea small molecule ligands and phosphoramidate small molecule ligands.
In some embodiments, the prostate-specific membrane antigen small mole cule ligands of the present application can be selected from the group consist ing of 2-[[methylhydroxyphosphinyl]methyl]glutaric acid; 2-[[ethylhydroxyphos phinyl]methyl]glutaric acid; 2-[[propylhydroxyphosphinyl]methyl]glutaric acid;
2-[[butylhydroxyphosphinyl]methyl]glutaric acid; 2-[[cyclohexylhydroxyphosphin yl]methyl] glutaric acid; 2- [[phenylhydroxyphosphinyl]methyl] glutaric acid;
2- [ [2 -(tetrahydrofuranyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[(2-tetrahydropyra nyl)hydroxyphosphinyl]methyl] glutaric acid; 2 - [ [((4-pyridyl)methyl)hydroxyphos phinyl]methyl] glutaric acid; 2 - [ [((2-pyridyl)methyl)hydroxyphosphinyl]methyl] gl utaric acid; 2-[[(phenylmethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[((2-p henylethyl)methyl)hydroxyphosphinyl]methyl] glutaric acid; 2- [ [((3 -phenylpropyl) methyl)hydroxyphosphinyl]methyl] glutaric acid; 2- [ [((3 -phenylbutyl)methyl)hydr oxyphosphinyl]methyl]glutaric acid; 2-[[((2-phenylbutyl)methyl)hydroxyphosphin yl]methyl] glutaric acid; 2- [ [(4-phenylbutyl)hydroxypho sphinyl] methyl]
glutaric ac id; and 2-[[(aminomethyl)hydroxyphosphinyl]methyl]glutaric acid; 2-[[methyl h ydroxyphosphinyl] oxy] glutaric acid; 2- [ [ethyl hydroxyphosphinyl] oxy]
glutaric a cid; 2-[[propyl hydroxyphosphinyl]oxy]glutaric acid; 2-[[butyl hydroxyphosphin yl] oxy] glutaric acid; 2- [ [phenyl hydroxypho sphinyl] oxy] glutaric acid; 2-[ [((4-py ridyl)methyl)hydroxyphosphinyl] oxy] glutaric acid; 2- [ [((2-pyridyl)methyl)hydrox yphosphinyl] oxy] glutaric acid; 2- [ [(phenylmethyl)hydroxyphosphinyl] oxy]
glutaric acid; and 2[[((2-phenylethyl)methyl)hydroxyphosphinyl]oxy]glutaric acid; 2-[[(n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-methyl)carbamo yl]methyl]glutaric acid; 2-[[(n-butyl-n-hydroxyl)carbamoyl]methyl]glutaric acid;
2-[[(n-benzyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-phen yl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-2-phenylethyl)carbamoyl]m ethyl]glutaric acid; 2-[[(n-ethyl-n-hydroxyl)carbamoyl]methyl]glutaric acid; 2-[ [(n-hydroxyl-n-propyl)carbamoyl]methyl] glutaric acid; 2- [ [(n-hydroxyl-n-3 -phen Date Recue/Date Received 2021-07-27 ylpropyl)carbamoyl]methyl]glutaric acid; 2-[[(n-hydroxyl-n-4-pyridyl)carbamoyl]
methyl]glutaric acid; 2-[[(n-hydroxyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl (methyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(benzyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(phenyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(2-pheny lethyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(ethyl)amide]methyl]glutaric aci d; 2-[[n-hydroxyl(propyl)amide]methyl]glutaric acid; 2-[[n-hydroxyl(3-phenylpro pyl)amide]methyl]glutaric acid; and 2-[[n-hydroxyl(4-pyridyl)amide]methyl]gluta ric acid; 2-[(thionyl)methyl]glutaric acid; 2-[(methylthionyl)methyl]glutaric acid;
2-[(ethylthionyl)methyl]glutaric acid; 2-[(propylthionyl)methyl]glutaric acid;
[(butylthionyl)methyl]glutaric acid; 2-[(phenylthionyl]methyl]glutaric acid; 2-[[(2-phenylethyl)thionyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)thionyl]methyl]
glutaric acid; 2-[[(4-pyridyl)thionyl]methyl]glutaric acid; 2-[(benzylthionyl)meth yl]glutaric acid; 2-[(sulfonyl)methyl]glutaric acid; 2-[(methylsulfonyl)methyl]glu taric acid; 2-[(ethylsulfonyl)methyl]glutaric acid; 2-[(propylsulfonyl)methyl]gluta ric acid; 2-[(butylsulfonyl)methyl]glutaric acid; 2-[(phenylsulfonyl]methyl]glutar ic acid; 2-[[(2-phenylethyl)sulfonyl]methyl]glutaric acid; 2-[[(3-phenylpropyl)sul fonyl]methyl]glutaric acid; 2-[[(4-pyridyl)sulfonyl]methyl]glutaric acid; 2-[(benz ylsulfonyl)methyl]glutaric acid; 2-[(sulfoximinyl)methyl]glutaric acid; 2-[(methy lsulfoximinyl)methyl]glutaric acid; 2-[(ethylsulfoximinyl)methyl]glutaric acid; 2-[(propylsulfoximinyl)methyl]glutaric acid; 2-[(butylsulfoximinyl)methyl]glutaric acid; 2-[(phenylsulfoximinyl]methyl]glutaric acid; 2-[[(2-phenylethyl)sulfoximiny l]methyl]glutaric acid; 2-[[(3-phenylpropyl)sulfoximinyl]methyl]glutaric acid;
[[(4-pyridyl)sulfoximinyl]methyl]glutaric acid; and 2-[(benzylsulfoximinyl)methy l]glutaric acid; n- [methyl hydroxyphosphinyl]glutamic acid; n- [ethyl hydroxyph osphinyl]glutamic acid; n-[propyl hydroxyphosphinyl]glutamic acid; n4butyl hy droxyphosphinyl]glutamic acid; n- [phenyl hydroxyphosphinyl]glutamic acid; n-[(phenylmethyl)hydroxyphosphinyl]glutamic acid; n-[((2-phenylethyl)methyl)hydr oxyphosphinyl]glutamic acid; and N-methyl-N4phenylhydroxyphosphinyl]gluta mic acid. The prostate-specific membrane antigen ligands of the present appli cation also include all prostate-specific membrane antigen small molecule liga nds disclosed in PCT applications WO 2010/108125 and WO 2006/093991, t Date Recue/Date Received 2021-07-27 he above-mentioned two patent applications are incorporated herein in their e ntirety.
In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application is a glutaric acid derivative. In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application is an aminocarbonyl derivative of glutaric acid.
In some embodiments, the prostate-specific membrane antigen small molecule ligand of the present application has the following structure:
H0( (s) NAN1/4 H H
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO
(s) NANiNC., H H H
0 .
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO 00 0, _OH
HO (S) NANNAN 0 H H H H
In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
HO 0 0 , -OH
H 0 N AN : NA N 0 (s) (S) 0 H H H H
N (S) H
0 \
H 0 .
Date Recue/Date Received 2021-07-27 In some embodiments, the prostate-specific membrane antigen ligand comprised in the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
_ HO
(S) NANNAN 0 i_i 0 H H H H
0 j(isj,(sA
H i H
0 \
HO
In some embodiments, one targeting molecule in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is a ligand moiety represented by formula (I):
> _____________________ (D-Phe)-Cys-Tyr-(D-Trp)-Lys-Thr-Cys-Thr A¨
/
(I), or a ligand moiety having at least 70%, at least 80%, at least 85% or at least 90% amino acid sequence homology thereto or having at most 3, 2 or 1 amino acid substitutions (for example, conservative substitutions) therewith.
In some embodiments, the one targeting molecule in the conjugate compound or the pharmaceutically acceptable salt thereof of the present application is P10 or a ligand moiety having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92% or at least 93% amino acid sequence homology thereto or having at most 3, 2 or 1 amino acid substitutions (for example, conservative substitutions) therewith.
The term "P10" as used herein refers to a peptide having an amino acid sequence Cys-Lys-Glu-Phe-Leu-His-Pro-Ser-Lys-Val-Asp-Leu-Pro-Arg.
In some embodiments, the conjugate compound of the present application has targeting molecules selected from the group consisting of (1) a folate ligand and a prostate-specific membrane antigen ligand; (2) a TRPV6 ligand and a Date Recue/Date Received 2021-07-27 prostate-specific membrane antigen ligand; (3) a GNRHR ligand and a prostate-specific membrane antigen ligand; (4) an SSTR2 ligand and a prostate-specific membrane antigen ligand; (5) a folate ligand and an SSTR2 ligand;
or (6) a TRPV6 ligand and a folate ligand.
In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a folate or an analog thereof and a prostate-specific membrane antigen ligand moiety, respectively. Without wishing to be limited by the theory, a specific folate or an analog thereof and a prostate-specific membrane antigen ligand moiety that have a better stability than the ligand combinations in the prior art are selected.
In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a synergistic molecule moiety and a ligand moiety represented by formula (I), respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules in the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a folate or an analog thereof and a ligand moiety represented by formula (I), respectively.
In some embodiments, the two targeting molecules of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a synergistic molecule moiety and P10, respectively. In some embodiments, the synergistic molecule is capable of mediating endocytosis. In some embodiments, the two targeting molecules of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application are a folate or an analog thereof and P10, respectively.
Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound provided in the present application only comprises a single payload conjugated with the two targeting molecules. In some embodiments, the conjugate compound provided in the present application comprises multiple payloads conjugated with the two targeting molecules.
The teini "conjugated" as used herein refer to the linking through a covalent bond of two chemical groups, either directly founing a covalent bond between the two chemical groups, or indirectly linking the two chemical groups via a linker.
In some embodiments, the conjugate compound or the phannaceutically acceptable salt thereof comprises a payload(s) (for example, 1 payload) and two targeting molecules, wherein the payload is directly covalently linked to at least one of the targeting molecules. In some embodiments, the payload is directly covalently linked to the two targeting molecules.
In some embodiments, the conjugate compound or the phannaceutically acceptable salt thereof comprises a payload(s) (for example, 1 payload) and two targeting molecules, wherein the payload is covalently linked to at least one of the targeting molecules via a linker. In some embodiments, the payload is covalently linked to the two targeting molecules via a linker.
The teini "linker" as used herein refers to a molecule or moiety that covalently links a payload to a targeting molecule. The linkers include a functional group for linking a payload to at least one targeting molecule. In some embodiments, the functional group may comprise two reactive moieties, one for linking to a payload and the other for linking to a targeting molecule. In some embodiments, the functional groups are different from each other. In some embodiments, the functional groups include a group containing a thiol-reacting moiety and an amine-reacting moiety. In some embodiments, the functional groups are identical to each other. In some embodiments, the functional groups are maleimide groups.
In some embodiments, the linker contains an amino acid. In some embodiments, the carboxylic acid in the amino acid contained in the linker is amidated. In some embodiments, the linker contains a short chain polyethylene glycol (for example, comprising 2-10, 2-8, 3-8, 4-8, 4-7, 4-6, or 5 repeating units).
Date Recue/Date Received 2021-07-27 In some embodiments, the linker of the present application is a multivalent linker capable of binding to at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and at least one targeting molecule. The payloads bound to the multivalent linker may be identical or different, and the targeting molecules bound to the multivalent linker may be identical or different.
In one aspect, the linker shall be sufficiently stable to avoid from unintended release of payloads during a blood circulation to increase an effective amount of payloads delivered to target cells or tissues and avoid toxicity. In another aspect, the linker shall be capable of releasing the payloads around or within target cells to efficiently kill target cells or block functions of target cells. In some embodiments, the linker comprises at least one cleavable functional group. Preferably, the cleavable functional group is sufficiently stable outside a target cell, but upon entry into the target cell, is cleaved to release a payload(s). In some embodiments, the cleavable functional group is cleaved at least 10, 20, 30, 50, 100 times or more efficiently in target cells than in the blood or serum.
The cleavable linker may be cleaved by a hydrolysis, an enzymatic reaction, or a reduction reaction, or by a pH change. In some embodiments, the linker is cleavable under a certain physiological environment (for example, under an appropriate pH environment). In some embodiments, the linker is cleavable in an acidic environment with a pH of about 6.5 or lower, or by reagents such as enzymes.
In some embodiments, the linker is susceptible to cleavage agents, for example, pH, redox potential or the presence of degradative molecules.
In some embodiments, the linker is non-cleavable. Non-cleavable linkers as used herein refer to linkers which remain basically intact during intracellular metabolism.
In some embodiments, the linker is a peptide linker consisting of a straight or branched chain amino acids linked by peptide bonds. In some embodiments, the peptide linker is cleavable by a protease that is highly or specifically expressed around or in target cells, for example, cathepsin B in the lysosome or endosome.
The peptide linker as used herein can be of varying lengths. Typically, the peptide linker of the present application is from 1 to 50 amino acids in length. In some Date Recue/Date Received 2021-07-27 embodiments, the peptide liker is from 1 to 45, from 1 to 40, from 1 to 35, from 1 to 30, from 1 to 25, from 1 to 20, from 1 to 15, from 1 to 10, from 1 to 9, from 1 to 8, from 1 to 7, from 1 to 6, from 1 to 5, from 1 to 4, from 1 to 3, from 1 to 2 or 1 amino acids in length. In some embodiments, the peptide liker is from 2 to 45, from 2 to 40, from 2 to 35, from 2 to 30, from 2 to 25, from 2 to 20, from 2 to 15, from 2 to 10, from 2 to 9, from 2 to 8, from 2 to 7, from 2 to 6, from 2 to 5, from 2 to 4, from 2 to 3 or 2 amino acids in length. The number of amino acids of the peptide linker as described in the present application can be equal to any integer value within the above numerical range, including the end values of this range. In some embodiments, the peptide linker is preferred to be 1, 2, 3, 4 or 5 amino acids in length. In some embodiments, the peptide linker is cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino acids is amidated.
In some embodiments, the linker is a disulfide linker containing a disulfide bond. The disulfide bond may be cleaved under an intracellular reductive environment, while remains stable in a circular system. The disulfide linker of the present application may be DSDM, DMDS, MDS, or NDMDS. The structures of these disulfide linkers are shown in Table 1 below.
Table 1: Structures of DSDM, DMDS, MDS and NDMDS
Name Structure DSDM NcS,s0.1\j I
DMDS N S -S7).L ,11-Date Recue/Date Received 2021-07-27 MDS I
,)-L _II?
I H
NS'70-11\i'l\r el 0 0.---.................---).(0, In some embodiments, the linker is a pH-dependent linker. The pH-dependent linker as described in the present application is cleavable under a certain pH
environment. In some embodiments, the pH-dependent linker may be stable under an alkaline condition, while cleavable under an acidic condition, for example, under a pH value of 6.5 or lower. In some embodiments, the pH-dependent linker is a cis-aconitic anhydride.
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof is NH
H H
N,(sA N 4 40 H
0 0 Oy\
0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
Date Recue/Date Received 2021-07-27 NH
\N(R) s 0 NtLz HN (S) 0 N 110 Oy\
0 0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
(S) N
- (R) 0 H 0 =
01r\
(S) or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
J,r1\1 N (S) = H
0 0 Oy\
HOs, Date Recue/Date Received 2021-07-27 or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
N-11\j'LSAN (S) N
= H
110 Olf\
HO
(s) 0 or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
NH
N N (s) N
- H
(S) 0 a 0 N H2 0 (S) HN N,CSAN
= H H
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
Date Recue/Date Received 2021-07-27 In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
4N -rN 1,c!
0 (:) ) H H
NI-r-rNi 0()) le 10 0 0 0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
--2r0 (30 ) H H
N 10r N 023) 0 , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
Date Recue/Date Received 2021-07-27 s I
) 3.L0 110 0 0 H H
HN
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
/NH N,N
Ari\Aõ..N 0 ) H
N ) Ny,,cy...---)i,N 0C).õõ=-i H
HN
H2N , or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
Date Recue/Date Received 2021-07-27 /NH N,N
0 H2N 00 0) 5,?,,H 0 (S) N
HN
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the conjugate compound or the pharmaceutically acceptable salt thereof has the following structure:
N(S = " \/\ Nss (s) H N
or a combination of the above-mentioned structure and a peptide linker (for example, binding to a targeting molecule through a peptide linker containing 1-amino acids).
In some embodiments, the linker of the present application may comprise any one of or a combination of the linkers as described above.
In some embodiments, the payload is conjugated with a first targeting molecule directly or indirectly, and the first targeting molecule is conjugated with a second targeting molecule directly or indirectly. In some embodiments, the payload is conjugated with each of the first and the second targeting molecule directly. In some embodiments, the payload is conjugated with each of the first and the second targeting molecule indirectly. In some embodiments, the payload is conjugated with the first targeting molecule indirectly, e.g. via a linker, and the first targeting molecule is conjugated with the second targeting molecule directly or indirectly. In some embodiments, the payload is conjugated with the first targeting molecule via a Date Recue/Date Received 2021-07-27 first linker, and the payload is conjugated with the second targeting molecule via a second linker. In some embodiments, the linker is a multivalent linker that binds to at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload and two targeting molecules.
In some embodiments, the two targeting molecules are linked to each other via a spacer. In some embodiments, the spacer is cleavable by a protease that is specifically expressed in target cells or to be expressed in target cells.
Such proteases include, for example, the proteases as listed in Table 2 below. In some embodiments, the spacer comprises the amino acid sequence selected from any one of the amino acid sequences as listed in Table 2 below.
Table 2: List of Enzymatically Cleavable Sequences Amino acid sequence of Protease SEQ ID NO.
recognition site Cathepsin B RR -Legumain ASN -Matripase KSRAEDE SEQ ID NO: 1 MMP-2 PLGLAG SEQ ID NO: 2 Prostate-specific antigen SSLY SEQ ID NO: 3 Stromelysin-3 AAA -TMPRSS2 LLRSLIG SEQ ID NO: 4 Urokinase-type SSR -plasminogen activator Activated protein C LVKR SEQ ID NO: 5 Factor Ixa LVVR SEQ ID NO: 6 Factor VIIa QLTR SEQ ID NO: 7 Factor Xa LEGR SEQ ID NO: 8 Thrombin PR -Calpain-a PLFAEP SEQ ID NO: 9 Calpain-2 GLGSEP SEQ ID NO: 10 Enteropeptidase DDDDK SEQ ID NO: 11 MMP-8 GPSG SEQ ID NO: 12 Cathepsin L PLG -Date Recue/Date Received 2021-07-27 Proprotein convertase 5 RSKR SEQ ID
NO: 13 Calpain-3 VGVF SEQ ID
NO: 14 The Wails "cleavable" or "cleaved" as used herein refer to a metabolic process or reaction process on the conjugate compound provided in the present application, whereby a linker between a payload and a targeting molecule, or a spacer between targeting molecules are broken to release free payload or targeting molecule.
The linker and spacer is either cleaved by a protease or cleaved under a certain physiological environment, e.g. a pH environment.
In some embodiments, the conjugate compound has a structure of foimula I, II, III, or IV shown as follows, wherein n, m, p and q are independently 0 or 1, which represent that the linker and spacer are present or absent independently. The "molecule" in the following formula is an abbreviation for "targeting molecule".
Payload Linker )0 __ Molecule 14.J ( Spacer )03 ¨.Molecule 2 ( Formula i) ,N10 ec ule 1 4.0v Payload (Li nicer " Molecule Formula II) Date Recue/Date Received 2021-07-27 Molecule 1 4-Payload I, Multivalent linker Pa-y-1-o- ad 2 Molecule 2 ( Formula 0) Molecule I +I
.09**
Multivalent Payload linker Molecule 2 ( Formula M
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises at least one (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) payload as provided in the present application, two targeting molecules as provided in the present application and optionally a linker or spacer as provided in the present application. In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises one payload as provided in the present application, one ligand that specifically binds to a cell surface protein or marker as provided in the present application, one synergistic molecule as provided in the present application, and a linker or spacer as provided in the present application.
In some embodiments, the conjugate compound has a structure of formula V, VI, VII, or VIII shown as follows, wherein n, m, p, q and s are independently 0 or 1, which represent that the linker, multivalent linker and spacer are present or absent independently.
Date Recue/Date Received 2021-07-27 Payload _______________ ( Linker )n Ligand __ ( Spacer )m Synergistic molecule ( Formula V) Synergistic Payload ( Linker ) ¨ _________________ molecule ( Spacer )mn Ligand ( Formula VI) _________________________________________________ Ligand Payload .ver 2.4 _______________________________________ Synergistic molecule ( Formula VII) Ligand C
Payload __________________________________ Multivalent linker ¨^ Synergistic molecule Formula VIII) In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively, for example, CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SMO9 and CB-20R.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises one or more payloads and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a ligand moiety represented by formula (I), respectively, for example, CB-18G, CB-1820 and CR19426.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application comprises one payload Date Recue/Date Received 2021-07-27 and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof, such as CB-10S, CR19425 and CB-50S.
In some embodiments, the conjugate compound of the present application is selected from the group consisting of the following compounds: CB-20B, CB-20BK, CB-60S, CB-60SK, CB-20C, CB-1020, CB-1320, CB-1820, CR19428, 20R-SM09, CB-20R, CB-18G, CR19426, CB-10S, CR19425 and CB-50S (the specific structure of each conjugate compound is shown in Fig. 1). In some embodiments, the conjugate compound of the present application is fottned by linking a linker-drug moiety to a ligand moiety via a covalent bond. The linker-drug moiety of the present application comprises a payload and a linker, and the ligand moiety of the present application comprises two targeting molecules and an optional spacer or linker, wherein the two moieties foal" the conjugate compound of the present application by reacting and fottning a covalent bond, and the covalent bond can be fottned between the linker in the linker-drug moiety and the ligand molecule in the ligand moiety, or can be formed between the linker in the linker-drug moiety and the spacer or linker in the ligand moiety.
The conjugate compound of the present application, CB-20B, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety 20B-SMO9 via a covalent bond. The conjugate compound of the present application, CB-20BK, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety 20BK-SMO9 via a covalent bond. The conjugate compound of the present application, CB-60S, is fottned by linking a linker-drug moiety LT2000C to a ligand moiety 60S-SM09 via a covalent bond. The conjugate compound of the present application, CB-60SK, is fottned by linking a linker-drug moiety LT2000C to a ligand moiety 60SK-SM09 via a covalent bond. The conjugate compound of the present application, CB-20C, is fottned by linking a linker-drug moiety LD1001 to a ligand moiety 20BK-SM09 via a covalent bond. The conjugate compound of the present application, CB-1020, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety 1020BK-SMO9 via a covalent bond. The conjugate compound of the present application, CB-1320, is fottned by linking a linker-drug moiety LT1002 to a ligand moiety Date Recue/Date Received 2021-07-27 1320BK-SMO9 via a covalent bond. The conjugate compound of the present application, CB-1820, is fonned by linking a linker-drug moiety LT1002 to a ligand moiety 1820BK-SMO9 via a covalent bond. The conjugate compound of the present application, CR19428, is fonned by linking a linker-drug moiety CR19423 to a ligand moiety 20BK-SMO9 via a covalent bond. The conjugate compound of the present application. CB-20R. is fonned by complexing 20R-SMO9 with a radionuclide ion M. The conjugate compound of the present application, CB-18G, is fonned by linking a linker-drug moiety LT1002 to a ligand moiety 18G-SMO9 via a covalent bond. The conjugate compound of the present application, CR19426, is fonned by linking a linker-drug moiety CR19423 to a ligand moiety 18G-SMO9 via a covalent bond. The conjugate compound of the present application, CB-10S, is fonned by linking a linker-drug moiety LT1000 to a ligand moiety CBSMO9 via a covalent bond. The conjugate compound of the present application, CR19425, is fonned by linking a linker-drug moiety CR19423 to a ligand moiety CBSMO9 via a covalent bond. The conjugate compound of the present application, CB-50S, is fonned by linking a linker-drug moiety LT1000N3 to a ligand moiety 50S-SMO9 via a covalent bond. Each structure is shown in Table 3 below.
Table 3: Structures of Linker-drug Moiety and Ligand Moiety Abbreviation Structure NH
LD1001 N¨
H
N
I N
OH
Hµ!`i \
0,0 N
(s) 0 Date Recue/Date Received 2021-07-27 Abbreviation Structure OH
H H
cN ,ro.iN
NI \
() 0 y N
(s) 0 ¨) 0 0) \ / 0 0 o NH
LT1002 H ii N Ali ? I V ? H
' H
R OH
0 y (e) N (S) (R) (s) 010 0 õA., 0 ,0 0 HO
\ /
I
/
A (s) LT2000C H H ii N, 0 iNlrOr - N
H
(K--) 0 0¨? NH2 \ /
HN
HO Hr_N 9 H \- 0 = H H
s) Ni,,,,,, s)N
. s) N .
(s) N lifiRN (s) N yrsl'N IfISI'N
Nlr'NH
H H H H H H H
(R) 0 ao N NH
N)?r N 'N 0 _, I
H
Date Recue/Date Received 2021-07-27 Abbreviation Structure 18G-SMO9 o Ho it 0 FIN 0 J\
HN (Rs 0,$) 'S
0, ,NH2 HN H 0 0 H r-o-,,..$) (R) ki (si s) ki 0 0) NH -HN60 0 õ H H
---7" 0 HN (s) H) ;--- 0 0 N
,..) .(R) HO HO (S) /
(R) õ SH
OOH
/ /
NH
N"..----"Thr H
N)-,NN 0 H2N Nle H
20B-SMO9 HO 0 (:),OH
Vli N 0 H 0 0 N,@-L
N (s) NH
H
HO
(R) SH
OOH
H
N NH
0 N(-r H
NJ,NN 0 0=,N H2 H
Date Recue/Date Received 2021-07-27 Abbreviation Structure HO 0 0, _OH
HO (s) N)N(N)-1\1 r I
H i H
HO
(R) SH
0 0, _OH
r /H
NiNNH
1\1)NN
H2N NIµl H
D 0 0 0,0H 0 HO
(s) N N N N
H H H H 0 ,$) CI?
(s) 'LNH2 H : H
0 - 0 -) \O
HO
COOH
,--4 0 /----..---\
0 la [\ilfil HN-Q - ) 1\1)?rN 0 N N-\
I H c-NJ b 0H
H
Hooc) NH HO X
HN N0 0 ¨( 0 , H \ 0 ---q ro o HrH, HolomAvli,i,,N)sN-1.0N S) NINA(c.)s)NN s)N Ir r., (s) N N'NH
0 H 0 H 0 H 0 H 0 H 0 0 \, 0, ,OH /
NH
0 el 11 ic) r-N
N N
1 __ H
Date Recue/Date Received 2021-07-27 Abbreviation Structure 60S-SMO9 HO 0 0, _OH
HO (S) N -LINI(.N)-NI 0 H
H H H H
N (s) . NH
H 0 \c) HO
(s) 0,0H
H
Nriµl-,QSNIH
H
N, NN 0 H2N NIµl H
yEiof 0 (:),OH HO
HO .
(s)NA NIN1j-LN 0 9 H H H H
0 N.(sP kil N (s) H = H
0 -\ 0 HO
(s) \
H
N _iN,a NH
H
NJ-,NN 0 H
H
HN N
HO H
0 ---( 0 H 0 H ,0 0H2OH,0 HONNN NN.k....--..,o, 01 1 H HH (:) NH2 HNC) (S) HO 0 0 0 ,OH HO
HO A
(s)N N NA N 0 LSHH 0 H H H H
N (s) N (s) H
0 -\ 0 HO
Date Recue/Date Received 2021-07-27 Abbreviation Structure H
HN
0 0 y H2NN s) N11.,4 .õ
,--,IH
OH H
, 0 i 0 HN N 0 O(R), s) Q H
11 li-rsiN ysiN (2) (:) o " o "
o) ¨
NH A HN
OH
(s) SH
;JO 0 0OH HO
p H H H H
0 N p NkSAN kl1)-LN
H i H H
HO
1820BK-SMO9 o 41 ',, NH
,t) HO do 0 HN 0 ,, HN-3\
os) ss 0 NH2 HN 0 H r-o-õ....s: (,õi-I, ? (-S) N 0 (21 HN r NH i H H
3- 0 0 (157 0 HN (s) (5/4 - ' 0 'OH , j ) 0 ki ) I-K/(-17 HO _c _.....1s) NH
HO
H2N) 0 H2N \O
(R) HO (s) r\l)N1N)N 0 .,,,(0 0 0 H H H H H , H
/
0 NkS),.)-L.
....i.kl.õ....¨..õ--,}___ N (s) NH
N (s) 0 HO
H H H
N )-L N ,(s-L N
0 0 b 0 , N
N
\
¨ OH
:
p -o-Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application enters the blood circulation and the outside of cells (in an intercellular substance). Since the linker is very stable in an extracellular environment and drug molecules cannot be released, the toxicity of the drug molecules is blocked. The conjugate is a drug with no cytotoxicity or with a low toxicity and will not have a toxic effect on normal cells.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application binds to multiple receptors or antigens and other acting molecules that are simultaneously highly expressed on diseased cells, and the synergistic effect thereof greatly increases the affinity of the conjugate compound to target cells, reducing the possibility of binding to normal cells. Therefore, a highly effective toxin drug such as MMAE/Dxd/5N38/a radionuclide complex can be carried to enhance the efficacy of drugs, broaden a therapeutic window and avoid drug side effects.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application enters the interior of targeted cells, and then the linker can be cleaved through changes of the internal environment of the cells (by a specific enzyme digestion, a pH change, a disulfide bond reduction, etc.) to release drug molecules (equivalent to removing modifying groups of drug molecules), which has a therapeutic effect on tumor cells.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application can be used to specifically deliver a payload to target cells in a target tissue environment. Generally, the two targeting molecules of the conjugate compound or the pharmaceutically acceptable salt thereof have three advantages. Firstly, the two targeting molecules can act in multiple modes (often synergistically), resulting in improved therapeutic effects while reducing side effects. Secondly, the binding of the two targeting molecules increases the affinity and avidity of the conjugate compound or the pharmaceutically acceptable salt thereof to target receptors or target cells, thereby enhancing its specificity and avoiding off-target toxicity. Finally, when properly Date Recue/Date Received 2021-07-27 designed, a combination of the two targeting molecules can fulfill multi-functional properties required for a drug conjugate.
The conjugate compound or the pharmaceutically acceptable salt thereof of the present application achieves unexpected technical effects, including but not limited to: (1) a combination of a ligand capable of binding to cell surface receptors and a endocytosis molecule capable of mediating endocytosis enable the conjugate compound to specifically enter target cells; (2) the conjugate compound or the pharmaceutically acceptable salt thereof enhances an affinity and targeting specificity of drug compounds, thereby delivering highly effective chemotherapeutic agents such as MMAE to a patient, broadening the therapeutic window of such agents and avoiding side effects; (3) a linker can prevent the release of a payload outside of target cells (for example, in a blood circulation system, intercellular substance, etc.), which ensures the stability of the conjugate compound during the blood circulation, and reduces the toxicity of the drug; after entering target cells, the linker is cleaved to release the payload and exert the effect of the drug, and meanwhile multiple drug resistance (MDR) can be avoided; and (4) a wide variety of drugs may be delivered in a form of the conjugate compound of the present application, and therefore, the application scopes of relevant drugs are widened. Therefore, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application not only broadens the targeting scope and therapeutic window of LDC-based drugs, but also reduces toxicity and side effects of some drugs.
The terms "polypeptide", "protein" and "peptide" as used herein can be a single amino acid or a polymer of amino acids. The polypeptide, protein or peptide as described in the present application may contain naturally-occurring amino acids and non-naturally-occurring amino acids, or analogs and mimetics thereof The polypeptide, protein or peptide can be obtained by any method well known in the art, for example, but not limited to, by an isolation and a purification from natural materials, a recombinant expression, a chemical synthesis, etc.
In another aspect, the present application discloses a pharmaceutical composition comprising the conjugate compound or the pharmaceutically Date Recue/Date Received 2021-07-27 acceptable salt thereof provided in the present application, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means, within the scope of sound medical judgment, being suitable for contact with cells of human beings and other animals without undue toxicity, irritation, allergic response, etc., and being commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" as used herein refers to a relatively non-toxic, inorganic and organic acid addition salt and base addition salt, of the conjugate compound of the present application. Representative acid addition salts include hydrobromides, hydrochlorides, sulfates, bisulfates, phosphates, nitrates, acetates, oxalates, valerates, oleates, palmitates, stearates, laurates, borates, benzo ate s, lactates, phosphates, to sylate s, citrates, male ate s, fumarates, succinates, tartrates, naphthylates, me sylate s, glucoheptonates, lactiobionates, sulphamates, malonates, salicylates, propionates, methylene-b is -b-hydroxynaphthoate s, genti s ate s, is ethionate s, di-p-toluoyltartrates, methane sulphonate s, ethane sulphonate s, benzene sulphonate s, p-toluenesulphonates, cyclohexylsulphamates, quinateslaurylsulphonate salts, and the like. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. In some embodiments, sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide.
Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include the following amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use: ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, Date Recue/Date Received 2021-07-27 benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, dicyclohexylamine, and the like.
The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable solvent, suspension or any other pharmacologically inert vehicle for delivering the conjugate compound provided in the present application to a subject, without interfering with the structures and properties of the conjugate compound. Such carriers enable the conjugate compound to be formulated as, for example, tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and pastilles, for oral ingestion by a subject. Such carriers enable the conjugate compound to be formulated as injections, infusions or preparations for local administration.
The pharmaceutically acceptable carriers for use in the pharmaceutical composition provided in the present application may include, but are not limited to, for example, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous vehicles (such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), nonaqueous vehicles (such as fixed oils derived from vegetables, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), anesthetics (such as procaine hydrochloride), suspensions/dispersions (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifying agents (such as polysorbate 80 (Tween-80)), diluents, adjuvants, excipients or non-toxic auxiliary substances, other components well known in the art, or various combinations thereof Suitable components may include, for example, fillers, binders, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, or emulsifying agents.
In some embodiments, the pharmaceutical composition is an injection preparation. The injection preparations include sterile water solutions or dispersions, Date Recue/Date Received 2021-07-27 suspensions or emulsions. In all cases, the injection preparations should be sterile and be a fluid for easy injection. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carriers can be solvents or dispersion mediums containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof and/or vegetable oils. The injection preparations should maintain appropriate fluidity. The appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by the use of surfactants, and the like.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
In some embodiments, the pharmaceutical composition is an oral preparation.
The oral preparations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (using an insert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes.
In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, pulvis, granules and the like), the conjugate compound is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the followings: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatins, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, Date Recue/Date Received 2021-07-27 solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.
In liquid dosage forms for oral administration, the conjugate compound is mixed with any of the followings: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the conjugate compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as, water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butylene glycol, oils (in particular, cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof Besides inert diluents, an oral composition can also include adjuvants such as wetting agents, emulsifying agents and suspensions, sweetening agents, flavoring agents, coloring agents, perfuming agents and preservatives.
In some embodiments, the pharmaceutical composition is a mouth spray preparation or a nasal spray preparation. The spray preparations include, but are not limited to, aqueous aerosols, nonaqueous suspensions, lipidosome preparations or solid granular preparations. Aqueous aerosols are prepared by mixing aqueous solutions or suspensions of agents with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary according to the requirements of specific compounds, but in general, they include nonionic surfactants (Tweens or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solution, salts, sugar or sugar alcohol. Aerosols are generally prepared from isotonic solutions, and can be delivered by sprayers.
In some embodiments, the pharmaceutical composition can be used by mixing with one or more other drugs. In some embodiments, the pharmaceutical composition comprises at least one other drug. In some embodiments, the other drugs are antineoplastic drugs, cardiovascular drugs, anti-inflammatory drugs, antiviral drugs, digestive system drugs, nervous system drugs, respiratory system drugs, immune system drugs, dermatologic drugs, metabolic drugs, and the like.
Date Recue/Date Received 2021-07-27 In some embodiments, the pharmaceutical compositions can be administered to a subject in need thereof by appropriate routes, including without limitation, oral, injection (such as intravenous, intramuscular, subcutaneous, intracutaneous, intracardiac, intrathecal, intrapleural and intraperitoneal injection), mucosal (such as nasal and intraoral administration), sublingual, rectal, percutaneous, intraocular, and pulmonary administration. In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly.
Due to the properties of some payloads, such as high toxicity and high hydrophilicity, it is desired to deliver the payloads more specifically and more efficiently to a subject in need thereof For example, in cancer treatment, it is desired to specifically deliver chemotherapeutic agents to cancer cells without toxicity to normal cells. Therefore, in another aspect, the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application, or the pharmaceutical composition provided in the present application.
The payloads described in the present application may be any pharmaceutical agent that elicits biological or medicinal responses in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinicians in preventing, inhibiting, ameliorating or treating a disease.
The term "object" as used herein refers to human and non-human animals.
Non-human animals include all vertebrates, for example, mammals and non-mammals. The subject may also be a livestock animal such as cattle, swine, sheep, poultry and horse, or a domestic animal such as dog and cat. The subject may be male (e.g. man) or female (e.g. woman), may be elderly, and may be an adult, adolescent, child, or infant. A human subject may be Caucasian, African, Asian, Semitic, or human with other racial backgrounds or a mixture of such racial backgrounds.
The term "therapeutically effective amount" as used herein refers to an amount of the conjugate compound or the pharmaceutically acceptable salt thereof, or the Date Recue/Date Received 2021-07-27 pharmaceutical composition which relieves to some extent one or more symptoms of a disease or disorder in a subject, returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or disorder, and/or reduces the likelihood of the onset of the disease or disorder. Such amounts generally vary according to a number of factors which can be detettnined and explained, according to the scope of the specification provided in the present application, by those of ordinary skill in the art.
These factors include, without limitation: the particular subject and the age, weight, height, general physical condition, and medical history thereof, the particular compound used, the carrier of the preparation and the administration route selected, and the nature and severity of the condition being treated.
In some embodiments, the amount of the conjugate compound, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition is sufficient to inhibit a disease or disorder in a subject, or prophylactically inhibit or prevent the onset of a disease or disorder in a subject. Although the therapeutically effective amount may vary in different subjects, it is generally ranged from 0.01 to 100 mg/kg, for example, 0.01 to 90 mg/kg, 0.01 to 80 mg/kg, 0.01 to 70 mg/kg, 0.01 to 60 mg/kg, 0.01 to 50 mg/kg, 0.01 to 40 mg/kg, 0.01 to 30 mg/kg, 0.01 to 20 mg/kg, 0.01 to 10 mg/kg, 0.01 to 5 mg/kg, 0.01 to 4 mg/kg, 0.01 to 3 mg/kg, 0.01 to 2 mg/kg, 0.01 to 1 mg/kg, and 0.01 to 0.1 mg/kg. The therapeutically effective amount as described in the present application can be equal to any value within the above numerical range, including the end values of this range.
In another aspect, the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application, or the pharmaceutical composition provided in the present application.
In another aspect, the present application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt Date Recue/Date Received 2021-07-27 thereof provided in the present application, or the pharmaceutical composition provided in the present application.
In some embodiments, the disease is a cancer, including but not limited to, prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.
In some embodiments, cancer cells of the cancers have an expression of the cell surface receptors or antigens mentioned in the present application. In some embodiments, cancer cells of the cancers have a high expression (for example, according to data from Depmap (see: https://depmap.org/portal/), the corresponding gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) of cell surface receptors or antigens mentioned in the present application.
In some embodiments, cancer cells of the cancers have a high expression of and FOLH1, TRPV6 and FOLH1, GNRHR and FOLH1, SSTR2 and FOLH1, FOLR1 and SSTR2, or TRPV6 and FOLR1. In some embodiments, the disease is an immunological disease, for example, an autoimmune disease, including but not limited to, connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
In some embodiments, the disease is a cardiovascular disease, including but not limited to, angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital heart disease.
In some embodiments, the disease is a metabolic disease, including but not limited to, diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
In some embodiments, the disease is a neurological disease, including but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
In some embodiments, the method provided in the present application further comprises administering one or more therapeutic agents in combination with the conjugate compound or the pharmaceutically acceptable salt thereof, or the Date Recue/Date Received 2021-07-27 pharmaceutical composition. In some embodiments, the therapeutic agents target an anti-cancer therapeutic target, induce or boost an immune response against cancer, or are chemotherapeutic agents.
The present application will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. A person skilled in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
EXAMPLES
The following examples are intended to further illustrate the present application. The advantages and features of the present application will become clear with the descriptions. However, these illustrations are merely exemplary, and should not be constructed as limitations to the scope of the present application.
Example 1: Preparation of Conjugate Compounds Synthesis of conjugate compounds CB-20BK, CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE, CB-20AK, CB-1020, CB-1320 and CB-1820 1. 10 g of Rink amide-am resin (hereafter referred to as "Rink Resin", from Sunresin New Materials Co. Ltd., Cat#: 183599-10-2) with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM
was added, and nitrogen gas was bubbled through the solvent to swell the resin for minutes; the solvent was pumped off; and Fmoc protecting groups on the resin were removed by DBLK, and then the resin was washed 5 times with DMF. 4.79 g (9 mmol) of Fmoc-Lys(Dde)-OH and 1.47 g (10.8 mmol) of HOBt were weighed 25 and dissolved in DMF. To the above-mentioned solution at 0 C in an ice-water bath, 1.67 ml (10.8 mmol) of DIC was added and mixed to activate for 5 minutes. The solution was added to the above-mentioned reaction column and reacted for 3 hours, and then the solvent was pumped off; and the resin in the reaction column was washed 3 times. Then Fmoc protecting groups were removed by DBLK.
Date Recue/Date Received 2021-07-27 2. The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H, Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate 108 were successively conjugated according to the structures.
tBuO 0 0 ,OtB u tBuO (s) N N N 0 H H H H
OH
(Intel _______ me diate 108) 3. Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated successively. Finally, the resin was condensed twice with methanol, and the solvent was pumped off to obtain 17.4 g of a protected peptide resin.
4. 17.4 g of the peptide resin obtained in the previous step was added to a 250 ml single-necked flask; 139 ml of lysis buffer (TFA : H20 : TIS = 95 : 3 :
(volume ratio)) was previously prepared, and 2.1 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the above-mentioned flask, and the mixture was reacted at room temperature for 2.5 hours and filtered; the resin was continued to be washed with 30 ml of TFA; the above-mentioned filtrate was combined, and the mixture was added to 834 ml of absolute ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 6.4 g of a crude product as a yellow solid and with a yield of 93.4% and an HPLC purity of 82.3%. The product was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60 minutes;
fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 4.73 g of 20BK-SM09, with a purity of 98.8%.
5. 4.09 g (3.11 mmol) of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a 1000 ml single-necked flask, and 500 ml of phosphate buffer and 100 ml of acetonitrile were added; the solution was stirred and maintained at pH =
7.2 until a clear solution was obtained; and 4.73 g (3.11 mmol) of inteimediate Date Recue/Date Received 2021-07-27 20BK-SM09 was added, and the mixture was reacted at room temperature for 2 hours, during which the reaction was monitored by HPLC. After the reaction was completed, the mixture was filtered, and the filtrate was separated by prep-HPLC
(condition: C18 column, mobile phase A: ammonium bicarbonate solution (pH =
7.2), B: acetonitrile, elution gradient: (25-35)%B, elution time: 60 minutes;
fractions were collected); the fraction containing the qualified product was lyophilized to obtain 6.96 g of CB-20BK product, with a purity of 98.8% and a yield of 78.8%.
In the same way, conjugate compounds CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE (with a structure shown as follows), CB-20AK (with a structure shown as follows), CB-1020, CB-1320 and CB-1820 can be obtained through similar steps in the above-mentioned methods.
H2NyJN
, NH
0 OH 0 oH 0 0 4N NH N LC)JL1 N
(R) S 0 H 8 H 0 H I
N
,OH
(s) (FA-MMAE) HO
0 C) HO H Y N Cri4õ))))),,1 -101 (s) J(rA N (s) NF-o o r_f(.ti 0 H
HO
0 OyN/S,LLN N r 0 H 0 (s) }D
(CB-20AK) Synthesis of conjugate compounds CB-50S, CB-60S and CB-60SK
1. 10 g of Wang Resin (from Sunresin New Materials Co. Ltd., Cat#:
1365700-43-1) with a degree of substitution of 1.1 mmolig was weighed and loaded onto a solid phase reaction column; DMF was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes; 14.3 g (22 mmol) of Date Recue/Date Received 2021-07-27 Fmoc-Arg(pbf)-0H, 3.56 g (26.4 mmol) of HOBt, and 0.27 g (2.2 mmol) of DMAP
were weighed and dissolved in DMF; at 0 C in an ice-water bath, 4.1 ml (26.4 mmol) of DIC was added and mixed to activate for 5 minutes; the solution was added to the reaction column and reacted for 3 hours, and then the solvent was pumped off; and the resin was washed 3 times.
2. 10.4 ml of acetic oxide and 8.9 ml of pyridine were dissolved in 50 ml of DMF and mixed, and the resin after washing in the above steps was added; the mixture was blocked at room temperature for 5 hours and washed three times with DMF; and the resin was condensed with methanol, and then the solvent was pumped off to obtain Fmoc-Arg(pbf)-Wang Resin, which has a degree of substitution detet __ mined to be 0.53 mmol/g.
3. 3.8 g (2 mmol) of Fmoc-Arg(pbf)-Wang Resin (Sub = 0.53 mmol/g) was weighed and loaded onto a reaction column; the resin was washed 3 times with DMF and then was swelled for 30 minutes by means of adding DMF. Then Fmoc protecting groups were removed by DBLK, and the resin was washed 6 times with DMF. 2.0 g (6 mmol) of Fmoc-Pro-OH and 0.97 g (7.2 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 1.1 ml (7.2 mmol) of DIC was added and mixed to activate for 5 minutes; the mixture was added to the reaction column and reacted for 2 hours; and then Fmoc protecting groups were removed by DBLK.
4. The above-mentioned operations were repeated, and Fmoc-Leu-OH, Fmoc-Asp(OtBu)-0H, Fmoc-Val-OH, Fmoc-Lys(Boc)-0H, Fmoc-Ser(tBu)-0H, Fmoc-Pro-OH, Fmoc-His(Trt)-0H, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-0H, Fmoc-Lys(Boc)-0H, Fmoc-propargyl-Gly-OH, Fmoc-Glu-OtBu and pteroic acid were conjugated successively according to the structures. The resin was condensed twice with methanol, and the solvent was pumped off to obtain 8.4 g of a peptide resin.
5. 8.4 g of the peptide resin obtained in the previous step was added to a ml single-necked flask; 67 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 0.92 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was reacted at Date Recue/Date Received 2021-07-27 room temperature for 2.5 hours; the resin was filtered and washed with 20 ml of TFA; the filtrate was combined, and the mixture was added to 402 ml of absolute ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 4.06 g of a crude product as a yellow solid and with a yield of 97.3% and an HPLC purity of 84.6%.
The product was separated by prep-HPLC (condition: C18 column, mobile phase A:
0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-29)%B, elution time: 60 minutes; fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 2.86 g of 50S-SM09, with a purity of 97.6%.
6. 1.29 g (1.37 mmol) of LT1000N3 was weighed and added to a 500 ml single-necked flask, and 270 ml of a mixed solvent (CAN : H20 = 1 : 4) and 393 mg (2.74 mmol) of CuBr were added and stirred. 2.86 g (1.37 mmol) of intermediate 50S-SMO9 was added, and the mixture was reacted at room temperature for 2-3 hours, during which the reaction was monitored by HPLC.
After the reaction was completed, the mixture was filtered, and the filtrate was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (22-40)%B, elution time: 60 minutes;
fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 3.17 g of CB-50S, with a purity of 98.6% and a yield of 76.4%.
In the same way, conjugate compounds CB-60S and CB-60SK can be obtained through similar steps in the above-mentioned methods.
Synthesis of CB-20R
1. 5 g of Rink Resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes; the solvent was pumped off; Fmoc protecting groups on the resin were removed by DBLK; and then the resin was washed 5 times with DMF. 2.4 g (4.5 mmol) of Fmoc-Lys(Dde)-OH and 0.74 g (5.4 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.84 ml (5.4 mmol) of DIC was added and mixed Date Recue/Date Received 2021-07-27 to activate for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped off, and the resin was washed 3 times.
Then Fmoc protecting groups were removed by DBLK.
2. The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H, Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate 108 were successively conjugated according to the structures.
3. Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. DOTA-tris(tBu) ester was conjugated. Then Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated successively, and finally, the resin was condensed twice with methanol and the solvent was pumped off to obtain 9.2 g of a protected peptide resin.
4. 9.2 g of the peptide resin obtained in the previous step was added to a ml single-necked flask; 74 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 1.05 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml of TFA; the filtrate was combined, and the mixture was added to 560 ml of absolute ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 3.8 g of a crude product as a yellow solid and with a yield of 87.4% and an HPLC purity of 81.2%.
The product was separated by prep-HPLC (condition: C18 column, mobile phase A:
0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60 minutes; fractions were collected); and the fraction containing a synthetic product was lyophilized to obtain 2.9 g of 20R-SM09, with a purity of 97.8%.
5. 20R-SMO9 was complexed with radionuclide ion M to obtain CB-20R.
Specifically, the radioactive label 177Lu (about 50 MBq) was mixed with 100 [t.1 of 0.5 M sodium acetate buffer (pH = 5). 40 [t.1 of aqueous solution of 1 mM CB-dissolved in 10% DMSO, 2 1 of saturated ascorbic acid solution and 100 [t.1 of Date Recue/Date Received 2021-07-27 solution containing 177Lu were mixed, and the mixture was heated to 95 C for minutes. The label was detected by radio-HPLC (within 5 minutes; 0%-100% ACN
in water; C18 column).
Synthesis of compound CR19425 --- `-..'11112 44080311 11 9 g *õ..õ.=-- ..1 i N..., ...)..,.....),. ,,,,, 1 Molecular weight : 324.396 (7i's 'NH F N
tis ,---, Si... ` CR19420 . , . 4:t , Piped(MK
....., b simi HATUDIPEA Y X 114_,(P
., HO-,:'-"-µ,N ".=, = -.'", -,, , ,)---sil.õ,i,J
Molecular weight : 435.46 0N19418 FICrts, \ b Molecular weight : 801,8.284 11''r"'il i 1 rt, 0 ? H Y
Q
0 - soH
Molecular weight : 472.60 ---l',..%, ..-, HATU,DIPEA
F '' 'N
A.µ,..,..
P
\ b Molecular weight : 57'9.5854 eft18422 ., ri n ri i N ,,..,..i.õ . , . N. ,.." = i- , ,,..F
'0' 0 N µ!,;coshio9 H " , -. . Ni __ o= CR19425 , . ...
Molecular we i dm :1 034,01 N ..:=f ., Molecular weight: 3126.43/4 Cil1-113423, .0-0:
1. Under N2 protection, to a reaction flask, 441.4 mg of CR19420 (with a structure as shown in the above reaction step) and 8.0 mL of DMF were added, 10 stirred, dissolved and cooled in an ice bath; then 459.2 mg of HATU and 380 [IL of Date Recue/Date Received 2021-07-27 DIPEA were added and stirred for half an hour; and then 500.0 mg of CR19419 (with a structure as shown in the above reaction step) and 190 [IL of DIPEA
were added, and the mixture was reacted at room temperature until the reaction was completed. After the reaction was completed, the reaction solution was poured into an acetic acid aqueous solution; a solid was precipitated and filtered, and the filter cake was washed with an acetic acid aqueous solution and water, and dried in vacuo to obtain 835.7 mg of CR19421 (with a structure as shown in the above reaction step) as a brown powder and with an HPLC purity of 90.0% and a yield of 90.6%.
2. Under N2 protection, to a reaction flask, 835.7 mg of CR19421 and 16 mL
of 10% piperidine DMF solution were added and reacted at room temperature for half an hour. After the reaction was completed, the reaction solution was poured into TFA/MTBE; a solid was precipitated and filtered, and the filter cake was washed with MTBE and dried in vacuo to obtain 599.7 mg of CR19422 (with a structure as shown in the above reaction step) as a field gray powder and with an HPLC
purity of 84.7% and a yield of 83.0%.
3. Under N2 protection, to a reaction flask, 599.7 mg of CR19422, 586.7 mg of CR19424 (with a structure as shown in the above reaction step) and 15 mL of DMF were added, stirred, dissolved and cooled in an ice bath; then 495.7 mg of HATU and 410 [IL of DIPEA were added and reacted at room temperature. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the acetonitrile was removed under reduced pressure from the pure product solution; the residue was extracted with a mixed solvent of dichloromethane and methanol, concentrated and dried to obtain 674.9 mg of CR19423 (with a structure as shown in the above reaction step) as a yellow powder and with an HPLC purity of 88.4% and a yield of 63.1%.
4. Under N2 protection, to a reaction flask, 14.8 mg of CR19423, 3.0 mL of PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and dissolved;
then 32.9 mg of CBSMO9 was added, and the mixture was adjusted to pH 6.6-6.8 with Na2HPO4 and reacted for half an hour. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the pure product Date Recue/Date Received 2021-07-27 was lyophilized to obtain 21.8 mg of CR19425 as a yellow powder and with an HPLC purity of 95.6% and a yield of 48.8%.
Synthesis of compound CR19426 H ' 1 , e`ir = 'N y'"*. 0 e' =:' :
H
t4: N = =L, Molecular weight :10t)7 N
Molecular weight : 3312.63 CRI.S4.0 ,OH
1. Under N2 protection, to a reaction flask, 25.2 mg of CR19423, 3.0 mL of PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and dissolved;
then 55.5 mg of 18G-5M09 was added, and the mixture was adjusted to pH 6.6-6.8 with Na2HPO4 and reacted for half an hour. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the pure product 113 was lyophilized to obtain 44.6 mg of CR19426 as a yellow powder and with an HPLC purity of 95.4% and a yield of 55.2%.
Synthesis of compound CR19428 .0 t rt - z H. " CR19428 4 ?
MO leCUlar t:tglit. )3.4 07 Molecular weight : 25b,6384 0114043 .0H
.(;) 1. Under N2 protection, to a reaction flask, 486 mg of CR19423, 3.0 mL of PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and dissolved;
then 712 mg of 20BK-SMO9 was added, and the mixture was adjusted to pH 6.6-6.8 with Na2HPO4 and reacted for half an hour. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the pure product Date Recue/Date Received 2021-07-27 was lyophilized to obtain 508 mg of CR19428 as a yellow powder and with an HPLC purity of 96.7% and a yield of 42.3%.
Example 2: Determination of Affinity of Conitmate Compounds to Tamet Proteins 1. Determination of binding affinity of CB-20BK to protein FOLR1 Experimental instruments, materials and reagents:
BIAcore T200 (GE) CMS chip (GE, Cat#: 29104988) Buffer: HBS-EP+ buffer 10X (GE, Cat#: BR100669), diluted 10 folds with deionized water before use.
Amino coupling kit (GE, Cat#: BR100050) Regeneration reagents: 10 mM Glycine 2.0 (GE, Cat#: BR100355) 10 mM Glycine 3.0 (GE, Cat#: BR100357) Experimental steps The experiment was carried out according to BIAcore T200 (GE) instruction manual to determine the affinity of analytes CB-20BK, CB-20AK, folate (FA) and FA-MMAE to FOLR1. In the experiment, CMS chip was conjugated with ligand FOLR1 (R&D System, Cat#: 5646-FR). The experimental results are as shown in Table 4.
Table 4. Binding Affinity of CB-20BK and Related Compounds to FOLR1 ka kd KD
FA (folate) 3.34 x 106 M's' 2.26 x 10-4 s-1 6.77 x FA-MMAE 1.79 x 10 M's' 1.15 x 10-4 s-1 6.43 x CB-20BK 1.03 x 10 M's' 1.31 x 10-4 s-1 1.27 x "N/D" means that no specific binding was detected.
Table 4 shows that CB-20BK specifically binds to FOLR1 with a good affinity.
The binding affinity of CB-20BK to FOLR1 is slightly weaker than the binding affinity of FA or FA-MMAE to FOLR1. CB-20AK does not comprise the folate Date Recue/Date Received 2021-07-27 moiety of CB-20BK and has not been detected to specifically bind to FOLR1 in the experiment.
2. Determination of bindin2 affinity of CB-20BK to protein FOLH1 Experimental instruments, materials and reagents:
GatorTM (Probe Life) SA probe (Probe Life, Cat#: 1906018) Buffer: Q buffer (Probe Life), 10 mM, pH = 7.4 Experimental steps (1) Synthesis steps of analyte Biotin-CB-20BK
1) 5.1 g of Rink Resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM was added, and nitrogen gas was bubbled to swell the resin for 30 minutes; the solvent was pumped off; Fmoc protecting groups were removed by DBLK; and then the resin was washed 5 times with DMF. 2.45 g of Fmoc-Lys(Dde)-OH and 0.75 g of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.83 ml of DIC was added to activate for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped off, and the resin was washed times. Then Fmoc protecting groups were removed by DBLK.
2) The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H, Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate 108 were successively conjugated according to the structures.
3) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF.
Fmoc-Lys(Biotin)-0H, Fmoc-Glu-OtBu and pteroic acid were conjugated successively, and after pteroic acid was conjugate, the resin was washed twice with DMF; and finally, the resin was condensed with methanol, and the solvent was pumped off to obtain 9.7 g of a protected peptide resin.
4) 9.7 g of the peptide resin obtained in the previous step was added to a 250 ml single-necked flask; 77.6 ml of lysis buffer (TFA : H20 : TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 1.1 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was Date Recue/Date Received 2021-07-27 reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml of TFA; the filtrate was combined, and the mixture was added to 466 ml of methyl tert-butyl ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with methyl tert-butyl ether and dried in vacuo to obtain 4.6 g of a yellow solid with an HPLC purity of 83.2%. The product was separated by prep-HPLC and lyophilized to obtain 2.1 g of Biotin-20BK-SM09 with a purity of no less than 95%.
5) 500 mg of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a 250 ml single-necked flask, and 65 ml of phosphate buffer and 20 ml of acetonitrile were added; the solution was stirred and maintained at pH = 7.2 until a clear solution was obtained; and 785 mg of intermediate Biotin-20BK-SMO9 was added, and the mixture was reacted at room temperature for 2 hours, during which the reaction was monitored by HPLC. After the reaction was completed, the mixture was filtered, separated by prep-HPLC, and lyophilized to obtain 723 mg of Biotin-CB-20BK, with a purity of 96.8% and a yield of 59.4%.
(2) The experiment was carried out according to GatorTM (Probe Life) instruction manual to determine the binding affinity of Biotin-CB-20BK to FOLH1.
In the experiment, SA probe binds to ligand Biotin-CB-20BK, and the analyte is FOLH1 (Sino Biologicals, Cat#: 15877-H07H). The experimental results are as shown in Table 5.
Table 5. Binding Affinity of CB-20BK to FOLH1 ka kd KD
FOLH1 1.19 x 104 A4-1s-1 1.70 x 10-4 s-1 6.98 x 10-9 M
Table 5 shows that CB-20BK binds to FOLH1 with a good affinity.
3. FOLH1 binds to CB-20BK-FOLR1 complex Experimental materials and reagents:
BIAcore T200 (GE) CMS chip (GE, Cat#: 29104988) Date Recue/Date Received 2021-07-27 Buffer: HBS-EP+ buffer 10X (GE, Cat#: BR100669), diluted 10 folds with deionized water before use.
Amino coupling kit (GE, Cat#: BR100050) Regeneration reagents: 10 mM Glycine 2.0 (GE, Cat#: BR100355) 10 mM Glycine 3.0 (GE, Cat#: BR100357) Experimental steps CM5 chip was conjugated with a ligand: FOLR1 (R&D System, Cat#:
5646-FR) Analyte: CB-20BK, FA-MMAE and FOLH1 (Sino Biologicals, Cat#:
15877-H07H) According to BIAcore T200 (GE) operating manual, FOLR1 was conjugated to the CMS chip; CB-20BK or FA-MMAE was loaded first, and then FOLH1 was loaded; and the binding of FOLH1 to the CB-20BK-FOLR1 complex was detected.
The experimental results are as shown in Table 6.
Table 6. Binding of CB-20BK-FOLR1 Complex to FOLH1 Solution at Different Concentrations Concentration (04) FA-MMAE (RU) CB-20BK (RU) 0.5 9.8 23.9 0.25 8.4 14.1 0.125 4.2 4.6 0.0625 -1 -0.9 Table 6 shows that with regard to an FOLH1 solution at a high concentration, the amount of FOLH1 bound to a CB-20BK-FOLR1 complex is significantly higher than that bound to an FA-MMAE-FOLR1 complex, which shows a continuous rising trend. The binding of FOLH1 to an FA-MMAE-FOLR1 complex tends to saturate at a high concentration. This experiment has proved that CB-20BK can bind to both FOLR1 and FOLH1 receptors with a good affinity.
Example 3: Study on Binding and Endocytosis of Ligand Conjugates to Target Cells Date Recue/Date Received 2021-07-27 1. Cell binding and endocytosis experiment of conjugate compound Synthesis of labeled samples Cy5-pep-20BK, Cy5-FA and Cy5-pep-20AK
(1) 2 g of Rink Resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes; the solvent was pumped off; Fmoc protecting groups were removed by DBLK; and then the resin was washed 5 times with DMF. 0.96 g (1.8 mmol) of Fmoc-Lys(Dde)-OH
and 0.3 g (2.2 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.33 ml (2.2 mmol) of DIC was added and mixed to activate for minutes; the mixture was added to the reaction column and reacted for 3 hours;
and the solvent was pumped off, and the resin was washed 3 times. Then Fmoc protecting groups were removed by DBLK.
(2) The above-mentioned operations were repeated, and Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and intermediate 108 were conjugated successively according to the structures:
(3) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. Fmoc-Lys(Dde)-0H, Fmoc-Glu-OtBu and pteroic acid were conjugated successively.
(4) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF; the resin was conjugated with Cy5-COOH and then washed twice with DMF;
and finally, the resin was condensed twice with methanol and the solvent was pumped off to obtain 3.6 g of a protected peptide resin.
(5) 3.6 g of the peptide resin obtained in the previous step was added to a 50 ml single-necked flask; 29 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 0.4 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 8 ml of TFA;
the filtrate was combined, and the mixture was added to 173 ml of absolute ether, Date Recue/Date Received 2021-07-27 with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 1.9 g of a crude product as a blue solid and with a yield of 89.6% and an HPLC purity of 76.3%. The product was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1%
trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-28)%B, elution time: 50 minutes; fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 736 mg of Cy5-pep-20BK, with a purity of 93.4%.
In the same way, compounds Cy5-FA and Cy5-pep-20AK can be obtained through similar steps in the above-mentioned methods.
HO 0 0, _OH
0 '03S HO (s) NAN 0 0 ,.rH 0 H H H H
N (s) (s) N NH2 H H
N
HO
0, _OH HN -03S
I H
SOS-(Cy5-pep-20BK) -03s HO 0 0,0H
HO (s)NANNAN 0 (rH 0 0 H H H H
0 NksA N,)-LN H2 -03s N (S) N (S) H H
0 \(:) 0 \
\_H
HO
SOB-(Cy5-pep-20AK) Date Recue/Date Received 2021-07-27 -03s N-frN
I
(Cy5 -FA) Experiment for detecting cell binding and endocytosis of samples Cy5-pep-20AK/Cy5-pep-20BK by flow cytometry method Sample infoimation: Cy5-pep-20AK and Cy5-pep-20BK
Cell lines: LNCaP human prostate cancer cells, Du145 human prostate cancer cells, SKOV3 human ovarian cancer cells and NCI-H460 human lung cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and PBS
Experimental operations:
(1) Cell culture: the cells were digested with trypsin, collected and counted, and then the cells were added to several culture flasks containing a complete medium; the culture flasks were placed in an incubator at 37 C, 5% CO2 for culturing the cells, and finally about 5 x 106 cells were collected into a centrifuge tube.
(2) Sample incubation: Cy5-pep-20AK/Cy5-pep-20BK samples were diluted to 8 nmol/L with PBS; the cell suspension was centrifuged at 1000 rpm for 5 minutes;
the supernatant was removed, and the residue was washed once with PBS; the cells were suspended uniformly and divided equally into different centrifuge tubes according to the experimental scheme; PBS was removed by centrifugation; 200 [t.1 of a sample working solution was added to each tube and incubated at 37 C for 15, 30, 60, and 90 minutes, respectively, and 1 tube to which only PBS was added was used as a blank control; all operations were performed avoiding light exposure.
Date Recue/Date Received 2021-07-27 (3) Washing and loading: the working solution was removed by centrifugation at 1000 rpm for 5 minutes, and the cells were washed 3 times with PBS, and an appropriate amount of PBS was added to suspend the cells to 1 x 106 cells/ml;
Beckman CytoFLEX flow cytometer was turned on in advance to complete the startup cleaning process; and the cell samples were loaded successively, and the fluorescence of 10,000 living cells was read in the APC channel; all operations were performed avoiding light exposure.
(4) Data analysis: the absolute MFI (mean fluorescence intensity) of the APC
fluorescence of each cell sample and the relative MFI of the relative blank control were obtained, and a data line graph was completed according to the relative MFI.
Results and analysis Table 7. Expression Levels of FOLR1 and FOLH1 in Different Cell Lines (with reference to data from Depmap, https://depmap.org/portal/) RNA-Seq LNCaP SKOV3 Du145 NCI-H460 FOLR1 +/- 6+ +/- +1-FOLH1 10+ + +/- +/-According to data from Depmap, the data being 0 or negative is expressed as "-", the data being 0.001-0.499 is expressed as "+/-", the data being 0.500-1.499 is expressed as "+", the data being 1.500-2.499 is expressed as "2+", and so on.
The fluorescence intensity of cells was respectively detected at 15 minutes, minutes, 60 minutes, and 90 minutes of incubation, and the results were plotted and shown in Figs. 2A and 2B. The binding and endocytosis of the sample Cy5-pep-20BK cells can be seen from the figures. Compared with a DU145 cell line with a relatively low expression of FOLR1 and a NCI-H460 cell line with a relatively low expression of FOLH1, an LNCaP cell line with a relatively high expression of FOLR1 and an SKOV3 cell line with a relatively high expression of FOLH1 have a significantly higher fluorescence intensity of CB-20BK that was bond and endocytosed. Since Cy5-pep-20AK only comprises an FOLH1 ligand and does not comprise the FOLR1 ligand, FA, Cy5-pep-20AK shows a high level of binding and endocytosis in LNCaP cells with a high expression of FOLH1, and Date Recue/Date Received 2021-07-27 shows a moderate level of binding and endocytosis in SKOV3 cells with a moderate expression of FOLH1; in the two cell lines, the binding and endocytosis level of Cy5-pep-20AK was significantly lower than that of Cy5-pep-CB-20BK. The degree of binding and internalization of a ligand conjugate to cells is positively correlated with expression levels of cell-related receptors.
2. Binding and endocytosis experiment of single-ligand conjugate Cy5-FA to cells Sample infoimation: Cy5-FA
Cell lines: Hela cervical cancer cells, SKOV3 human ovarian cancer cells and A549 human lung cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, PBS, and anti-fluorescence quenching mounting medium containing DAPI
Experimental operations:
(1) Cells growing on coverslips: coverslips were placed in a 24-well plate;
the cells were digested with trypsin, collected and counted, and then the cells were diluted with a complete cell culture medium to about 2 x 105 cells/ml; 500 IA
of the diluted cell solution was added to each well of the 24-well plate; and the plate was placed in an incubator at 37 C, 5% CO2 for culturing 48 hours.
(2) Sample incubation: the Cy5-FA sample was diluted in an IMDM medium to 73 nmol/L; the culture medium in the 24-well plate was discarded; 200 IA of the sample working solution was added to the cell coverslip in each well and incubated at 37 C for 15, 30, and 60 minutes, respectively, and 1 well to which only an IMDM
medium was added was used as a blank control; all operations were perfoinied avoiding light exposure.
(3) Washing and mounting: the working solution in the 24-well plate was discarded, and the plate was washed 3 times with preheated PBS at 37 C; the cell coverslips were taken out, and 5 [t.1 of anti-fluorescence quenching mounting medium containing DAPI was added dropwise onto the slides and then covered with the cell coverslips to complete the mounting; all operations were perfoinied avoiding light exposure.
Date Recue/Date Received 2021-07-27 (4) Image interpretation for samples: Leica fluorescence microscope DM2500 was turned on to preheat the fluorescence exciter for 15 minutes; at the same position, cell nucleus and Cy5 fluorescein were photographed at the corresponding fluorescence channel, and the image fusion was completed in the software.
The binding and endocytosis of the sample Cy5-FA to cells at 15 minutes and 30 minutes can be seen from Fig. 3, and the fluorescence intensity in cell lines Hela and SKOV-3 with a high expression of FOLR1 is significantly higher than that in a cell line A549 with a relatively low expression of FOLR1. The degree of binding and internalization of a ligand conjugate to cells is positively correlated with expression levels of cell-related receptors.
Example 4: Experiment of Inhibitin2 Cell Expansion in Conjugate Compounds 1. Experiment of inhibiting tumor cell expansion in CB-20BK
Sample infoimation: CB-20BK
Cell lines: KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 2.5 x cells/ml, T-47D cells were diluted to 1.3 x 104 cells/ml, NCI-H460 cells were diluted to 3.5 x 104 cells/ml, CALU-3 cells were diluted to 4.0 x 104 cells/ml, HuH-7 cells were diluted to 3.0 x 104 cells/ml, and LNCaP cells were diluted to 8.0 x 104 cells/ml; 100 IA of the diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were set on each plate.
The 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples Date Recue/Date Received 2021-07-27 The samples were diluted with a culture medium to desired concentrations (see Table 8). To each well of the 96-well plates that were cultured overnight, 50 1.11 of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 8: Concentration of Sample (CB-20BK) After Dilution Concentration after Tube number dilution (mon) 4 3.1 5 0.8 6 0.2 7 0.05 8 0.01 9 0.003 0.0008 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing 10 solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4A, wherein C value corresponds to IC5o), CB-20BK has an inhibitory effect on the growth and Date Recue/Date Received 2021-07-27 expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines.
Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell lines T-47D, KB, LNCaP, HuH-7 and CALU-3 with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-20BK shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
2. Experiment of conjugate compound CB-20BK and related compounds inhibiting expansion of tumor cell lines LNCaP (human prostate cancer cells) and 22RV1 (human prostate cancer cells) Since different cells have different sensitivity to a cell proliferation inhibitory toxicity of the payload in a ligand conjugate, the quantitative analysis may be interfered occasionally. A series of cell lines with known expression levels of receptors of different ligands were selected and tested for the response to inhibitory effects of the cell proliferation of a ligand conjugate and a single payload.
The ICso ratio of a single payload to that of a ligand conjugate in same cell line were taken to remove such interference.
Related samples: MMAE, CB-20BK, CB-20AK and FA-MMAE
Experimental operations: LNCaP and 22RV1 cells were prepared in advance, counted, and plated in 96-well cell culture plates at a cell density of 4 x 104 cells/ml and 2.0 x 104 cells/ml, respectively (100 [a/well); negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 [d/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10%
of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The calculated data is shown in Table 9.
Table 9. Inhibitory Effects of MMAE, FA-MMAE, CB-20BK and CB-20AK on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) Date Recue/Date Received 2021-07-27 FOL FOL
ICso ICso ratio gene gene IC50 IC50 ratio ratio Cell IC50 (FA- (MM
expr expr (CB-2 (CB-20 (MMA (MMA
line (MMAE) MMA AE/F
essio essio OAK) BK) E) A-M
n n OAK) OBK) MAE) level level LNCaP +/- 10+ 0.00255 0.264 0.102 0.0958 0.010 0.025 0.0267 22RV1 +/- 6+ 0.00648 1.01 1.05 0.502 0.006 0.006 0.013 It can be seen from the above table that there is not much difference in the expression level of FOLR1 between LNCaP and 22RV1, and the expression level of FOLH1 in LNCaP is significantly higher than the expression level of FOLH1 in 22RV1. CB-20BK (with FOLH1 ligand and FOLR1 ligand) and CB-20AK (only with FOLH1 ligand) have inhibitory effects on the expansion of both LNCaP and 22RV1 tumor cell lines. The inhibitory effects of CB-20BK and CB-20AK on the cells with different expression levels of receptor FOLH1 are different. The ICso ratio in cell line LNCaP with a relatively high expression of receptor FOLH1 is 2-4 times higher than the ICso ratio in cell line 22RV1 with a relatively low expression of the receptor. FA-MMAE also has an inhibitory effect on the expansion of LNCaP and 22RV1 tumor cell lines. Since the expression level of FOLR1 in LNCaP and 22RV1 is very low, the expression in LNCaP cells (FOLR1 gene expression level is 0.3219, with reference to data from Depmap) is slightly higher than that in 22RV1 (FOLR1 gene expression level is 0.0704, with reference to data from Depmap), and the ICso ratio of FA-MMAE between LNCaP and 22RV1 only differs by a factor of 1.5. The above data indicates that inhibitory effects on cell proliferation is positively correlated with expression levels of cell expression receptors FOLH1 and FOLR1.
3. Experiment of conjugate compound CB-20BK and related compounds inhibiting expansion of tumor cell lines PANC-1 (human pancreatic cancer cells) and CFPAC-1 (human pancreatic cancer cells) Related samples: MMAE and CB-20BK
Date Recue/Date Received 2021-07-27 Experimental operations: PANC-1 and CFPAC-1 cells were prepared in advance, counted, and plated in 96-well cell culture plates at a cell density of 4 x 104 cells/ml (100 [d/well); negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 [d/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader.
The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The calculated data is shown in the following table:
Table 10. Inhibitory Effects of CB-20BK on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) FOLR1 gene FOLH1 gene ICso ICso ICso (MMAE)/
Cell line expression expression (MMAE) (CB-20BK) ICso (CB-20BK) level level CFPAC-1 4+ +1- 0.015 0.557 0.027 PANC-1 +1- +1- 0.002 0.126 0.020 It can be seen from Table 10 that the expression level of FOLH1 in CFPAC-1 is very low, and the expression level of FOLR1 in CFPAC-1 is significantly higher than that in PANC1 cell lines. CB-20BK has an inhibitory effect on the expansion of PANC-1 and CFPAC-1 tumor cell lines. The inhibitory effects on the cells with different expression levels of receptor FOLR1 are different. The ICso ratio in cell line CFPAC-1 with a relatively high expression of receptor FOLR1 is significantly higher than the ICso ratio in the PANC-1 with a relatively low expression of receptor FOLR1, and the inhibitory effects on cell proliferation is positively correlated with expression levels of cell-related receptor FOLR1.
Experiments 2 and 3 prove that the two ligands in CB-20BK and the receptors thereof (FOLR1 and FOLH1) expressed on the cell surface play an important role in the compound inhibiting tumor cell proliferation.
Date Recue/Date Received 2021-07-27 4. Experiment of inhibiting tumor cell expansion in CB-20B
Sample infoimation: CB-20B
Cell lines: A549 human lung cancer cells, HuH-7 human liver cancer cells, KB
human oral epideituoid carcinoma cells, LNCaP human prostate cancer cells, DU145 human prostate cancer cells and T-47D human breast cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; diluted with a complete cell culture medium, A549 cells, HuH-7 cells, KB
cells and DU145 cells were plated at a density of 2 x 104 cells/ml, T-47D
cells were plated at a density of 1 x 105 cells/ml, and LNCaP cells were plated at a density of 6 x 104 cells/ml in 96-well plates; 100 1.11 of the diluted cell solution was added to each well; and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 11). To each well of the 96-well plates that were cultured overnight, 50 1.11 of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 11: Concentration of Sample (CB-20B) After Dilution Concentration after Tube number dilution (mon) 2 66.7 3 22.2 4 7.4 5 2.5 Date Recue/Date Received 2021-07-27 6 0.8 7 0.3 8 0.09 9 0.03 0.01 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an 5 appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was 10 plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4B, wherein C value corresponds to IC5o), CB-20B has an inhibitory effect on the growth and expansion of A549, HuH-7, KB, LNcap, DU145 and T-47D tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The IC5o for cell lines T-47D, LNcap, KB, HuH-7 and DU145 with a relatively high expression of the receptors is significantly lower than that for cell line A549 with a relatively low expression of the receptors. CB-20B shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
5. Experiment of inhibiting tumor cell expansion in CB-10S
Sample info' _______ "nation: CB-10S
Cell lines: KB human oral epideimoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells and LNcap human prostate cancer cells Date Recue/Date Received 2021-07-27 Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 3.5 x cells/ml, LNCaP cells were diluted to 8.0 x 104 cells/ml, T-47D cells were diluted to 1.2 x 104 cells/ml, NCI-H460 cells were diluted to 2.5 x 104 cells/ml, and RT4 cells were diluted to 1.2 x 104 cells/ml; to each well of 96-well plates, 100 [11 of the diluted cell solution was added; and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 12). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 12: Concentration of Sample (CB-10S) After Dilution Concentration after Tube number dilution (junol/L) 4 0.9 5 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 10 0.000007 Date Recue/Date Received 2021-07-27 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4C, wherein C value corresponds to ICso), CB-10S has an inhibitory effect on the growth and expansion of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell lines KB, LNcap, T-47D and with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-10S
shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
6. Experiment of inhibiting tumor cell expansion in CB-60S
Sample infoimation: CB-605 Cell lines: KB human oral epideimoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating Date Recue/Date Received 2021-07-27 The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 2.0 x cells/ml, T-47D cells were diluted to 1.5 x 104 cells/ml, NCI-H460 cells were diluted to 2.0 x 104 cells/ml, CALU-3 cells were diluted to 3.0 x 104 cells/ml, HuH-7 cells were diluted to 4.0 x 104 cells/ml, and LNCaP cells were diluted to 8.0 x 104 cells/ml; 100 IA of the diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were set on each plate.
The 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 13). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 13: Concentration of Sample (CB-60S) After Dilution Concentration after Tube number dilution (mon) 5 1.3 6 0.3 7 0.08 8 0.02 9 0.005 10 0.001 3) Color development and reading plate Date Recue/Date Received 2021-07-27 To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4D, wherein C value corresponds to IC5o), CB-605 has an inhibitory effect on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The IC50 for cell lines LNCaP
and HuH-7 with a relatively high expression of the receptors is significantly lower than that for cell lines NCI-H460, KB, T-47D and CALU-3 with a relatively low expression of the receptors. CB-605 shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
7. Experiment of inhibiting tumor cell expansion in CB-60SK
Sample infoimation: CB-605K
Cell lines: KB human oral epideimoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 2.5 x cells/ml, T-47D cells were diluted to 1.3 x 104 cells/ml, NCI-H460 cells were Date Recue/Date Received 2021-07-27 diluted to 3.5 x 104 cells/ml, CALU-3 cells were diluted to 4.0 x 104 cells/ml, HuH-7 cells were diluted to 3.0 x 104 cells/ml, and LNCaP cells were diluted to 8.0 x 104 cells/ml; 100 [t.1 of the diluted cell solution was added to each well;
and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 14). To each well of the 96-well plates that were cultured overnight, 50 [11 of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 14: Concentration of Sample (CB-60SK) After Dilution Concentration after Tube number dilution (mon) 4 4.5 5 1.1 6 0.3 7 0.07 8 0.02 9 0.004 10 0.001 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the Date Recue/Date Received 2021-07-27 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4E, wherein C value corresponds to IC5o), CB-605K has an inhibitory effect on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines.
Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The IC50 for cell lines LNCaP and HuH-7 with a relatively high expression of the receptors is significantly lower than that for cell lines T-47D, NCI-H460, CALU-3 and KB with a relatively low expression of the receptors. CB-605K shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
8. Experiment of inhibiting tumor cell expansion in CB-18G
Sample info' _______ "nation: CB-18G
Cell lines: A549 human lung cancer cells, Hela human cervical cancer cells, SCLC-21H small cell lung cancer cells, U-2 OS human osteosarcoma cells, T-47D
human breast cancer cells and NCI-H460 human lung cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; diluted with a complete cell culture medium, A549 cells, Hela cells, SCLC-21H cells and U-2 OS cells were plated at a density of 2 x 104 cells/ml, and T-47D cells and NCI-H460 cells were plated at a density of 3 x 104 cells/ml in 96-well plates; to each well of the 96-well plates, 100 [t.1 of the diluted cell solution was added; and negative control and blank control wells were set on each plate. The Date Recue/Date Received 2021-07-27 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 15). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 15: Concentration of Sample (CB-18G) After Dilution Concentration after Tube number dilution (mon) 3 6.25 4 1.56 5 0.39 6 0.10 7 0.02 8 0.006 9 0.002 0.0004 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
Date Recue/Date Received 2021-07-27 5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4F, wherein C value corresponds to IC50), CB-18G has an inhibitory effect on the growth and expansion of A549, Hela, SCLC-21H, U-2 OS, T-47D and NCI-H460 tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell line Hela with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-18G shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
9. Experiment of inhibiting tumor cell expansion in CB-50S
Sample infoimation: CB-505 Cell lines: KB human oral epideimoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 3.5 x cells/ml, LNCaP cells were diluted to 8.0 x 104 cells/ml, T-47D cells were diluted to 1.2 x 104 cells/ml, NCI-H460 cells were diluted to 2.5 x 104 cells/ml, and RT4 cells were diluted to 1.2 x 105 cells/ml; to each well of 96-well plates, 100 [1.1 of the diluted cell solution was added; and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 16). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank Date Recue/Date Received 2021-07-27 controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 16: Concentration of Sample (CB-50S) After Dilution Concentration after Tube number dilution (umol/L) 4 0.9 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 0.000008 5 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate 10 reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4G, wherein C value corresponds to IC5o), CB-505 has an inhibitory effect on the growth and expansion of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell lines KB, LNcap, T-47D and Date Recue/Date Received 2021-07-27 with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-50S
shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
10. Experiment of inhibiting tumor cell expansion in conjugate compound CB-1020 Experiment purpose: to test inhibitory effects of CB-1020 and related compounds on the expansion of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), NCI-H226 (human lung cancer cells), CFPAC-1 (pancreatic cancer cells) and PANC -1 (pancreatic cancer cells).
Related samples: MMAE and CB-1020 Experimental operations: LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 cells were prepared in advance and counted; with a culture medium (IMDM + 10% FBS + 1X L-Glutamine + 1X P/S), the cells LNCaP, SK-BR-3 and CFPAC-1 were diluted to 4 x 104 cells/ml, NCI-H226 was diluted to 2.0 x 104 cells/ml and PANC-1 was diluted to 3.0 x 104 cells/ml; the diluted cell solution was plated in 96-well plates at 100 [11/well; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 1/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P
fitting curve was plotted. The data is shown in the following table:
Table 17. Inhibitory Effects of CB-1020 on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) TRPV6 gene FOLH1 gene ICso ICso ICso Cell line expression expression (MMAE)/ICso (MMAE) (CB-1020) level level (CB-1020) LNCap 5+ 10+ 0.003 0.168 0.0179 Date Recue/Date Received 2021-07-27 SK-BR-3 4+ 2+ 0.002 0.287 0.0070 CFPAC -1 +/- 0.015 3.000 0.005 PANC-1 +/- +/- 0.002 0.458 0.0044 NCI-H226 +/- 0.006 1.570 0.00384 It can be seen from the above table that both LNCaP and SK-BR-3 express TRPV6 and FOLRH1, wherein LNCap has a relatively higher expression level of the two receptors. NCI-H226, CFPAC-1 and PANC-1 express the two receptors at a low or weak level. CB-1020 has an inhibitory effect on the growth and expansion of LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 tumor cell lines. The ICso ratio of CB-1020 for cell line LNCaP with a relatively high expression of the two receptors is higher than the IC50 ratio for cell line SK-BR-3 with a medium expression of the receptors; the ICso ratio for SK-BR-3 is higher than the IC50 ratios for CFPAC-1 with a relatively low expression of the receptors and cell lines NCI-H226 and PANC-1 with a weak expression of the two receptors. The inhibitory effect on cell proliferation is positively correlated with the expression level of the two receptors in the cells.
11. Experiment of inhibiting tumor cell expansion in conjugate compound CB-1320 Experiment purpose: to test inhibitory effects of CB-1320 and related compounds on the expansion of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), MDA-MB-468 (human breast cancer cells) and CFPAC-1 (pancreatic cancer cells).
Related samples: MMAE and CB-1320 Experimental operations: LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 cells were prepared in advance and counted; with a culture medium (IMDM + 10%
FBS + lx L-Glutamine + 1X P/S), the cells LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 were diluted to 4 x 104 cells/ml; the diluted cell solution was plated in 96-well plates at 100 ul/well; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 1.11/well. The plates were placed in an incubator at 37 C, 5%
Date Recue/Date Received 2021-07-27 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The specific data is shown in the following table:
Table 18. Inhibitory Effects of CB-1320 on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) FOLH1 gene ICso GNRHR gene ICso ICso Cell line expression (MMAE)/1C50 expression level (MMAE) (CB-1320) level (CB-1320) LNCaP +/- 10+ 0.003 0.060 0.043 SK-BR-3 +/- 2+ 0.002 0.093 0.016 CFPAC-1 +/- +/- 0.015 1.090 0.014 MDA-MB-468 +/- +/- 0.002 0.212 0.011 It can be seen from the above table that the expression levels of GNRHR in the 4 cell lines are roughly equivalent, and the expression level of FOLH1 in LNCaP is significantly higher than that in other cell lines. CB-1320 has an inhibitory effect on the expansion of 4 tumor cell lines. The inhibitory effects of CB-1320 on the cells with different expression levels of receptor FOLH1 are different. The ICso ratio in cell line LNCap with a relatively high expression of receptor FOLH1 is significantly higher than the ICso ratio in other cell lines with a relatively low expression of the receptor.
o) ¨
NH A HN
OH
(s) SH
;JO 0 0OH HO
p H H H H
0 N p NkSAN kl1)-LN
H i H H
HO
1820BK-SMO9 o 41 ',, NH
,t) HO do 0 HN 0 ,, HN-3\
os) ss 0 NH2 HN 0 H r-o-õ....s: (,õi-I, ? (-S) N 0 (21 HN r NH i H H
3- 0 0 (157 0 HN (s) (5/4 - ' 0 'OH , j ) 0 ki ) I-K/(-17 HO _c _.....1s) NH
HO
H2N) 0 H2N \O
(R) HO (s) r\l)N1N)N 0 .,,,(0 0 0 H H H H H , H
/
0 NkS),.)-L.
....i.kl.õ....¨..õ--,}___ N (s) NH
N (s) 0 HO
H H H
N )-L N ,(s-L N
0 0 b 0 , N
N
\
¨ OH
:
p -o-Date Recue/Date Received 2021-07-27 In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application enters the blood circulation and the outside of cells (in an intercellular substance). Since the linker is very stable in an extracellular environment and drug molecules cannot be released, the toxicity of the drug molecules is blocked. The conjugate is a drug with no cytotoxicity or with a low toxicity and will not have a toxic effect on normal cells.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application binds to multiple receptors or antigens and other acting molecules that are simultaneously highly expressed on diseased cells, and the synergistic effect thereof greatly increases the affinity of the conjugate compound to target cells, reducing the possibility of binding to normal cells. Therefore, a highly effective toxin drug such as MMAE/Dxd/5N38/a radionuclide complex can be carried to enhance the efficacy of drugs, broaden a therapeutic window and avoid drug side effects.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application enters the interior of targeted cells, and then the linker can be cleaved through changes of the internal environment of the cells (by a specific enzyme digestion, a pH change, a disulfide bond reduction, etc.) to release drug molecules (equivalent to removing modifying groups of drug molecules), which has a therapeutic effect on tumor cells.
In some embodiments, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application can be used to specifically deliver a payload to target cells in a target tissue environment. Generally, the two targeting molecules of the conjugate compound or the pharmaceutically acceptable salt thereof have three advantages. Firstly, the two targeting molecules can act in multiple modes (often synergistically), resulting in improved therapeutic effects while reducing side effects. Secondly, the binding of the two targeting molecules increases the affinity and avidity of the conjugate compound or the pharmaceutically acceptable salt thereof to target receptors or target cells, thereby enhancing its specificity and avoiding off-target toxicity. Finally, when properly Date Recue/Date Received 2021-07-27 designed, a combination of the two targeting molecules can fulfill multi-functional properties required for a drug conjugate.
The conjugate compound or the pharmaceutically acceptable salt thereof of the present application achieves unexpected technical effects, including but not limited to: (1) a combination of a ligand capable of binding to cell surface receptors and a endocytosis molecule capable of mediating endocytosis enable the conjugate compound to specifically enter target cells; (2) the conjugate compound or the pharmaceutically acceptable salt thereof enhances an affinity and targeting specificity of drug compounds, thereby delivering highly effective chemotherapeutic agents such as MMAE to a patient, broadening the therapeutic window of such agents and avoiding side effects; (3) a linker can prevent the release of a payload outside of target cells (for example, in a blood circulation system, intercellular substance, etc.), which ensures the stability of the conjugate compound during the blood circulation, and reduces the toxicity of the drug; after entering target cells, the linker is cleaved to release the payload and exert the effect of the drug, and meanwhile multiple drug resistance (MDR) can be avoided; and (4) a wide variety of drugs may be delivered in a form of the conjugate compound of the present application, and therefore, the application scopes of relevant drugs are widened. Therefore, the conjugate compound or the pharmaceutically acceptable salt thereof of the present application not only broadens the targeting scope and therapeutic window of LDC-based drugs, but also reduces toxicity and side effects of some drugs.
The terms "polypeptide", "protein" and "peptide" as used herein can be a single amino acid or a polymer of amino acids. The polypeptide, protein or peptide as described in the present application may contain naturally-occurring amino acids and non-naturally-occurring amino acids, or analogs and mimetics thereof The polypeptide, protein or peptide can be obtained by any method well known in the art, for example, but not limited to, by an isolation and a purification from natural materials, a recombinant expression, a chemical synthesis, etc.
In another aspect, the present application discloses a pharmaceutical composition comprising the conjugate compound or the pharmaceutically Date Recue/Date Received 2021-07-27 acceptable salt thereof provided in the present application, and a pharmaceutically acceptable carrier.
The term "pharmaceutically acceptable" as used herein means, within the scope of sound medical judgment, being suitable for contact with cells of human beings and other animals without undue toxicity, irritation, allergic response, etc., and being commensurate with a reasonable benefit/risk ratio.
The term "pharmaceutically acceptable salt" as used herein refers to a relatively non-toxic, inorganic and organic acid addition salt and base addition salt, of the conjugate compound of the present application. Representative acid addition salts include hydrobromides, hydrochlorides, sulfates, bisulfates, phosphates, nitrates, acetates, oxalates, valerates, oleates, palmitates, stearates, laurates, borates, benzo ate s, lactates, phosphates, to sylate s, citrates, male ate s, fumarates, succinates, tartrates, naphthylates, me sylate s, glucoheptonates, lactiobionates, sulphamates, malonates, salicylates, propionates, methylene-b is -b-hydroxynaphthoate s, genti s ate s, is ethionate s, di-p-toluoyltartrates, methane sulphonate s, ethane sulphonate s, benzene sulphonate s, p-toluenesulphonates, cyclohexylsulphamates, quinateslaurylsulphonate salts, and the like. Base addition salts include pharmaceutically acceptable metal and amine salts. Suitable metal salts include sodium, potassium, calcium, barium, zinc, magnesium, and aluminum salts. In some embodiments, sodium and potassium salts are preferred. Suitable inorganic base addition salts are prepared from metal bases which include, for example, sodium hydride, sodium hydroxide, potassium hydroxide, calcium hydroxide, aluminum hydroxide, lithium hydroxide, magnesium hydroxide, and zinc hydroxide.
Suitable amine base addition salts are prepared from amines which have sufficient basicity to form a stable salt, and preferably include the following amines which are frequently used in medicinal chemistry because of their low toxicity and acceptability for medical use: ammonia, ethylenediamine, N-methyl-glucamine, lysine, arginine, ornithine, choline, N,N' -dibenzylethylenediamine, chloroprocaine, diethanolamine, procaine, N-benzylphenethylamine, diethylamine, piperazine, tris(hydroxymethyl)-aminomethane, tetramethylammonium hydroxide, triethylamine, dibenzylamine, ephenamine, dehydroabietylamine, N-ethylpiperidine, Date Recue/Date Received 2021-07-27 benzylamine, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, ethylamine, basic amino acids, e.g., lysine and arginine, dicyclohexylamine, and the like.
The term "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable solvent, suspension or any other pharmacologically inert vehicle for delivering the conjugate compound provided in the present application to a subject, without interfering with the structures and properties of the conjugate compound. Such carriers enable the conjugate compound to be formulated as, for example, tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and pastilles, for oral ingestion by a subject. Such carriers enable the conjugate compound to be formulated as injections, infusions or preparations for local administration.
The pharmaceutically acceptable carriers for use in the pharmaceutical composition provided in the present application may include, but are not limited to, for example, pharmaceutically acceptable liquids, gels, or solid carriers, aqueous vehicles (such as sodium chloride injection, Ringer's injection, isotonic dextrose injection, sterile water injection, or dextrose and lactated Ringer's injection), nonaqueous vehicles (such as fixed oils derived from vegetables, cottonseed oil, corn oil, sesame oil, or peanut oil), antimicrobial agents, isotonic agents (such as sodium chloride or dextrose), buffers (such as phosphate or citrate buffers), antioxidants (such as sodium bisulfate), anesthetics (such as procaine hydrochloride), suspensions/dispersions (such as sodium carboxymethylcellulose, hydroxypropyl methylcellulose, or polyvinylpyrrolidone), chelating agents (such as EDTA (ethylenediamine tetraacetic acid) or EGTA (ethylene glycol tetraacetic acid)), emulsifying agents (such as polysorbate 80 (Tween-80)), diluents, adjuvants, excipients or non-toxic auxiliary substances, other components well known in the art, or various combinations thereof Suitable components may include, for example, fillers, binders, buffers, preservatives, lubricants, flavoring agents, thickening agents, coloring agents, or emulsifying agents.
In some embodiments, the pharmaceutical composition is an injection preparation. The injection preparations include sterile water solutions or dispersions, Date Recue/Date Received 2021-07-27 suspensions or emulsions. In all cases, the injection preparations should be sterile and be a fluid for easy injection. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carriers can be solvents or dispersion mediums containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof and/or vegetable oils. The injection preparations should maintain appropriate fluidity. The appropriate fluidity can be maintained, for example, by the use of coatings such as lecithin, by the use of surfactants, and the like.
Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
In some embodiments, the pharmaceutical composition is an oral preparation.
The oral preparations include, but are not limited to, capsules, cachets, pills, tablets, lozenges (using a flavored basis, usually sucrose and acacia or tragacanth), powders, granules, or as solutions or suspensions in aqueous or non-aqueous liquids, or as oil-in-water or water-in-oil liquid emulsions, or as elixirs or syrups, or as pastilles (using an insert base, such as gelatin and glycerin, or sucrose and acacia) and/or as mouth washes.
In solid dosage forms for oral administration (e.g., capsules, tablets, pills, dragees, pulvis, granules and the like), the conjugate compound is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and/or any of the followings: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and/or silicic acid; (2) binders, such as carboxymethylcellulose, alginates, gelatins, polyvinyl pyrrolidone, sucrose and/or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain silicates, and sodium carbonate; (5) solution retarding agents, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as acetyl alcohol and glycerol monostearate; (8) absorbents, such as kaolin and bentonite clay; (9) lubricants, such as talc, calcium stearate, magnesium stearate, Date Recue/Date Received 2021-07-27 solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.
In liquid dosage forms for oral administration, the conjugate compound is mixed with any of the followings: pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups and elixirs. In addition to the conjugate compound, the liquid dosage forms may contain inert diluents commonly used in the art, such as, water or other solvents, solubilizing agents and emulsifying agents, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, isopropanol, 1,3-butylene glycol, oils (in particular, cottonseed oil, peanut oil, corn oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofurfuryl alcohol, polyethylene glycol and fatty acid esters of sorbitan, and mixtures thereof Besides inert diluents, an oral composition can also include adjuvants such as wetting agents, emulsifying agents and suspensions, sweetening agents, flavoring agents, coloring agents, perfuming agents and preservatives.
In some embodiments, the pharmaceutical composition is a mouth spray preparation or a nasal spray preparation. The spray preparations include, but are not limited to, aqueous aerosols, nonaqueous suspensions, lipidosome preparations or solid granular preparations. Aqueous aerosols are prepared by mixing aqueous solutions or suspensions of agents with conventional pharmaceutically acceptable carriers and stabilizers. The carriers and stabilizers vary according to the requirements of specific compounds, but in general, they include nonionic surfactants (Tweens or polyethylene glycol), oleic acid, lecithin, amino acids such as glycine, buffer solution, salts, sugar or sugar alcohol. Aerosols are generally prepared from isotonic solutions, and can be delivered by sprayers.
In some embodiments, the pharmaceutical composition can be used by mixing with one or more other drugs. In some embodiments, the pharmaceutical composition comprises at least one other drug. In some embodiments, the other drugs are antineoplastic drugs, cardiovascular drugs, anti-inflammatory drugs, antiviral drugs, digestive system drugs, nervous system drugs, respiratory system drugs, immune system drugs, dermatologic drugs, metabolic drugs, and the like.
Date Recue/Date Received 2021-07-27 In some embodiments, the pharmaceutical compositions can be administered to a subject in need thereof by appropriate routes, including without limitation, oral, injection (such as intravenous, intramuscular, subcutaneous, intracutaneous, intracardiac, intrathecal, intrapleural and intraperitoneal injection), mucosal (such as nasal and intraoral administration), sublingual, rectal, percutaneous, intraocular, and pulmonary administration. In some embodiments, the pharmaceutical composition can be administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly.
Due to the properties of some payloads, such as high toxicity and high hydrophilicity, it is desired to deliver the payloads more specifically and more efficiently to a subject in need thereof For example, in cancer treatment, it is desired to specifically deliver chemotherapeutic agents to cancer cells without toxicity to normal cells. Therefore, in another aspect, the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application, or the pharmaceutical composition provided in the present application.
The payloads described in the present application may be any pharmaceutical agent that elicits biological or medicinal responses in a tissue, system, animal, individual or human that is being sought by a researcher, veterinarian, medical doctor or other clinicians in preventing, inhibiting, ameliorating or treating a disease.
The term "object" as used herein refers to human and non-human animals.
Non-human animals include all vertebrates, for example, mammals and non-mammals. The subject may also be a livestock animal such as cattle, swine, sheep, poultry and horse, or a domestic animal such as dog and cat. The subject may be male (e.g. man) or female (e.g. woman), may be elderly, and may be an adult, adolescent, child, or infant. A human subject may be Caucasian, African, Asian, Semitic, or human with other racial backgrounds or a mixture of such racial backgrounds.
The term "therapeutically effective amount" as used herein refers to an amount of the conjugate compound or the pharmaceutically acceptable salt thereof, or the Date Recue/Date Received 2021-07-27 pharmaceutical composition which relieves to some extent one or more symptoms of a disease or disorder in a subject, returns to normal either partially or completely one or more physiological or biochemical parameters associated with or causative of the disease or disorder, and/or reduces the likelihood of the onset of the disease or disorder. Such amounts generally vary according to a number of factors which can be detettnined and explained, according to the scope of the specification provided in the present application, by those of ordinary skill in the art.
These factors include, without limitation: the particular subject and the age, weight, height, general physical condition, and medical history thereof, the particular compound used, the carrier of the preparation and the administration route selected, and the nature and severity of the condition being treated.
In some embodiments, the amount of the conjugate compound, or the pharmaceutically acceptable salt thereof, or the pharmaceutical composition is sufficient to inhibit a disease or disorder in a subject, or prophylactically inhibit or prevent the onset of a disease or disorder in a subject. Although the therapeutically effective amount may vary in different subjects, it is generally ranged from 0.01 to 100 mg/kg, for example, 0.01 to 90 mg/kg, 0.01 to 80 mg/kg, 0.01 to 70 mg/kg, 0.01 to 60 mg/kg, 0.01 to 50 mg/kg, 0.01 to 40 mg/kg, 0.01 to 30 mg/kg, 0.01 to 20 mg/kg, 0.01 to 10 mg/kg, 0.01 to 5 mg/kg, 0.01 to 4 mg/kg, 0.01 to 3 mg/kg, 0.01 to 2 mg/kg, 0.01 to 1 mg/kg, and 0.01 to 0.1 mg/kg. The therapeutically effective amount as described in the present application can be equal to any value within the above numerical range, including the end values of this range.
In another aspect, the present application discloses a method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof provided in the present application, or the pharmaceutical composition provided in the present application.
In another aspect, the present application discloses a method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt Date Recue/Date Received 2021-07-27 thereof provided in the present application, or the pharmaceutical composition provided in the present application.
In some embodiments, the disease is a cancer, including but not limited to, prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, thyroid cancer, pancreatic cancer, colon cancer, colorectal cancer, esophageal cancer, skin cancer, lymphoma, and multiple myeloma.
In some embodiments, cancer cells of the cancers have an expression of the cell surface receptors or antigens mentioned in the present application. In some embodiments, cancer cells of the cancers have a high expression (for example, according to data from Depmap (see: https://depmap.org/portal/), the corresponding gene expression is at least 0, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more than 10) of cell surface receptors or antigens mentioned in the present application.
In some embodiments, cancer cells of the cancers have a high expression of and FOLH1, TRPV6 and FOLH1, GNRHR and FOLH1, SSTR2 and FOLH1, FOLR1 and SSTR2, or TRPV6 and FOLR1. In some embodiments, the disease is an immunological disease, for example, an autoimmune disease, including but not limited to, connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
In some embodiments, the disease is a cardiovascular disease, including but not limited to, angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital heart disease.
In some embodiments, the disease is a metabolic disease, including but not limited to, diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
In some embodiments, the disease is a neurological disease, including but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
In some embodiments, the method provided in the present application further comprises administering one or more therapeutic agents in combination with the conjugate compound or the pharmaceutically acceptable salt thereof, or the Date Recue/Date Received 2021-07-27 pharmaceutical composition. In some embodiments, the therapeutic agents target an anti-cancer therapeutic target, induce or boost an immune response against cancer, or are chemotherapeutic agents.
The present application will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes only, and are not intended to limit the invention in any manner. A person skilled in the art will readily recognize a variety of noncritical parameters which can be changed or modified to yield essentially the same results.
EXAMPLES
The following examples are intended to further illustrate the present application. The advantages and features of the present application will become clear with the descriptions. However, these illustrations are merely exemplary, and should not be constructed as limitations to the scope of the present application.
Example 1: Preparation of Conjugate Compounds Synthesis of conjugate compounds CB-20BK, CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE, CB-20AK, CB-1020, CB-1320 and CB-1820 1. 10 g of Rink amide-am resin (hereafter referred to as "Rink Resin", from Sunresin New Materials Co. Ltd., Cat#: 183599-10-2) with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM
was added, and nitrogen gas was bubbled through the solvent to swell the resin for minutes; the solvent was pumped off; and Fmoc protecting groups on the resin were removed by DBLK, and then the resin was washed 5 times with DMF. 4.79 g (9 mmol) of Fmoc-Lys(Dde)-OH and 1.47 g (10.8 mmol) of HOBt were weighed 25 and dissolved in DMF. To the above-mentioned solution at 0 C in an ice-water bath, 1.67 ml (10.8 mmol) of DIC was added and mixed to activate for 5 minutes. The solution was added to the above-mentioned reaction column and reacted for 3 hours, and then the solvent was pumped off; and the resin in the reaction column was washed 3 times. Then Fmoc protecting groups were removed by DBLK.
Date Recue/Date Received 2021-07-27 2. The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H, Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate 108 were successively conjugated according to the structures.
tBuO 0 0 ,OtB u tBuO (s) N N N 0 H H H H
OH
(Intel _______ me diate 108) 3. Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated successively. Finally, the resin was condensed twice with methanol, and the solvent was pumped off to obtain 17.4 g of a protected peptide resin.
4. 17.4 g of the peptide resin obtained in the previous step was added to a 250 ml single-necked flask; 139 ml of lysis buffer (TFA : H20 : TIS = 95 : 3 :
(volume ratio)) was previously prepared, and 2.1 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the above-mentioned flask, and the mixture was reacted at room temperature for 2.5 hours and filtered; the resin was continued to be washed with 30 ml of TFA; the above-mentioned filtrate was combined, and the mixture was added to 834 ml of absolute ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 6.4 g of a crude product as a yellow solid and with a yield of 93.4% and an HPLC purity of 82.3%. The product was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60 minutes;
fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 4.73 g of 20BK-SM09, with a purity of 98.8%.
5. 4.09 g (3.11 mmol) of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a 1000 ml single-necked flask, and 500 ml of phosphate buffer and 100 ml of acetonitrile were added; the solution was stirred and maintained at pH =
7.2 until a clear solution was obtained; and 4.73 g (3.11 mmol) of inteimediate Date Recue/Date Received 2021-07-27 20BK-SM09 was added, and the mixture was reacted at room temperature for 2 hours, during which the reaction was monitored by HPLC. After the reaction was completed, the mixture was filtered, and the filtrate was separated by prep-HPLC
(condition: C18 column, mobile phase A: ammonium bicarbonate solution (pH =
7.2), B: acetonitrile, elution gradient: (25-35)%B, elution time: 60 minutes;
fractions were collected); the fraction containing the qualified product was lyophilized to obtain 6.96 g of CB-20BK product, with a purity of 98.8% and a yield of 78.8%.
In the same way, conjugate compounds CB-18G, CB-20B, CB-10S, CB-20C, FA-MMAE (with a structure shown as follows), CB-20AK (with a structure shown as follows), CB-1020, CB-1320 and CB-1820 can be obtained through similar steps in the above-mentioned methods.
H2NyJN
, NH
0 OH 0 oH 0 0 4N NH N LC)JL1 N
(R) S 0 H 8 H 0 H I
N
,OH
(s) (FA-MMAE) HO
0 C) HO H Y N Cri4õ))))),,1 -101 (s) J(rA N (s) NF-o o r_f(.ti 0 H
HO
0 OyN/S,LLN N r 0 H 0 (s) }D
(CB-20AK) Synthesis of conjugate compounds CB-50S, CB-60S and CB-60SK
1. 10 g of Wang Resin (from Sunresin New Materials Co. Ltd., Cat#:
1365700-43-1) with a degree of substitution of 1.1 mmolig was weighed and loaded onto a solid phase reaction column; DMF was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes; 14.3 g (22 mmol) of Date Recue/Date Received 2021-07-27 Fmoc-Arg(pbf)-0H, 3.56 g (26.4 mmol) of HOBt, and 0.27 g (2.2 mmol) of DMAP
were weighed and dissolved in DMF; at 0 C in an ice-water bath, 4.1 ml (26.4 mmol) of DIC was added and mixed to activate for 5 minutes; the solution was added to the reaction column and reacted for 3 hours, and then the solvent was pumped off; and the resin was washed 3 times.
2. 10.4 ml of acetic oxide and 8.9 ml of pyridine were dissolved in 50 ml of DMF and mixed, and the resin after washing in the above steps was added; the mixture was blocked at room temperature for 5 hours and washed three times with DMF; and the resin was condensed with methanol, and then the solvent was pumped off to obtain Fmoc-Arg(pbf)-Wang Resin, which has a degree of substitution detet __ mined to be 0.53 mmol/g.
3. 3.8 g (2 mmol) of Fmoc-Arg(pbf)-Wang Resin (Sub = 0.53 mmol/g) was weighed and loaded onto a reaction column; the resin was washed 3 times with DMF and then was swelled for 30 minutes by means of adding DMF. Then Fmoc protecting groups were removed by DBLK, and the resin was washed 6 times with DMF. 2.0 g (6 mmol) of Fmoc-Pro-OH and 0.97 g (7.2 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 1.1 ml (7.2 mmol) of DIC was added and mixed to activate for 5 minutes; the mixture was added to the reaction column and reacted for 2 hours; and then Fmoc protecting groups were removed by DBLK.
4. The above-mentioned operations were repeated, and Fmoc-Leu-OH, Fmoc-Asp(OtBu)-0H, Fmoc-Val-OH, Fmoc-Lys(Boc)-0H, Fmoc-Ser(tBu)-0H, Fmoc-Pro-OH, Fmoc-His(Trt)-0H, Fmoc-Leu-OH, Fmoc-Phe-OH, Fmoc-Glu(OtBu)-0H, Fmoc-Lys(Boc)-0H, Fmoc-propargyl-Gly-OH, Fmoc-Glu-OtBu and pteroic acid were conjugated successively according to the structures. The resin was condensed twice with methanol, and the solvent was pumped off to obtain 8.4 g of a peptide resin.
5. 8.4 g of the peptide resin obtained in the previous step was added to a ml single-necked flask; 67 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 0.92 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was reacted at Date Recue/Date Received 2021-07-27 room temperature for 2.5 hours; the resin was filtered and washed with 20 ml of TFA; the filtrate was combined, and the mixture was added to 402 ml of absolute ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 4.06 g of a crude product as a yellow solid and with a yield of 97.3% and an HPLC purity of 84.6%.
The product was separated by prep-HPLC (condition: C18 column, mobile phase A:
0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-29)%B, elution time: 60 minutes; fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 2.86 g of 50S-SM09, with a purity of 97.6%.
6. 1.29 g (1.37 mmol) of LT1000N3 was weighed and added to a 500 ml single-necked flask, and 270 ml of a mixed solvent (CAN : H20 = 1 : 4) and 393 mg (2.74 mmol) of CuBr were added and stirred. 2.86 g (1.37 mmol) of intermediate 50S-SMO9 was added, and the mixture was reacted at room temperature for 2-3 hours, during which the reaction was monitored by HPLC.
After the reaction was completed, the mixture was filtered, and the filtrate was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (22-40)%B, elution time: 60 minutes;
fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 3.17 g of CB-50S, with a purity of 98.6% and a yield of 76.4%.
In the same way, conjugate compounds CB-60S and CB-60SK can be obtained through similar steps in the above-mentioned methods.
Synthesis of CB-20R
1. 5 g of Rink Resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes; the solvent was pumped off; Fmoc protecting groups on the resin were removed by DBLK; and then the resin was washed 5 times with DMF. 2.4 g (4.5 mmol) of Fmoc-Lys(Dde)-OH and 0.74 g (5.4 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.84 ml (5.4 mmol) of DIC was added and mixed Date Recue/Date Received 2021-07-27 to activate for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped off, and the resin was washed 3 times.
Then Fmoc protecting groups were removed by DBLK.
2. The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H, Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate 108 were successively conjugated according to the structures.
3. Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. DOTA-tris(tBu) ester was conjugated. Then Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. Fmoc-Glu-OtBu and pteroic acid were conjugated successively, and finally, the resin was condensed twice with methanol and the solvent was pumped off to obtain 9.2 g of a protected peptide resin.
4. 9.2 g of the peptide resin obtained in the previous step was added to a ml single-necked flask; 74 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 1.05 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml of TFA; the filtrate was combined, and the mixture was added to 560 ml of absolute ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 3.8 g of a crude product as a yellow solid and with a yield of 87.4% and an HPLC purity of 81.2%.
The product was separated by prep-HPLC (condition: C18 column, mobile phase A:
0.1% trifluoroacetic acid in water, B: acetonitrile, elution gradient: (15-25)%B, elution time: 60 minutes; fractions were collected); and the fraction containing a synthetic product was lyophilized to obtain 2.9 g of 20R-SM09, with a purity of 97.8%.
5. 20R-SMO9 was complexed with radionuclide ion M to obtain CB-20R.
Specifically, the radioactive label 177Lu (about 50 MBq) was mixed with 100 [t.1 of 0.5 M sodium acetate buffer (pH = 5). 40 [t.1 of aqueous solution of 1 mM CB-dissolved in 10% DMSO, 2 1 of saturated ascorbic acid solution and 100 [t.1 of Date Recue/Date Received 2021-07-27 solution containing 177Lu were mixed, and the mixture was heated to 95 C for minutes. The label was detected by radio-HPLC (within 5 minutes; 0%-100% ACN
in water; C18 column).
Synthesis of compound CR19425 --- `-..'11112 44080311 11 9 g *õ..õ.=-- ..1 i N..., ...)..,.....),. ,,,,, 1 Molecular weight : 324.396 (7i's 'NH F N
tis ,---, Si... ` CR19420 . , . 4:t , Piped(MK
....., b simi HATUDIPEA Y X 114_,(P
., HO-,:'-"-µ,N ".=, = -.'", -,, , ,)---sil.õ,i,J
Molecular weight : 435.46 0N19418 FICrts, \ b Molecular weight : 801,8.284 11''r"'il i 1 rt, 0 ? H Y
Q
0 - soH
Molecular weight : 472.60 ---l',..%, ..-, HATU,DIPEA
F '' 'N
A.µ,..,..
P
\ b Molecular weight : 57'9.5854 eft18422 ., ri n ri i N ,,..,..i.õ . , . N. ,.." = i- , ,,..F
'0' 0 N µ!,;coshio9 H " , -. . Ni __ o= CR19425 , . ...
Molecular we i dm :1 034,01 N ..:=f ., Molecular weight: 3126.43/4 Cil1-113423, .0-0:
1. Under N2 protection, to a reaction flask, 441.4 mg of CR19420 (with a structure as shown in the above reaction step) and 8.0 mL of DMF were added, 10 stirred, dissolved and cooled in an ice bath; then 459.2 mg of HATU and 380 [IL of Date Recue/Date Received 2021-07-27 DIPEA were added and stirred for half an hour; and then 500.0 mg of CR19419 (with a structure as shown in the above reaction step) and 190 [IL of DIPEA
were added, and the mixture was reacted at room temperature until the reaction was completed. After the reaction was completed, the reaction solution was poured into an acetic acid aqueous solution; a solid was precipitated and filtered, and the filter cake was washed with an acetic acid aqueous solution and water, and dried in vacuo to obtain 835.7 mg of CR19421 (with a structure as shown in the above reaction step) as a brown powder and with an HPLC purity of 90.0% and a yield of 90.6%.
2. Under N2 protection, to a reaction flask, 835.7 mg of CR19421 and 16 mL
of 10% piperidine DMF solution were added and reacted at room temperature for half an hour. After the reaction was completed, the reaction solution was poured into TFA/MTBE; a solid was precipitated and filtered, and the filter cake was washed with MTBE and dried in vacuo to obtain 599.7 mg of CR19422 (with a structure as shown in the above reaction step) as a field gray powder and with an HPLC
purity of 84.7% and a yield of 83.0%.
3. Under N2 protection, to a reaction flask, 599.7 mg of CR19422, 586.7 mg of CR19424 (with a structure as shown in the above reaction step) and 15 mL of DMF were added, stirred, dissolved and cooled in an ice bath; then 495.7 mg of HATU and 410 [IL of DIPEA were added and reacted at room temperature. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the acetonitrile was removed under reduced pressure from the pure product solution; the residue was extracted with a mixed solvent of dichloromethane and methanol, concentrated and dried to obtain 674.9 mg of CR19423 (with a structure as shown in the above reaction step) as a yellow powder and with an HPLC purity of 88.4% and a yield of 63.1%.
4. Under N2 protection, to a reaction flask, 14.8 mg of CR19423, 3.0 mL of PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and dissolved;
then 32.9 mg of CBSMO9 was added, and the mixture was adjusted to pH 6.6-6.8 with Na2HPO4 and reacted for half an hour. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the pure product Date Recue/Date Received 2021-07-27 was lyophilized to obtain 21.8 mg of CR19425 as a yellow powder and with an HPLC purity of 95.6% and a yield of 48.8%.
Synthesis of compound CR19426 H ' 1 , e`ir = 'N y'"*. 0 e' =:' :
H
t4: N = =L, Molecular weight :10t)7 N
Molecular weight : 3312.63 CRI.S4.0 ,OH
1. Under N2 protection, to a reaction flask, 25.2 mg of CR19423, 3.0 mL of PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and dissolved;
then 55.5 mg of 18G-5M09 was added, and the mixture was adjusted to pH 6.6-6.8 with Na2HPO4 and reacted for half an hour. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the pure product 113 was lyophilized to obtain 44.6 mg of CR19426 as a yellow powder and with an HPLC purity of 95.4% and a yield of 55.2%.
Synthesis of compound CR19428 .0 t rt - z H. " CR19428 4 ?
MO leCUlar t:tglit. )3.4 07 Molecular weight : 25b,6384 0114043 .0H
.(;) 1. Under N2 protection, to a reaction flask, 486 mg of CR19423, 3.0 mL of PBS buffer (pH = 6.6) and 3.0 mL of acetonitrile were added, stirred and dissolved;
then 712 mg of 20BK-SMO9 was added, and the mixture was adjusted to pH 6.6-6.8 with Na2HPO4 and reacted for half an hour. After the reaction was completed, the reaction solution was subjected to a preparative purification, and the pure product Date Recue/Date Received 2021-07-27 was lyophilized to obtain 508 mg of CR19428 as a yellow powder and with an HPLC purity of 96.7% and a yield of 42.3%.
Example 2: Determination of Affinity of Conitmate Compounds to Tamet Proteins 1. Determination of binding affinity of CB-20BK to protein FOLR1 Experimental instruments, materials and reagents:
BIAcore T200 (GE) CMS chip (GE, Cat#: 29104988) Buffer: HBS-EP+ buffer 10X (GE, Cat#: BR100669), diluted 10 folds with deionized water before use.
Amino coupling kit (GE, Cat#: BR100050) Regeneration reagents: 10 mM Glycine 2.0 (GE, Cat#: BR100355) 10 mM Glycine 3.0 (GE, Cat#: BR100357) Experimental steps The experiment was carried out according to BIAcore T200 (GE) instruction manual to determine the affinity of analytes CB-20BK, CB-20AK, folate (FA) and FA-MMAE to FOLR1. In the experiment, CMS chip was conjugated with ligand FOLR1 (R&D System, Cat#: 5646-FR). The experimental results are as shown in Table 4.
Table 4. Binding Affinity of CB-20BK and Related Compounds to FOLR1 ka kd KD
FA (folate) 3.34 x 106 M's' 2.26 x 10-4 s-1 6.77 x FA-MMAE 1.79 x 10 M's' 1.15 x 10-4 s-1 6.43 x CB-20BK 1.03 x 10 M's' 1.31 x 10-4 s-1 1.27 x "N/D" means that no specific binding was detected.
Table 4 shows that CB-20BK specifically binds to FOLR1 with a good affinity.
The binding affinity of CB-20BK to FOLR1 is slightly weaker than the binding affinity of FA or FA-MMAE to FOLR1. CB-20AK does not comprise the folate Date Recue/Date Received 2021-07-27 moiety of CB-20BK and has not been detected to specifically bind to FOLR1 in the experiment.
2. Determination of bindin2 affinity of CB-20BK to protein FOLH1 Experimental instruments, materials and reagents:
GatorTM (Probe Life) SA probe (Probe Life, Cat#: 1906018) Buffer: Q buffer (Probe Life), 10 mM, pH = 7.4 Experimental steps (1) Synthesis steps of analyte Biotin-CB-20BK
1) 5.1 g of Rink Resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM was added, and nitrogen gas was bubbled to swell the resin for 30 minutes; the solvent was pumped off; Fmoc protecting groups were removed by DBLK; and then the resin was washed 5 times with DMF. 2.45 g of Fmoc-Lys(Dde)-OH and 0.75 g of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.83 ml of DIC was added to activate for 5 minutes; the mixture was added to the reaction column and reacted for 3 hours; and the solvent was pumped off, and the resin was washed times. Then Fmoc protecting groups were removed by DBLK.
2) The above-mentioned operations were repeated, and Fmoc-Cys(Trt)-0H, Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and inteitnediate 108 were successively conjugated according to the structures.
3) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF.
Fmoc-Lys(Biotin)-0H, Fmoc-Glu-OtBu and pteroic acid were conjugated successively, and after pteroic acid was conjugate, the resin was washed twice with DMF; and finally, the resin was condensed with methanol, and the solvent was pumped off to obtain 9.7 g of a protected peptide resin.
4) 9.7 g of the peptide resin obtained in the previous step was added to a 250 ml single-necked flask; 77.6 ml of lysis buffer (TFA : H20 : TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 1.1 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was Date Recue/Date Received 2021-07-27 reacted at room temperature for 2.5 hours; the resin was filtered and washed with 20 ml of TFA; the filtrate was combined, and the mixture was added to 466 ml of methyl tert-butyl ether, with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with methyl tert-butyl ether and dried in vacuo to obtain 4.6 g of a yellow solid with an HPLC purity of 83.2%. The product was separated by prep-HPLC and lyophilized to obtain 2.1 g of Biotin-20BK-SM09 with a purity of no less than 95%.
5) 500 mg of Mc-Val-Cit-PAB-MMAE (LT1002) was weighed and added to a 250 ml single-necked flask, and 65 ml of phosphate buffer and 20 ml of acetonitrile were added; the solution was stirred and maintained at pH = 7.2 until a clear solution was obtained; and 785 mg of intermediate Biotin-20BK-SMO9 was added, and the mixture was reacted at room temperature for 2 hours, during which the reaction was monitored by HPLC. After the reaction was completed, the mixture was filtered, separated by prep-HPLC, and lyophilized to obtain 723 mg of Biotin-CB-20BK, with a purity of 96.8% and a yield of 59.4%.
(2) The experiment was carried out according to GatorTM (Probe Life) instruction manual to determine the binding affinity of Biotin-CB-20BK to FOLH1.
In the experiment, SA probe binds to ligand Biotin-CB-20BK, and the analyte is FOLH1 (Sino Biologicals, Cat#: 15877-H07H). The experimental results are as shown in Table 5.
Table 5. Binding Affinity of CB-20BK to FOLH1 ka kd KD
FOLH1 1.19 x 104 A4-1s-1 1.70 x 10-4 s-1 6.98 x 10-9 M
Table 5 shows that CB-20BK binds to FOLH1 with a good affinity.
3. FOLH1 binds to CB-20BK-FOLR1 complex Experimental materials and reagents:
BIAcore T200 (GE) CMS chip (GE, Cat#: 29104988) Date Recue/Date Received 2021-07-27 Buffer: HBS-EP+ buffer 10X (GE, Cat#: BR100669), diluted 10 folds with deionized water before use.
Amino coupling kit (GE, Cat#: BR100050) Regeneration reagents: 10 mM Glycine 2.0 (GE, Cat#: BR100355) 10 mM Glycine 3.0 (GE, Cat#: BR100357) Experimental steps CM5 chip was conjugated with a ligand: FOLR1 (R&D System, Cat#:
5646-FR) Analyte: CB-20BK, FA-MMAE and FOLH1 (Sino Biologicals, Cat#:
15877-H07H) According to BIAcore T200 (GE) operating manual, FOLR1 was conjugated to the CMS chip; CB-20BK or FA-MMAE was loaded first, and then FOLH1 was loaded; and the binding of FOLH1 to the CB-20BK-FOLR1 complex was detected.
The experimental results are as shown in Table 6.
Table 6. Binding of CB-20BK-FOLR1 Complex to FOLH1 Solution at Different Concentrations Concentration (04) FA-MMAE (RU) CB-20BK (RU) 0.5 9.8 23.9 0.25 8.4 14.1 0.125 4.2 4.6 0.0625 -1 -0.9 Table 6 shows that with regard to an FOLH1 solution at a high concentration, the amount of FOLH1 bound to a CB-20BK-FOLR1 complex is significantly higher than that bound to an FA-MMAE-FOLR1 complex, which shows a continuous rising trend. The binding of FOLH1 to an FA-MMAE-FOLR1 complex tends to saturate at a high concentration. This experiment has proved that CB-20BK can bind to both FOLR1 and FOLH1 receptors with a good affinity.
Example 3: Study on Binding and Endocytosis of Ligand Conjugates to Target Cells Date Recue/Date Received 2021-07-27 1. Cell binding and endocytosis experiment of conjugate compound Synthesis of labeled samples Cy5-pep-20BK, Cy5-FA and Cy5-pep-20AK
(1) 2 g of Rink Resin with a degree of substitution of 0.45 mmol/g was weighed and loaded onto a solid phase reaction column; DCM was added, and nitrogen gas was bubbled through the solvent to swell the resin for 30 minutes; the solvent was pumped off; Fmoc protecting groups were removed by DBLK; and then the resin was washed 5 times with DMF. 0.96 g (1.8 mmol) of Fmoc-Lys(Dde)-OH
and 0.3 g (2.2 mmol) of HOBt were weighed and dissolved in DMF; at 0 C in an ice-water bath, 0.33 ml (2.2 mmol) of DIC was added and mixed to activate for minutes; the mixture was added to the reaction column and reacted for 3 hours;
and the solvent was pumped off, and the resin was washed 3 times. Then Fmoc protecting groups were removed by DBLK.
(2) The above-mentioned operations were repeated, and Fmoc-Lys(Boc)-0H, Fmoc-Asp(OtBu)-0H, Fmoc-Asp(OtBu)-OH and intermediate 108 were conjugated successively according to the structures:
(3) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF. Fmoc-Lys(Dde)-0H, Fmoc-Glu-OtBu and pteroic acid were conjugated successively.
(4) Dde protecting groups were removed twice with 2% hydrazine hydrate/DMF, for 10 minutes each time, and then the resin was washed 5 times with DMF; the resin was conjugated with Cy5-COOH and then washed twice with DMF;
and finally, the resin was condensed twice with methanol and the solvent was pumped off to obtain 3.6 g of a protected peptide resin.
(5) 3.6 g of the peptide resin obtained in the previous step was added to a 50 ml single-necked flask; 29 ml of lysis buffer (TFA : H20: TIS = 95 : 3 : 2 (volume ratio)) was previously prepared, and 0.4 g of DTT was weighed and added to the lysis buffer. The lysis buffer was added to the flask, and the mixture was reacted at room temperature for 2.5 hours; the resin was filtered and washed with 8 ml of TFA;
the filtrate was combined, and the mixture was added to 173 ml of absolute ether, Date Recue/Date Received 2021-07-27 with a yellow solid being precipitated, and centrifuged to obtain a solid, which was washed with absolute ether and dried in vacuo to obtain 1.9 g of a crude product as a blue solid and with a yield of 89.6% and an HPLC purity of 76.3%. The product was separated by prep-HPLC (condition: C18 column, mobile phase A: 0.1%
trifluoroacetic acid in water, B: acetonitrile, elution gradient: (20-28)%B, elution time: 50 minutes; fractions were collected); and the fraction containing the qualified product was lyophilized to obtain 736 mg of Cy5-pep-20BK, with a purity of 93.4%.
In the same way, compounds Cy5-FA and Cy5-pep-20AK can be obtained through similar steps in the above-mentioned methods.
HO 0 0, _OH
0 '03S HO (s) NAN 0 0 ,.rH 0 H H H H
N (s) (s) N NH2 H H
N
HO
0, _OH HN -03S
I H
SOS-(Cy5-pep-20BK) -03s HO 0 0,0H
HO (s)NANNAN 0 (rH 0 0 H H H H
0 NksA N,)-LN H2 -03s N (S) N (S) H H
0 \(:) 0 \
\_H
HO
SOB-(Cy5-pep-20AK) Date Recue/Date Received 2021-07-27 -03s N-frN
I
(Cy5 -FA) Experiment for detecting cell binding and endocytosis of samples Cy5-pep-20AK/Cy5-pep-20BK by flow cytometry method Sample infoimation: Cy5-pep-20AK and Cy5-pep-20BK
Cell lines: LNCaP human prostate cancer cells, Du145 human prostate cancer cells, SKOV3 human ovarian cancer cells and NCI-H460 human lung cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and PBS
Experimental operations:
(1) Cell culture: the cells were digested with trypsin, collected and counted, and then the cells were added to several culture flasks containing a complete medium; the culture flasks were placed in an incubator at 37 C, 5% CO2 for culturing the cells, and finally about 5 x 106 cells were collected into a centrifuge tube.
(2) Sample incubation: Cy5-pep-20AK/Cy5-pep-20BK samples were diluted to 8 nmol/L with PBS; the cell suspension was centrifuged at 1000 rpm for 5 minutes;
the supernatant was removed, and the residue was washed once with PBS; the cells were suspended uniformly and divided equally into different centrifuge tubes according to the experimental scheme; PBS was removed by centrifugation; 200 [t.1 of a sample working solution was added to each tube and incubated at 37 C for 15, 30, 60, and 90 minutes, respectively, and 1 tube to which only PBS was added was used as a blank control; all operations were performed avoiding light exposure.
Date Recue/Date Received 2021-07-27 (3) Washing and loading: the working solution was removed by centrifugation at 1000 rpm for 5 minutes, and the cells were washed 3 times with PBS, and an appropriate amount of PBS was added to suspend the cells to 1 x 106 cells/ml;
Beckman CytoFLEX flow cytometer was turned on in advance to complete the startup cleaning process; and the cell samples were loaded successively, and the fluorescence of 10,000 living cells was read in the APC channel; all operations were performed avoiding light exposure.
(4) Data analysis: the absolute MFI (mean fluorescence intensity) of the APC
fluorescence of each cell sample and the relative MFI of the relative blank control were obtained, and a data line graph was completed according to the relative MFI.
Results and analysis Table 7. Expression Levels of FOLR1 and FOLH1 in Different Cell Lines (with reference to data from Depmap, https://depmap.org/portal/) RNA-Seq LNCaP SKOV3 Du145 NCI-H460 FOLR1 +/- 6+ +/- +1-FOLH1 10+ + +/- +/-According to data from Depmap, the data being 0 or negative is expressed as "-", the data being 0.001-0.499 is expressed as "+/-", the data being 0.500-1.499 is expressed as "+", the data being 1.500-2.499 is expressed as "2+", and so on.
The fluorescence intensity of cells was respectively detected at 15 minutes, minutes, 60 minutes, and 90 minutes of incubation, and the results were plotted and shown in Figs. 2A and 2B. The binding and endocytosis of the sample Cy5-pep-20BK cells can be seen from the figures. Compared with a DU145 cell line with a relatively low expression of FOLR1 and a NCI-H460 cell line with a relatively low expression of FOLH1, an LNCaP cell line with a relatively high expression of FOLR1 and an SKOV3 cell line with a relatively high expression of FOLH1 have a significantly higher fluorescence intensity of CB-20BK that was bond and endocytosed. Since Cy5-pep-20AK only comprises an FOLH1 ligand and does not comprise the FOLR1 ligand, FA, Cy5-pep-20AK shows a high level of binding and endocytosis in LNCaP cells with a high expression of FOLH1, and Date Recue/Date Received 2021-07-27 shows a moderate level of binding and endocytosis in SKOV3 cells with a moderate expression of FOLH1; in the two cell lines, the binding and endocytosis level of Cy5-pep-20AK was significantly lower than that of Cy5-pep-CB-20BK. The degree of binding and internalization of a ligand conjugate to cells is positively correlated with expression levels of cell-related receptors.
2. Binding and endocytosis experiment of single-ligand conjugate Cy5-FA to cells Sample infoimation: Cy5-FA
Cell lines: Hela cervical cancer cells, SKOV3 human ovarian cancer cells and A549 human lung cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine, PBS, and anti-fluorescence quenching mounting medium containing DAPI
Experimental operations:
(1) Cells growing on coverslips: coverslips were placed in a 24-well plate;
the cells were digested with trypsin, collected and counted, and then the cells were diluted with a complete cell culture medium to about 2 x 105 cells/ml; 500 IA
of the diluted cell solution was added to each well of the 24-well plate; and the plate was placed in an incubator at 37 C, 5% CO2 for culturing 48 hours.
(2) Sample incubation: the Cy5-FA sample was diluted in an IMDM medium to 73 nmol/L; the culture medium in the 24-well plate was discarded; 200 IA of the sample working solution was added to the cell coverslip in each well and incubated at 37 C for 15, 30, and 60 minutes, respectively, and 1 well to which only an IMDM
medium was added was used as a blank control; all operations were perfoinied avoiding light exposure.
(3) Washing and mounting: the working solution in the 24-well plate was discarded, and the plate was washed 3 times with preheated PBS at 37 C; the cell coverslips were taken out, and 5 [t.1 of anti-fluorescence quenching mounting medium containing DAPI was added dropwise onto the slides and then covered with the cell coverslips to complete the mounting; all operations were perfoinied avoiding light exposure.
Date Recue/Date Received 2021-07-27 (4) Image interpretation for samples: Leica fluorescence microscope DM2500 was turned on to preheat the fluorescence exciter for 15 minutes; at the same position, cell nucleus and Cy5 fluorescein were photographed at the corresponding fluorescence channel, and the image fusion was completed in the software.
The binding and endocytosis of the sample Cy5-FA to cells at 15 minutes and 30 minutes can be seen from Fig. 3, and the fluorescence intensity in cell lines Hela and SKOV-3 with a high expression of FOLR1 is significantly higher than that in a cell line A549 with a relatively low expression of FOLR1. The degree of binding and internalization of a ligand conjugate to cells is positively correlated with expression levels of cell-related receptors.
Example 4: Experiment of Inhibitin2 Cell Expansion in Conjugate Compounds 1. Experiment of inhibiting tumor cell expansion in CB-20BK
Sample infoimation: CB-20BK
Cell lines: KB human oral epidermoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 2.5 x cells/ml, T-47D cells were diluted to 1.3 x 104 cells/ml, NCI-H460 cells were diluted to 3.5 x 104 cells/ml, CALU-3 cells were diluted to 4.0 x 104 cells/ml, HuH-7 cells were diluted to 3.0 x 104 cells/ml, and LNCaP cells were diluted to 8.0 x 104 cells/ml; 100 IA of the diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were set on each plate.
The 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples Date Recue/Date Received 2021-07-27 The samples were diluted with a culture medium to desired concentrations (see Table 8). To each well of the 96-well plates that were cultured overnight, 50 1.11 of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 8: Concentration of Sample (CB-20BK) After Dilution Concentration after Tube number dilution (mon) 4 3.1 5 0.8 6 0.2 7 0.05 8 0.01 9 0.003 0.0008 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing 10 solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4A, wherein C value corresponds to IC5o), CB-20BK has an inhibitory effect on the growth and Date Recue/Date Received 2021-07-27 expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines.
Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell lines T-47D, KB, LNCaP, HuH-7 and CALU-3 with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-20BK shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
2. Experiment of conjugate compound CB-20BK and related compounds inhibiting expansion of tumor cell lines LNCaP (human prostate cancer cells) and 22RV1 (human prostate cancer cells) Since different cells have different sensitivity to a cell proliferation inhibitory toxicity of the payload in a ligand conjugate, the quantitative analysis may be interfered occasionally. A series of cell lines with known expression levels of receptors of different ligands were selected and tested for the response to inhibitory effects of the cell proliferation of a ligand conjugate and a single payload.
The ICso ratio of a single payload to that of a ligand conjugate in same cell line were taken to remove such interference.
Related samples: MMAE, CB-20BK, CB-20AK and FA-MMAE
Experimental operations: LNCaP and 22RV1 cells were prepared in advance, counted, and plated in 96-well cell culture plates at a cell density of 4 x 104 cells/ml and 2.0 x 104 cells/ml, respectively (100 [a/well); negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 [d/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10%
of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The calculated data is shown in Table 9.
Table 9. Inhibitory Effects of MMAE, FA-MMAE, CB-20BK and CB-20AK on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) Date Recue/Date Received 2021-07-27 FOL FOL
ICso ICso ratio gene gene IC50 IC50 ratio ratio Cell IC50 (FA- (MM
expr expr (CB-2 (CB-20 (MMA (MMA
line (MMAE) MMA AE/F
essio essio OAK) BK) E) A-M
n n OAK) OBK) MAE) level level LNCaP +/- 10+ 0.00255 0.264 0.102 0.0958 0.010 0.025 0.0267 22RV1 +/- 6+ 0.00648 1.01 1.05 0.502 0.006 0.006 0.013 It can be seen from the above table that there is not much difference in the expression level of FOLR1 between LNCaP and 22RV1, and the expression level of FOLH1 in LNCaP is significantly higher than the expression level of FOLH1 in 22RV1. CB-20BK (with FOLH1 ligand and FOLR1 ligand) and CB-20AK (only with FOLH1 ligand) have inhibitory effects on the expansion of both LNCaP and 22RV1 tumor cell lines. The inhibitory effects of CB-20BK and CB-20AK on the cells with different expression levels of receptor FOLH1 are different. The ICso ratio in cell line LNCaP with a relatively high expression of receptor FOLH1 is 2-4 times higher than the ICso ratio in cell line 22RV1 with a relatively low expression of the receptor. FA-MMAE also has an inhibitory effect on the expansion of LNCaP and 22RV1 tumor cell lines. Since the expression level of FOLR1 in LNCaP and 22RV1 is very low, the expression in LNCaP cells (FOLR1 gene expression level is 0.3219, with reference to data from Depmap) is slightly higher than that in 22RV1 (FOLR1 gene expression level is 0.0704, with reference to data from Depmap), and the ICso ratio of FA-MMAE between LNCaP and 22RV1 only differs by a factor of 1.5. The above data indicates that inhibitory effects on cell proliferation is positively correlated with expression levels of cell expression receptors FOLH1 and FOLR1.
3. Experiment of conjugate compound CB-20BK and related compounds inhibiting expansion of tumor cell lines PANC-1 (human pancreatic cancer cells) and CFPAC-1 (human pancreatic cancer cells) Related samples: MMAE and CB-20BK
Date Recue/Date Received 2021-07-27 Experimental operations: PANC-1 and CFPAC-1 cells were prepared in advance, counted, and plated in 96-well cell culture plates at a cell density of 4 x 104 cells/ml (100 [d/well); negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 [d/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader.
The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The calculated data is shown in the following table:
Table 10. Inhibitory Effects of CB-20BK on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) FOLR1 gene FOLH1 gene ICso ICso ICso (MMAE)/
Cell line expression expression (MMAE) (CB-20BK) ICso (CB-20BK) level level CFPAC-1 4+ +1- 0.015 0.557 0.027 PANC-1 +1- +1- 0.002 0.126 0.020 It can be seen from Table 10 that the expression level of FOLH1 in CFPAC-1 is very low, and the expression level of FOLR1 in CFPAC-1 is significantly higher than that in PANC1 cell lines. CB-20BK has an inhibitory effect on the expansion of PANC-1 and CFPAC-1 tumor cell lines. The inhibitory effects on the cells with different expression levels of receptor FOLR1 are different. The ICso ratio in cell line CFPAC-1 with a relatively high expression of receptor FOLR1 is significantly higher than the ICso ratio in the PANC-1 with a relatively low expression of receptor FOLR1, and the inhibitory effects on cell proliferation is positively correlated with expression levels of cell-related receptor FOLR1.
Experiments 2 and 3 prove that the two ligands in CB-20BK and the receptors thereof (FOLR1 and FOLH1) expressed on the cell surface play an important role in the compound inhibiting tumor cell proliferation.
Date Recue/Date Received 2021-07-27 4. Experiment of inhibiting tumor cell expansion in CB-20B
Sample infoimation: CB-20B
Cell lines: A549 human lung cancer cells, HuH-7 human liver cancer cells, KB
human oral epideituoid carcinoma cells, LNCaP human prostate cancer cells, DU145 human prostate cancer cells and T-47D human breast cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; diluted with a complete cell culture medium, A549 cells, HuH-7 cells, KB
cells and DU145 cells were plated at a density of 2 x 104 cells/ml, T-47D
cells were plated at a density of 1 x 105 cells/ml, and LNCaP cells were plated at a density of 6 x 104 cells/ml in 96-well plates; 100 1.11 of the diluted cell solution was added to each well; and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 11). To each well of the 96-well plates that were cultured overnight, 50 1.11 of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 11: Concentration of Sample (CB-20B) After Dilution Concentration after Tube number dilution (mon) 2 66.7 3 22.2 4 7.4 5 2.5 Date Recue/Date Received 2021-07-27 6 0.8 7 0.3 8 0.09 9 0.03 0.01 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an 5 appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was 10 plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4B, wherein C value corresponds to IC5o), CB-20B has an inhibitory effect on the growth and expansion of A549, HuH-7, KB, LNcap, DU145 and T-47D tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The IC5o for cell lines T-47D, LNcap, KB, HuH-7 and DU145 with a relatively high expression of the receptors is significantly lower than that for cell line A549 with a relatively low expression of the receptors. CB-20B shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
5. Experiment of inhibiting tumor cell expansion in CB-10S
Sample info' _______ "nation: CB-10S
Cell lines: KB human oral epideimoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells and LNcap human prostate cancer cells Date Recue/Date Received 2021-07-27 Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 3.5 x cells/ml, LNCaP cells were diluted to 8.0 x 104 cells/ml, T-47D cells were diluted to 1.2 x 104 cells/ml, NCI-H460 cells were diluted to 2.5 x 104 cells/ml, and RT4 cells were diluted to 1.2 x 104 cells/ml; to each well of 96-well plates, 100 [11 of the diluted cell solution was added; and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 12). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 12: Concentration of Sample (CB-10S) After Dilution Concentration after Tube number dilution (junol/L) 4 0.9 5 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 10 0.000007 Date Recue/Date Received 2021-07-27 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4C, wherein C value corresponds to ICso), CB-10S has an inhibitory effect on the growth and expansion of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell lines KB, LNcap, T-47D and with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-10S
shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
6. Experiment of inhibiting tumor cell expansion in CB-60S
Sample infoimation: CB-605 Cell lines: KB human oral epideimoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating Date Recue/Date Received 2021-07-27 The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 2.0 x cells/ml, T-47D cells were diluted to 1.5 x 104 cells/ml, NCI-H460 cells were diluted to 2.0 x 104 cells/ml, CALU-3 cells were diluted to 3.0 x 104 cells/ml, HuH-7 cells were diluted to 4.0 x 104 cells/ml, and LNCaP cells were diluted to 8.0 x 104 cells/ml; 100 IA of the diluted cell solution was added to each well of 96-well plates; and negative control and blank control wells were set on each plate.
The 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 13). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 13: Concentration of Sample (CB-60S) After Dilution Concentration after Tube number dilution (mon) 5 1.3 6 0.3 7 0.08 8 0.02 9 0.005 10 0.001 3) Color development and reading plate Date Recue/Date Received 2021-07-27 To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4D, wherein C value corresponds to IC5o), CB-605 has an inhibitory effect on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The IC50 for cell lines LNCaP
and HuH-7 with a relatively high expression of the receptors is significantly lower than that for cell lines NCI-H460, KB, T-47D and CALU-3 with a relatively low expression of the receptors. CB-605 shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
7. Experiment of inhibiting tumor cell expansion in CB-60SK
Sample infoimation: CB-605K
Cell lines: KB human oral epideimoid carcinoma cells, T-47D human breast cancer cells, NCI-H460 human lung cancer cells, CALU-3 human lung adenocarcinoma cells, HuH-7 human liver cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 2.5 x cells/ml, T-47D cells were diluted to 1.3 x 104 cells/ml, NCI-H460 cells were Date Recue/Date Received 2021-07-27 diluted to 3.5 x 104 cells/ml, CALU-3 cells were diluted to 4.0 x 104 cells/ml, HuH-7 cells were diluted to 3.0 x 104 cells/ml, and LNCaP cells were diluted to 8.0 x 104 cells/ml; 100 [t.1 of the diluted cell solution was added to each well;
and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 14). To each well of the 96-well plates that were cultured overnight, 50 [11 of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 14: Concentration of Sample (CB-60SK) After Dilution Concentration after Tube number dilution (mon) 4 4.5 5 1.1 6 0.3 7 0.07 8 0.02 9 0.004 10 0.001 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the Date Recue/Date Received 2021-07-27 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4E, wherein C value corresponds to IC5o), CB-605K has an inhibitory effect on the growth and expansion of KB, T-47D, NCI-H460, CALU-3, HuH-7, and LNCaP tumor cell lines.
Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The IC50 for cell lines LNCaP and HuH-7 with a relatively high expression of the receptors is significantly lower than that for cell lines T-47D, NCI-H460, CALU-3 and KB with a relatively low expression of the receptors. CB-605K shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
8. Experiment of inhibiting tumor cell expansion in CB-18G
Sample info' _______ "nation: CB-18G
Cell lines: A549 human lung cancer cells, Hela human cervical cancer cells, SCLC-21H small cell lung cancer cells, U-2 OS human osteosarcoma cells, T-47D
human breast cancer cells and NCI-H460 human lung cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; diluted with a complete cell culture medium, A549 cells, Hela cells, SCLC-21H cells and U-2 OS cells were plated at a density of 2 x 104 cells/ml, and T-47D cells and NCI-H460 cells were plated at a density of 3 x 104 cells/ml in 96-well plates; to each well of the 96-well plates, 100 [t.1 of the diluted cell solution was added; and negative control and blank control wells were set on each plate. The Date Recue/Date Received 2021-07-27 96-well plates added with the cells were placed in an incubator at 37 C, 5%
CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 15). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 15: Concentration of Sample (CB-18G) After Dilution Concentration after Tube number dilution (mon) 3 6.25 4 1.56 5 0.39 6 0.10 7 0.02 8 0.006 9 0.002 0.0004 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
Date Recue/Date Received 2021-07-27 5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4F, wherein C value corresponds to IC50), CB-18G has an inhibitory effect on the growth and expansion of A549, Hela, SCLC-21H, U-2 OS, T-47D and NCI-H460 tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell line Hela with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-18G shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
9. Experiment of inhibiting tumor cell expansion in CB-50S
Sample infoimation: CB-505 Cell lines: KB human oral epideimoid carcinoma cells, NCI-H460 human lung cancer cells, RT4 human bladder cancer cells, T-47D human breast cancer cells and LNCaP human prostate cancer cells Main reagents: IMDM medium, fetal bovine serum, penicillin-streptomycin solution, L-glutamine and CCK8 Experimental operations:
1) Cell plating The cells were prepared in advance, digested with trypsin, collected and counted; with a complete cell culture medium, KB cells were diluted to 3.5 x cells/ml, LNCaP cells were diluted to 8.0 x 104 cells/ml, T-47D cells were diluted to 1.2 x 104 cells/ml, NCI-H460 cells were diluted to 2.5 x 104 cells/ml, and RT4 cells were diluted to 1.2 x 105 cells/ml; to each well of 96-well plates, 100 [1.1 of the diluted cell solution was added; and negative control and blank control wells were set on each plate. The 96-well plates added with the cells were placed in an incubator at 37 C, 5% CO2 for culturing overnight.
2) Diluting and adding samples The samples were diluted with a culture medium to desired concentrations (see Table 16). To each well of the 96-well plates that were cultured overnight, 50 [El of the diluted samples were added, with 3 replicate wells; negative controls and blank Date Recue/Date Received 2021-07-27 controls were set; and the plates were placed in an incubator at 37 C, 5% CO2 for culturing 72 hours.
Table 16: Concentration of Sample (CB-50S) After Dilution Concentration after Tube number dilution (umol/L) 4 0.9 0.1 6 0.02 7 0.003 8 0.0004 9 0.00005 0.000008 5 3) Color development and reading plate To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK-8 was added; the plates were incubated at 37 C for an appropriate time (try to keep the OD value in the range of 1.0-2.0); the covers of the 96-well plates were removed, and the plates were read at 450 nm on a microplate 10 reader (Molecular Devices Spectra MAX Plus).
4) Data processing The data was edited using SoftMax Pro and a four-parameter fitting curve was plotted.
5) Experimental results and analysis According to the four-parameter fitting curve graph (Fig. 4G, wherein C value corresponds to IC5o), CB-505 has an inhibitory effect on the growth and expansion of KB, LNCaP, T-47D, RT4 and NCI-H460 tumor cell lines. Since expression levels of the corresponding receptors of conjugate compounds in the cells are different, the inhibition levels are different. The ICso for cell lines KB, LNcap, T-47D and Date Recue/Date Received 2021-07-27 with a relatively high expression of the receptors is significantly lower than that for cell line NCI-H460 with a relatively low expression of the receptors. CB-50S
shows a correlation between tumor growth inhibition effects and expression levels of cell-related receptors.
10. Experiment of inhibiting tumor cell expansion in conjugate compound CB-1020 Experiment purpose: to test inhibitory effects of CB-1020 and related compounds on the expansion of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), NCI-H226 (human lung cancer cells), CFPAC-1 (pancreatic cancer cells) and PANC -1 (pancreatic cancer cells).
Related samples: MMAE and CB-1020 Experimental operations: LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 cells were prepared in advance and counted; with a culture medium (IMDM + 10% FBS + 1X L-Glutamine + 1X P/S), the cells LNCaP, SK-BR-3 and CFPAC-1 were diluted to 4 x 104 cells/ml, NCI-H226 was diluted to 2.0 x 104 cells/ml and PANC-1 was diluted to 3.0 x 104 cells/ml; the diluted cell solution was plated in 96-well plates at 100 [11/well; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 1/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P
fitting curve was plotted. The data is shown in the following table:
Table 17. Inhibitory Effects of CB-1020 on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) TRPV6 gene FOLH1 gene ICso ICso ICso Cell line expression expression (MMAE)/ICso (MMAE) (CB-1020) level level (CB-1020) LNCap 5+ 10+ 0.003 0.168 0.0179 Date Recue/Date Received 2021-07-27 SK-BR-3 4+ 2+ 0.002 0.287 0.0070 CFPAC -1 +/- 0.015 3.000 0.005 PANC-1 +/- +/- 0.002 0.458 0.0044 NCI-H226 +/- 0.006 1.570 0.00384 It can be seen from the above table that both LNCaP and SK-BR-3 express TRPV6 and FOLRH1, wherein LNCap has a relatively higher expression level of the two receptors. NCI-H226, CFPAC-1 and PANC-1 express the two receptors at a low or weak level. CB-1020 has an inhibitory effect on the growth and expansion of LNCaP, SK-BR-3, NCI-H226, CFPAC-1 and PANC-1 tumor cell lines. The ICso ratio of CB-1020 for cell line LNCaP with a relatively high expression of the two receptors is higher than the IC50 ratio for cell line SK-BR-3 with a medium expression of the receptors; the ICso ratio for SK-BR-3 is higher than the IC50 ratios for CFPAC-1 with a relatively low expression of the receptors and cell lines NCI-H226 and PANC-1 with a weak expression of the two receptors. The inhibitory effect on cell proliferation is positively correlated with the expression level of the two receptors in the cells.
11. Experiment of inhibiting tumor cell expansion in conjugate compound CB-1320 Experiment purpose: to test inhibitory effects of CB-1320 and related compounds on the expansion of tumor cell lines LNCaP (human prostate cancer cells), SK-BR-3 (human breast cancer cells), MDA-MB-468 (human breast cancer cells) and CFPAC-1 (pancreatic cancer cells).
Related samples: MMAE and CB-1320 Experimental operations: LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 cells were prepared in advance and counted; with a culture medium (IMDM + 10%
FBS + lx L-Glutamine + 1X P/S), the cells LNCaP, SK-BR-3, MDA-MB-468 and CFPAC-1 were diluted to 4 x 104 cells/ml; the diluted cell solution was plated in 96-well plates at 100 ul/well; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 1.11/well. The plates were placed in an incubator at 37 C, 5%
Date Recue/Date Received 2021-07-27 and cultured for 68-72 hours. To each well, 15 p1(10% of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The specific data is shown in the following table:
Table 18. Inhibitory Effects of CB-1320 on Proliferation of Cell Lines and Receptor Gene Expression Levels in Cell Lines (with reference to data from Depmap) FOLH1 gene ICso GNRHR gene ICso ICso Cell line expression (MMAE)/1C50 expression level (MMAE) (CB-1320) level (CB-1320) LNCaP +/- 10+ 0.003 0.060 0.043 SK-BR-3 +/- 2+ 0.002 0.093 0.016 CFPAC-1 +/- +/- 0.015 1.090 0.014 MDA-MB-468 +/- +/- 0.002 0.212 0.011 It can be seen from the above table that the expression levels of GNRHR in the 4 cell lines are roughly equivalent, and the expression level of FOLH1 in LNCaP is significantly higher than that in other cell lines. CB-1320 has an inhibitory effect on the expansion of 4 tumor cell lines. The inhibitory effects of CB-1320 on the cells with different expression levels of receptor FOLH1 are different. The ICso ratio in cell line LNCap with a relatively high expression of receptor FOLH1 is significantly higher than the ICso ratio in other cell lines with a relatively low expression of the receptor.
12. Experiment of inhibiting tumor cell expansion in conjugate compound CB-1820 Experiment purpose: to test inhibitory effects of CB-1820 and related compounds on the expansion of tumor cell lines LNCaP (human prostate cancer cells), MDA-MB-468 (human breast cancer cells), CFPAC-1 (pancreatic cancer cells) and PANC-1 (pancreatic cancer cells).
Related samples: MMAE and CB-1820 Date Recue/Date Received 2021-07-27 Experimental operations: LNCaP, MDA-MB-468, CFPAC-1 and PANC-1 cells were prepared in advance and counted; with a culture medium (IMDM + 10% FBS
+ lx L-Glutamine + lx P/S), the cells LNCaP, MDA-MB-468 and CFPAC-1 were diluted to 4 x 104 cells/ml, and PANC-1 was diluted to 3.0 x 104 cells/ml; the diluted cell solution was plated in 96-well plates at 100 [tl/well; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 p1/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, p1(10% of the liquid volume in a well) of a color developing solution from 10 CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The specific values are shown in the following table:
Table 19. Inhibitory Effects of CB-1820 on Proliferation of Cell Lines and Receptor 15 Gene Expression Levels in Cell Lines (with reference to data from Depmap) SSTR2 gene FOLH1 gene ICso ICso ICso (MMAE)/ICso Cell line expression expression (MMAE) (CB-1820) (CB-1820) level level LNCap +/- 10+ 0.003 0.021 0.1429 MDA-MB-468 +/- +/- 0.002 0.062 0.0323 CFPAC-1 +/- +/- 0.015 0.356 0.0421 PANC-1 +/- +/- 0.002 0.055 0.0364 It can be seen from the above table that the expression levels of SSTR2 in LNCap, MDA-MB-468, CFPAC-1 and PANC-1 are roughly equivalent, and the expression level of FOLH1 in LNCaP is significantly higher than the expression level of FOLH1 in other cell lines. CB-1820 has an inhibitory effect on the expansion of 4 tumor cell lines. The inhibitory effects of CB-1820 on the cells with different expression levels of receptor FOLH1 are different. The IC50 ratio in cell line LNCaP with a relatively high expression of the receptor is significantly higher Date Recue/Date Received 2021-07-27 than the ICso ratio in other cell lines with a relatively low expression of the receptor, and the inhibitory effects on cell proliferation is positively correlated with expression levels of cell-related receptor FOLH1.
Related samples: MMAE and CB-1820 Date Recue/Date Received 2021-07-27 Experimental operations: LNCaP, MDA-MB-468, CFPAC-1 and PANC-1 cells were prepared in advance and counted; with a culture medium (IMDM + 10% FBS
+ lx L-Glutamine + lx P/S), the cells LNCaP, MDA-MB-468 and CFPAC-1 were diluted to 4 x 104 cells/ml, and PANC-1 was diluted to 3.0 x 104 cells/ml; the diluted cell solution was plated in 96-well plates at 100 [tl/well; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 p1/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, p1(10% of the liquid volume in a well) of a color developing solution from 10 CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The specific values are shown in the following table:
Table 19. Inhibitory Effects of CB-1820 on Proliferation of Cell Lines and Receptor 15 Gene Expression Levels in Cell Lines (with reference to data from Depmap) SSTR2 gene FOLH1 gene ICso ICso ICso (MMAE)/ICso Cell line expression expression (MMAE) (CB-1820) (CB-1820) level level LNCap +/- 10+ 0.003 0.021 0.1429 MDA-MB-468 +/- +/- 0.002 0.062 0.0323 CFPAC-1 +/- +/- 0.015 0.356 0.0421 PANC-1 +/- +/- 0.002 0.055 0.0364 It can be seen from the above table that the expression levels of SSTR2 in LNCap, MDA-MB-468, CFPAC-1 and PANC-1 are roughly equivalent, and the expression level of FOLH1 in LNCaP is significantly higher than the expression level of FOLH1 in other cell lines. CB-1820 has an inhibitory effect on the expansion of 4 tumor cell lines. The inhibitory effects of CB-1820 on the cells with different expression levels of receptor FOLH1 are different. The IC50 ratio in cell line LNCaP with a relatively high expression of the receptor is significantly higher Date Recue/Date Received 2021-07-27 than the ICso ratio in other cell lines with a relatively low expression of the receptor, and the inhibitory effects on cell proliferation is positively correlated with expression levels of cell-related receptor FOLH1.
13. Experiment of inhibiting tumor cell expansion in conjugate compounds CR19425, CR19426 and CR19428 Experiment purpose: to test inhibitory effects of CR19428, CR19425, CR19426 and related compounds on the expansion of tumor cell lines NCI-H226 (human lung cancer cells), CFPAC-1 (human pancreatic cancer cells) and MDA-MB-468 (human breast cancer cells).
Related samples: SN-38, Dxd0017, CR19428, CR19425 and CR19426 Experimental operations: NCI-H226, CFPAC-1 and MDA-MB-468 cells were prepared in advance and counted; MDA-MB-468 and CFPAC-1 cells were plated at a density of 2 x 104 cells/ml (100 p1/well) and NCI-H226 cells were plated at a density of 1 x 104 cells/ml (100 1/well) in 96-well plates; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 1/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10%
of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The specific data is shown in the following table:
Table 20. Inhibitory Effects of CR19428, CR19425 and CR19426 on Proliferation of Cell Lines ICso ICso ICso ICso ICso Cell line (SN-38) (Dxd0017) (CR19428) (CR19425) (CR19426) CFPAC-1 0.0055 0.0064 0.9060 0.6210 1.6600 MDA-MB-468 0.0136 0.0061 1.0700 0.9510 2.3000 NCI-H226 0.0173 0.0080 1.6200 1.0100 4.3900 Example 5: Pharmacodynamic Study of Conjugate Compounds in CDX
Models Date Recue/Date Received 2021-07-27 1. Pharmacodynamic study 1 of CB-20BK in CDX models 1) Sample preparation:
CB-20BK lyophilized powder was weighed, dissolved with PBS and prepared into a sample mother solution, and the mother solution was diluted with a physiological saline for injection to a sample solution with a working concentration for later use.
2) CDX model construction Main cell lines: KB human oral epidermoid carcinoma cells, MIA paca-2 human pancreatic cancer cells, HCC1954 human breast cancer cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells.
Model construction: The cells were resuscitated, cultured, collected, counted, and subcutaneously injected into the right flank of BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping and administration were performed, or rapidly expanded tumor masses were transferred and injected to mice for scale up.
3) Grouping, administration and observation Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping was performed, and a model control group and different doses of administration groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the effects of tumor growth and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss (measuring weight twice a week), and other abnoinial conditions in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor mass were measured twice a week. The foimula for the calculation of tumor volume (TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis Date Recue/Date Received 2021-07-27 According to changes in tumor volumes of mice (Figs. 5A-5E), it can be seen that CB-20BK has a good inhibitory effect in CDX tumor models of KB, MIA aca-2, HCC1954, CALU-3 and DU145 cell lines in mice.
2. Pharmacodynamic study 2 of CB-20BK in CDX models Experiment purpose: to perfornt a phannacodynamic study of conjugate compound CB-20BK in a subcutaneous xenograft model (CDX) of humanized LNCaP, DU145 and NCI-H460 cell lines Main CDX models: LNCaP, DU145 and NCI-H460 Experimental scheme:
The cells or tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice. When the tumor volume expanded to 80-160 mm3, a randomized grouping was performed, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g. After grouping and administration, the effects of tumor growth and treatment on nothial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated. The formula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 21. Inhibitory Effects of CB-20BK on Growth in CDX models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) Date Recue/Date Received 2021-07-27 Tumor growth Model FOLR1 FOLH1 inhibition rate Administration dosage/time (TGI) (%) LNCaP 5+ 10+ 95.68 3 mg/kg D1, 8, 15 DU145 - - 52 5 mg/kg DI, 4, 7 NCI-H460 - - 32 10 mg/kg D1, 8, 15 It can be seen from the above table that the test sample CB-20BK shows different degrees of tumor growth inhibition effects with a regimen of administration via tail vein. For CDX models of humanized LNCaP, DU145 and NCI-H460 cell lines, the test sample has a certain anti-tumor growth effect compared with a negative control group. In the LNCaP model with dual expression of FOLR1 and FOLH1, the test sample was administered on day 1, day 8 and day (D1, D8 and D15) at a dose of 3 mg/kg, has a TGI value of 95.68%, shows an excellent anti-tumor growth effect, which is significantly better than that in 10 and NCI-H460 models with a relatively low expression of FOLR1 and FOLH1. The tumor growth inhibition effect has a certain correlation with the expression level of cell-related receptors.
3. Tumor growth inhibition experiment 1 of conjugate compound CB-20BK in HuPrime xenograft PDX models Experiment purpose: Phattnacodynamic study of conjugate compound CB-20BK in PDX models Main models: LU1206, LU1380 and LU0367 Experimental scheme: The tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g and an administration dosage of 3 mg/kg. After grouping and administration, the effects of tumor growth Date Recue/Date Received 2021-07-27 and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated.
The foimula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 22. Inhibitory Effects of CB-20BK on Growth in Lung Cancer PDX Models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) FOLR1 gene FOLH1 gene TGI (%) Model expression level expression level Dosage LU1206 4+ 2+ 90.89 3 mg/kg LU1380 - 4+ 30.02 3 mg/kg LU0367 - 5+ 71.34 3 mg/kg It can be seen from the data in Table 22 that the test sample CB-20BK (3 mg/kg) shows a certain anti-tumor effect on LU1206, LU1380 and LU0367 humanized lung cancer PDX models. The TGI value in the LU1206 model with dual expression of FOLR1 and FOLH1 is 90.89%, and the TGI values in LU1380 and LU0367 models with a single expression are 30.02% and 71.34%, respectively.
In the tumor growth inhibition experiment, the tumor growth inhibition effect has a certain correlation with the expression level of cell-related receptors.
4. Tumor growth inhibition experiment 2 of conjugate compound CB-20BK in HuPrime xenograft PDX models Experiment purpose: Phannacodynamic study of conjugate compound CB-20BK in PDX models Main models: BR1283 and BR0438 Date Recue/Date Received 2021-07-27 Experimental scheme: The tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 lig and an administration dosage of 3 mg/kg. After grouping and administration, the effects of tumor growth and treatment on nottnal behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnottnal conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated.
The fottnula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 23. Inhibitory Effects of CB-20BK on Growth in Breast Cancer PDX Models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) FOLR1 gene expression FOLH1 gene TGI (%) Model level expression level Dosage BR1283 7+ 6+ 96.49 3 mg/kg BR0438 5+ 4+ 70.74 3 mg/kg It can be seen from the above table that the TGI values of the test sample CB-20BK at a dose of 3 mg/kg in BR1283 and BR0438 models with dual expression of FOLR1 and FOLH1 are 96.49% and 70.74%, respectively, showing an excellent anti-tumor growth effect. Moreover, the TGI of CB-20BK is higher in BR1283 with a higher expression of FOLR1 and FOLH1.
5. Pharmacodynamic study of CB-20B in CDX models Date Recue/Date Received 2021-07-27 1) Sample preparation:
CB-20B lyophilized powder was weighed, dissolved with PBS and prepared into a sample mother solution, and the mother solution was diluted with a physiological saline for injection to a sample solution with a working concentration for later use.
2) CDX model construction Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells and DU145 human prostate cancer cells.
Model construction: The cells were resuscitated, cultured, collected, counted, and subcutaneously injected into the right flank of BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping and administration were performed, or rapidly expanded tumor masses were transferred and injected to mice for scale up.
3) Grouping, administration and observation Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping was performed, and a model control group and different doses of administration groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the effects of tumor growth and treatment on normal behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss (measuring weight twice a week), and other abnoinial conditions in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor mass were measured twice a week. The foimula for the calculation of tumor volume (TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis According to changes in tumor volume of mice (Figs. 6A-6C), it can be seen that CB-20B has a good inhibitory effect in CDX tumor models of KB, PC-9 and DU145 cell lines in mice.
6. Pharmacodynamic study of CB-18G in CDX models Date Recue/Date Received 2021-07-27 1) Sample preparation:
CB-18G lyophilized powder was weighed, dissolved with PBS and prepared into a sample mother solution, and the mother solution was diluted with a physiological saline for injection to a sample solution with a working concentration for later use.
2) CDX model construction Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells, SPC-Al human lung adenocarcinoma cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells Model construction: The cells were resuscitated, cultured, collected and counted, and the cell solution was subcutaneously injected into the right flank of BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping and administration were performed, or rapidly expanded tumor masses were transferred and injected to mice for scale up.
3) Grouping, administration and observation Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping was performed, and a model control group and different doses of administration groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the effects of tumor growth and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss (measuring weight twice a week), and other abnoinial conditions in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor mass were measured twice a week. The formula for the calculation of tumor volume (TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis According to changes in tumor volumes of mice (Figs. 7A-7E), it can be seen that CB-18G has a good inhibitory effect in CDX tumor models of KB, PC-9, SPC-Al, CALU-3 and DU145 cell lines in mice.
Date Recue/Date Received 2021-07-27 7. Tumor growth inhibition experiment of conjugate compounds CB-1020, CB-1320 and CB-1820 in subcutaneous xenograft model (CDX) of humanized HPAF-II, NCI-11226 and SCLC-211I cell lines Experiment purpose: Phannacodynamic study of ligand conjugates CB-1020, CB-1320 and CB-1820 in CDX models Main CDX models: HPAF-II (human pancreatic cancer cells), NCI-H226 (human lung cancer cells) and SCLC-21H (small cell lung cancer cells) Experimental scheme:
The cells or tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice. When the tumor volume expanded to 80-160 mm3, a randomized grouping was performed, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g. After grouping and administration, the effects of tumor growth and treatment on nothial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated. The formula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 24. Inhibitory Effects of CB-1020, CB-1320 and CB-1820 on Growth in CDX
Models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) Date Recue/Date Received 2021-07-27 gene gene gene gene TGI (%) TGI (%) TGI (%) P
Model Dosage expression expression expression expression (CB-1020) (CB-1320) (CB-1820) value level level level level 0.283 0.461 3 mg/kg HPAF-II + - +/- +/- 0.445 0.153 5 mg/kg 0.899 0.023 10 mg/kg 0.823 0.011 3 mg/kg NCIH226 +/- +/- +/- - 0.962 0.006 3 mg/kg 0.992 0.005 10 mg/kg 0.995 0.131 3 mg/kg SCLC-21H 2+ +/- 3+ - 0.361 0.610 3 mg/kg 0.985 0.134 10 mg/kg CB-1020 has an obvious anti-tumor growth effect in subcutaneous xenograft models of humanized HPAF-II cell lines; CB-1320 has an obvious anti-tumor growth effect in subcutaneous xenograft models of NCI-H226 and SCLC-21H cell lines; and CB-1820 has an obvious anti-tumor growth effect in subcutaneous xenograft models of SCLC-21H cell lines.
8. Tumor growth inhibition experiment of conjugate compound CR19428 in subcutaneous xenograft model (CDX) of humanized CALU-3, SCLC-2111 and SPC-Al cell lines Experimental scheme: The tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g and twice a week for Date Recue/Date Received 2021-07-27 consecutive 3 weeks. After grouping and administration, the effects of tumor growth and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated.
The foimula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 25. Inhibitory Effects of CR19428 in Lung Cancer PDX models Model TGI (%) Administration dosage/time CALU-3 51 50 mg/kg q2/w*3 SCLC-21H 66 50 mg/kg q2/w*3 SPC -A1 75 50 mg/kg q2/w*3 SPC -A1 91 100 mg/kg q2/w*3 CR-19428 is a drug conjugate of ligands FOLR1 and FOLH1 and a payload Dxd. It can be seen from the above table that the drug conjugate has an obvious anti-tumor growth effect in subcutaneous xenograft models of humanized CALU-3, SCLC-21H and SPC-Al cell lines.
9. Study on effectiveness of conjugate compound CBP-1018 in lung cancer LU2505 model (PDX model) The 3rd generation of lung cancer PDX model LU2505 (from Asian female patients), which is a rapid-growing tumor model, was used in this experiment.
BALB/c nude mice bore tumors subcutaneously and, when the tumor volume reached about 150 mm3, were grouped into: low-dose, medium-dose and high-dose test sample groups, a small molecule MMAE control group, a targeting polypeptide 20BK-SMO9 control group, and a blank control group (a total of 6 groups, and 8 mice per group); the mice were administered on day 1, day 8 and day 15 after the grouping; and the observation was continued for 14 days after the last Date Recue/Date Received 2021-07-27 administration. The active substance of test sample CBP-1018 is CB-20BK, which is obtained by mixing CB-20BK with auxiliary materials and then lyophilizing same.
Table 26. Phannacodynamic Results of CBP-1018 for Injection in Lung Cancer LU2505 models (first experiment) Tumor Test sample Tumor growth Dosage Weight Tumor weight /Control volume inhibition (mg/kg) (g) (mg) sample (mm3) rate TGI%
Blank control / 24.0 2219.62 / 2031.4 (D22) group 4.5 22.5 0.00 100.00 0.0 (D29) CBP-1018 1.5 22.1 386.16 82.60 400.7 (D29) 0.5 23.6 1634.50 26.36 2084.7 (D26) 20BK-SMO9 10 23.8 2024.75 8.78 1811.5 (D22) MMAE 0.375 23.5 1132.61 48.97 1597.1 (D29) Notes: 1) Due to requirements of animal welfare, animals in a group must be euthanized when the mean tumor volume in the group reaches 2000 mm3, leading to different execution time for each group. 2) The data of weight, tumor volume, and tumor growth inhibition rate is the data obtained on day 22 (D22).
As shown in Table 26 and Fig. 8A:
animals in all groups show no abnoinial clinical manifestations and no deaths after administration; and the weight of the animals in each group increases slowly.
The animals in the blank control group, 20BK-SMO9 group, and low-dose CBP-1018 group were euthanized on day 22, day 22, and day 26 (D26) because of the mean tumor volume exceeding 2000 mm3. Therefore, the data of weight, tumor volume, and tumor growth inhibition rate in Table 26 is the data obtained on D22.
The effectiveness of CBP-1018 for injection has an obvious dose correlation, wherein CBP-1018 for injection was ineffective in the low-dose group, and Date Recue/Date Received 2021-07-27 effective in the medium-dose group and the high-dose group. The tumor in the high-dose group was completely cured on day 19 (D19), and no signs of tumor growth were seen on day 29 (D29).
The polypeptide 20BK-SM09 group showed no obvious tumor growth inhibition effect, suggesting that a single targeting polypeptide is not enough to produce a clear anti-tumor effect.
The MMAE group showed a clear tumor growth inhibition effect. Equimolar MMAE is contained in MMAE at 0.375 mg/kg and in CBP-1018 at 1.5 mg/kg. It can be seen from the comparison that the tumor growth inhibition effect of CBP-1018 at 1.5 mg/kg is significantly better than that in the MMAE group, suggesting the advantage of ligand targeting (tumor growth inhibition rate:
82.60%
vs 48.97%).
10. Study on effectiveness of conjugate compound CBP-1018 in lung cancer LU1206 model (PDX model) The 5th generation of lung cancer PDX model LU1206 (from Asian female patients), which is a rapid-growing tumor model, was used in this experiment.
BALB/c nude mice bore tumors subcutaneously and, when the tumor volume reached about 150 mm3, were grouped into: low-dose, medium-dose and high-dose test sample groups, a small molecule MMAE control group, a targeting polypeptide 20BK-SMO9 control group, and a blank control group (a total of 6 groups, and 8 mice per group); the mice were administered on day 1, day 8 and day 15 after the grouping; and the observation was continued for 14 days after the last administration.
Table 27. Phannacodynamic Results of CBP-1018 for Injection in Lung Cancer LU1206 models (first experiment) Tumor Tumor Tumor volume Tumor weight Dosage Weight Groups volume Tumor growth weight Inhibition (mg/kg) (g) (mm3) inhibition rate (mg) ratio (%) (%) Blank control / 22.7 1216.67 / 856.14 /
Date Recue/Date Received 2021-07-27 group 4.5 23.4 31.44 97.42 13.55 98.42 CBP-1018 1.5 23.7 301.64 75.21 185.10 78.38 0.5 22.7 1118.30 8.08 852.19 0.46 20BK-SMO9 10 23.3 1158.80 4.76 914.36 -6.80 MMAE 0.375 23.6 778.32 36.03 437.73 54.53 As shown in Table 27 and Fig. 8B:
animals in all groups show no abnoinial clinical manifestations and no deaths after administration, and all animals were euthanized on day 29. The weight of the animals in each group remained basically unchanged, and is slightly higher than that on the first day of administration.
The effectiveness of CBP-1018 for injection has an obvious dose correlation, wherein CBP-1018 for injection was ineffective in the low-dose group (with a tumor growth inhibition rate of 8.08%), and effective in the medium-dose group and 113 the high-dose group, with a tumor growth inhibition rate of 75.21% and 97.42%, respectively. The difference in effectiveness between the low-dose group and the medium-dose group is relatively large.
The polypeptide 20BK-SM09 group showed no obvious tumor growth inhibition effect, suggesting that a single targeting polypeptide is not enough to produce a clear anti-tumor effect in this model.
The MMAE group showed a clear tumor growth inhibition effect. Equimolar MMAE is contained in MMAE at 0.375 mg/kg and in CBP-1018 at 1.5 mg/kg. It can be seen from the comparison that the tumor growth inhibition effect of CBP-1018 at 1.5 mg/kg is significantly better than that in the MMAE group, suggesting the advantage of ligand targeting (tumor volume inhibition rate:
75.21%
vs 36.03%; tumor weight inhibition rate: 78.38% vs 54.53%).
11. Tissue distribution of tumor-bearing mice (PDX model LU2505) 12 female tumor-bearing mice inoculated with tumor mass of an HuPrime lung cancer LU2505 model and 12 healthy male BALB/c nude mice were given 1.5 mg/140 fiCi/kg of [3fl]CBP-1018 (isotope labeling on MMAE) via single tail vein Date Recue/Date Received 2021-07-27 injection. At 0.5 hours, 2 hours, 6 hours and 24 hours respectively, 3 male mice and 3 female mice were placed in an induction box and anesthetized by inhaling an appropriate amount of carbon dioxide; and blood was collected by cardiac puncture, and then euthanasia was carried out immediately to collect samples.
Table 28. Total Radioactivity in Various Tissues at Different Time Points after Single Intravenous Administration of [3I-1]CBP-1018 Radioactivity concentration (ng Eq./g) Tissue 0.5 hours 2 hours 6 hours 24 hours (% C.) Tumor 763 / 643 / 566 / 413 Esophagus 915 784 628 508 521 172 286 91.7 Body fat 341 479 288 276 231 177 67.2 43.7 Skeletal muscle 321 311 207 137 188 95.5 90.8 61.5 Spleen 744 693 701 524 671 421 253 163 Stomach wall 658 623 414 370 460 259 211 Whole brain 66.3 67.9 53.8 39.5 82.0 48.2 76.4 68.3 Testicular epididymis 350 494 279 298 183 248 92.3 64.8 Heart 785 795 415 326 219 143 114 69.2 Lung 1493 1807 997 920 416 392 109 93.7 Kidney 5682 6118 3300 4197 1227 1391 241 238 Liver 1423 1306 1257 865 1138 599 712 518 Small intestine wall 726 778 641 816 490 334 210 90.9 Large intestine wall 550 520 672 823 1074 490 215 Whole blood 2087 3194 684 691 211 132 86.5 64.6 Plasma 7372 7610 1844 1760 495 306 214 140 It can be seen from the above table that the distribution in animal tissues shows no difference between male and female; after administration, the drug is mainly distributed in kidney, whole blood (mainly distributed in plasma), liver and lung; in all tissues, the highest concentration appears at 0.5 hours, and then the drug is eliminated rapidly, wherein the slowest elimination appears in liver and tumor; and Date Recue/Date Received 2021-07-27 the slow elimination in tumor can explain the advantages of CBP-1018 in terms of effectiveness.
12. Excretion experiment in rats Six noinial SD rats, half male and half female, were given 0.75 mg/70 fiCi/kg of [31-1]CBP-1018 via single tail vein injection. At designated time intervals, samples such as urine, feces, cage flushing/cleaning solution and corpses of integral rats were collected before administration and 0-168 hours after administration, and samples such as bile, urine, feces and cage flushing/cleaning solution of BDC
rats were collected before administration and 0-72 hours after administration. The above-mentioned samples were cryopreserved in a low-temperature refrigerator (-10 C to -30 C).
To each sample, an appropriate amount of scintillation fluid was added and uniformly mixed, and then the amount of radioactivity was measured using a liquid scintillation counter. The amount of radioactivity measured in samples such as bile, urine, feces, cage flushing/cleaning solution and corpses was used to calculate the percentage in the given dose. The amount of radioactivity in the plasma sample was used to calculate the total radioactivity in each gram of the sample. The main pharmacokinetic parameters of the total plasma radioactivity were calculated using WinNonLin software (version 7.0, Pharsight) according to a non-compartmental model.
Table 29. Results of Mass Balance Study on Integral Rats After Administration Cage Total Sex (number of Urine Feces Corpse flushing/cleaning recovery rats) (%) (%) (%) solution (%) (%) 50.26 23.93 4.51 84.40 Female (n = 3) 5.70 2.63 6.11 0.98 1.19 4.02 53.60 20.61 4.83 83.28 Male (n = 3) 4.24 2.16 3.68 1.74 0.42 0.71 Female and male 51.93 22.27 4.67 83.84 (n = 3 female and 4.97 2.30 4.87 2.21 0.82 2.65 3 male) Date Recue/Date Received 2021-07-27 It can be seen from the above table that CBP-1018 is mainly excreted in urine, which accounts for about 57% of the total given dose, and the amount excreted in feces accounts for less than 25%. The result of mainly excretion in urine through the kidney is consistent with the finding of large distribution in the kidney in the tissue distribution of nude mice.
13. Plasma stability CBP-1008 (the active substance thereof is LDC1OB described in WO
2017025047 Al, and CBP-1008 is obtained by mixing LDC1OB with auxiliary materials and lyophilizing same; WO 2017025047A1 is incorporated by reference in its entirety) and CBP-1018 at a concentration of 1 [tg/mL, 10 [tg/mL and [tg/mL were incubated with plasma of different species at 37 C for 2 hours to observe the stability of the two compounds. The results show (in Table 30) that CBP-1018 is stable in plasma of various species, and the remaining percentage thereof after 2 hours of incubation is above 90% relative to the concentration at 0 hours. However, CBP-1008 is only stable in plasma of rats (with 87.92%-91.82%
remaining), and is unstable in plasma of other species, with only 0.751%-24.52%
remaining.
The experimental results suggest that the plasma stability of CBP-1018 is better than that of CBP-1008.
Table 30. Summary of Plasma Stability Data of CBP-1008 and CBP-1018 at Different Concentrations in Various Species (% relative to 0 hours) Cynomolgus Compound Concentration Mouse Rat Human monkey 1 [tg/mL 0.805 87.92 0.961 0.751 CBP-1008 10 [tg/mL 1.27 91.47 0.988 0.807 100 [tg/mL 24.52 91.82 1.10 0.953 1 [tg/mL 94.4 92.2 97.3 99.1 CBP-1018 10 [tg/mL 96.5 95.0 95.8 102 100 [tg/mL 98.1 94.8 101 103 Date Recue/Date Received 2021-07-27 Notes:
1) The data in the table is the percentage of the compound concentration in plasma after plasma incubation for 2 hours relative to 0 hours.
2) Since the data in the table is obtained from two experiments, the effective digits after the decimal point are inconsistent. For the traceability and authenticity, the data in the report is directly referred without uniformity.
Related samples: SN-38, Dxd0017, CR19428, CR19425 and CR19426 Experimental operations: NCI-H226, CFPAC-1 and MDA-MB-468 cells were prepared in advance and counted; MDA-MB-468 and CFPAC-1 cells were plated at a density of 2 x 104 cells/ml (100 p1/well) and NCI-H226 cells were plated at a density of 1 x 104 cells/ml (100 1/well) in 96-well plates; and negative control and blank control wells were set on each plate. After adherent cell culture overnight, the diluted samples to be tested were added at 50 1/well. The plates were placed in an incubator at 37 C, 5% CO2 and cultured for 68-72 hours. To each well, 15 p1(10%
of the liquid volume in a well) of a color developing solution from CCK8 was added; and the plates were incubated at 37 C for 45-70 minutes and each plate was read at 450 nm on a microplate reader. The data was edited using SoftMax Pro and a 4-P fitting curve was plotted. The specific data is shown in the following table:
Table 20. Inhibitory Effects of CR19428, CR19425 and CR19426 on Proliferation of Cell Lines ICso ICso ICso ICso ICso Cell line (SN-38) (Dxd0017) (CR19428) (CR19425) (CR19426) CFPAC-1 0.0055 0.0064 0.9060 0.6210 1.6600 MDA-MB-468 0.0136 0.0061 1.0700 0.9510 2.3000 NCI-H226 0.0173 0.0080 1.6200 1.0100 4.3900 Example 5: Pharmacodynamic Study of Conjugate Compounds in CDX
Models Date Recue/Date Received 2021-07-27 1. Pharmacodynamic study 1 of CB-20BK in CDX models 1) Sample preparation:
CB-20BK lyophilized powder was weighed, dissolved with PBS and prepared into a sample mother solution, and the mother solution was diluted with a physiological saline for injection to a sample solution with a working concentration for later use.
2) CDX model construction Main cell lines: KB human oral epidermoid carcinoma cells, MIA paca-2 human pancreatic cancer cells, HCC1954 human breast cancer cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells.
Model construction: The cells were resuscitated, cultured, collected, counted, and subcutaneously injected into the right flank of BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping and administration were performed, or rapidly expanded tumor masses were transferred and injected to mice for scale up.
3) Grouping, administration and observation Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping was performed, and a model control group and different doses of administration groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the effects of tumor growth and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss (measuring weight twice a week), and other abnoinial conditions in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor mass were measured twice a week. The foimula for the calculation of tumor volume (TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis Date Recue/Date Received 2021-07-27 According to changes in tumor volumes of mice (Figs. 5A-5E), it can be seen that CB-20BK has a good inhibitory effect in CDX tumor models of KB, MIA aca-2, HCC1954, CALU-3 and DU145 cell lines in mice.
2. Pharmacodynamic study 2 of CB-20BK in CDX models Experiment purpose: to perfornt a phannacodynamic study of conjugate compound CB-20BK in a subcutaneous xenograft model (CDX) of humanized LNCaP, DU145 and NCI-H460 cell lines Main CDX models: LNCaP, DU145 and NCI-H460 Experimental scheme:
The cells or tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice. When the tumor volume expanded to 80-160 mm3, a randomized grouping was performed, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g. After grouping and administration, the effects of tumor growth and treatment on nothial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated. The formula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 21. Inhibitory Effects of CB-20BK on Growth in CDX models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) Date Recue/Date Received 2021-07-27 Tumor growth Model FOLR1 FOLH1 inhibition rate Administration dosage/time (TGI) (%) LNCaP 5+ 10+ 95.68 3 mg/kg D1, 8, 15 DU145 - - 52 5 mg/kg DI, 4, 7 NCI-H460 - - 32 10 mg/kg D1, 8, 15 It can be seen from the above table that the test sample CB-20BK shows different degrees of tumor growth inhibition effects with a regimen of administration via tail vein. For CDX models of humanized LNCaP, DU145 and NCI-H460 cell lines, the test sample has a certain anti-tumor growth effect compared with a negative control group. In the LNCaP model with dual expression of FOLR1 and FOLH1, the test sample was administered on day 1, day 8 and day (D1, D8 and D15) at a dose of 3 mg/kg, has a TGI value of 95.68%, shows an excellent anti-tumor growth effect, which is significantly better than that in 10 and NCI-H460 models with a relatively low expression of FOLR1 and FOLH1. The tumor growth inhibition effect has a certain correlation with the expression level of cell-related receptors.
3. Tumor growth inhibition experiment 1 of conjugate compound CB-20BK in HuPrime xenograft PDX models Experiment purpose: Phattnacodynamic study of conjugate compound CB-20BK in PDX models Main models: LU1206, LU1380 and LU0367 Experimental scheme: The tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g and an administration dosage of 3 mg/kg. After grouping and administration, the effects of tumor growth Date Recue/Date Received 2021-07-27 and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated.
The foimula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 22. Inhibitory Effects of CB-20BK on Growth in Lung Cancer PDX Models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) FOLR1 gene FOLH1 gene TGI (%) Model expression level expression level Dosage LU1206 4+ 2+ 90.89 3 mg/kg LU1380 - 4+ 30.02 3 mg/kg LU0367 - 5+ 71.34 3 mg/kg It can be seen from the data in Table 22 that the test sample CB-20BK (3 mg/kg) shows a certain anti-tumor effect on LU1206, LU1380 and LU0367 humanized lung cancer PDX models. The TGI value in the LU1206 model with dual expression of FOLR1 and FOLH1 is 90.89%, and the TGI values in LU1380 and LU0367 models with a single expression are 30.02% and 71.34%, respectively.
In the tumor growth inhibition experiment, the tumor growth inhibition effect has a certain correlation with the expression level of cell-related receptors.
4. Tumor growth inhibition experiment 2 of conjugate compound CB-20BK in HuPrime xenograft PDX models Experiment purpose: Phannacodynamic study of conjugate compound CB-20BK in PDX models Main models: BR1283 and BR0438 Date Recue/Date Received 2021-07-27 Experimental scheme: The tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 lig and an administration dosage of 3 mg/kg. After grouping and administration, the effects of tumor growth and treatment on nottnal behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnottnal conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated.
The fottnula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 23. Inhibitory Effects of CB-20BK on Growth in Breast Cancer PDX Models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) FOLR1 gene expression FOLH1 gene TGI (%) Model level expression level Dosage BR1283 7+ 6+ 96.49 3 mg/kg BR0438 5+ 4+ 70.74 3 mg/kg It can be seen from the above table that the TGI values of the test sample CB-20BK at a dose of 3 mg/kg in BR1283 and BR0438 models with dual expression of FOLR1 and FOLH1 are 96.49% and 70.74%, respectively, showing an excellent anti-tumor growth effect. Moreover, the TGI of CB-20BK is higher in BR1283 with a higher expression of FOLR1 and FOLH1.
5. Pharmacodynamic study of CB-20B in CDX models Date Recue/Date Received 2021-07-27 1) Sample preparation:
CB-20B lyophilized powder was weighed, dissolved with PBS and prepared into a sample mother solution, and the mother solution was diluted with a physiological saline for injection to a sample solution with a working concentration for later use.
2) CDX model construction Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells and DU145 human prostate cancer cells.
Model construction: The cells were resuscitated, cultured, collected, counted, and subcutaneously injected into the right flank of BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping and administration were performed, or rapidly expanded tumor masses were transferred and injected to mice for scale up.
3) Grouping, administration and observation Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping was performed, and a model control group and different doses of administration groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the effects of tumor growth and treatment on normal behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss (measuring weight twice a week), and other abnoinial conditions in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor mass were measured twice a week. The foimula for the calculation of tumor volume (TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis According to changes in tumor volume of mice (Figs. 6A-6C), it can be seen that CB-20B has a good inhibitory effect in CDX tumor models of KB, PC-9 and DU145 cell lines in mice.
6. Pharmacodynamic study of CB-18G in CDX models Date Recue/Date Received 2021-07-27 1) Sample preparation:
CB-18G lyophilized powder was weighed, dissolved with PBS and prepared into a sample mother solution, and the mother solution was diluted with a physiological saline for injection to a sample solution with a working concentration for later use.
2) CDX model construction Main cell lines: KB human oral epidermoid carcinoma cells, PC-9 human lung cancer cells, SPC-Al human lung adenocarcinoma cells, CALU-3 human lung adenocarcinoma cells and DU145 human prostate cancer cells Model construction: The cells were resuscitated, cultured, collected and counted, and the cell solution was subcutaneously injected into the right flank of BALB/c-nude mice. When the tumor grew and expanded to 80-160 mm3, grouping and administration were performed, or rapidly expanded tumor masses were transferred and injected to mice for scale up.
3) Grouping, administration and observation Grouping: When the tumor grew to about 80-160 mm3 on average, a grouping was performed, and a model control group and different doses of administration groups were set.
Administration: The mice were administrated via tail vein injection.
Experimental observation and measurement: After tumor inoculation, the effects of tumor growth and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss (measuring weight twice a week), and other abnoinial conditions in eyes, hair, etc., were conventionally monitored.
The weight of mice and the long diameter (a) and short diameter (b) of tumor mass were measured twice a week. The formula for the calculation of tumor volume (TV) is: TV= 1/2 x ax b2.
4) Experimental results and analysis According to changes in tumor volumes of mice (Figs. 7A-7E), it can be seen that CB-18G has a good inhibitory effect in CDX tumor models of KB, PC-9, SPC-Al, CALU-3 and DU145 cell lines in mice.
Date Recue/Date Received 2021-07-27 7. Tumor growth inhibition experiment of conjugate compounds CB-1020, CB-1320 and CB-1820 in subcutaneous xenograft model (CDX) of humanized HPAF-II, NCI-11226 and SCLC-211I cell lines Experiment purpose: Phannacodynamic study of ligand conjugates CB-1020, CB-1320 and CB-1820 in CDX models Main CDX models: HPAF-II (human pancreatic cancer cells), NCI-H226 (human lung cancer cells) and SCLC-21H (small cell lung cancer cells) Experimental scheme:
The cells or tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice. When the tumor volume expanded to 80-160 mm3, a randomized grouping was performed, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g. After grouping and administration, the effects of tumor growth and treatment on nothial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated. The formula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 24. Inhibitory Effects of CB-1020, CB-1320 and CB-1820 on Growth in CDX
Models and Receptor Gene Expression Levels in Each Model (with reference to data from Crown Bioscience Inc.) Date Recue/Date Received 2021-07-27 gene gene gene gene TGI (%) TGI (%) TGI (%) P
Model Dosage expression expression expression expression (CB-1020) (CB-1320) (CB-1820) value level level level level 0.283 0.461 3 mg/kg HPAF-II + - +/- +/- 0.445 0.153 5 mg/kg 0.899 0.023 10 mg/kg 0.823 0.011 3 mg/kg NCIH226 +/- +/- +/- - 0.962 0.006 3 mg/kg 0.992 0.005 10 mg/kg 0.995 0.131 3 mg/kg SCLC-21H 2+ +/- 3+ - 0.361 0.610 3 mg/kg 0.985 0.134 10 mg/kg CB-1020 has an obvious anti-tumor growth effect in subcutaneous xenograft models of humanized HPAF-II cell lines; CB-1320 has an obvious anti-tumor growth effect in subcutaneous xenograft models of NCI-H226 and SCLC-21H cell lines; and CB-1820 has an obvious anti-tumor growth effect in subcutaneous xenograft models of SCLC-21H cell lines.
8. Tumor growth inhibition experiment of conjugate compound CR19428 in subcutaneous xenograft model (CDX) of humanized CALU-3, SCLC-2111 and SPC-Al cell lines Experimental scheme: The tumor tissue mass were prepared and subcutaneously inoculated into the anterior right flank of BALB/c-nude mice.
When the tumor volume expanded to 80-160 mm3, a randomized grouping was perfottned, and a model control group and administration groups were set. The mice were administrated from the first day of grouping, wherein the administration dose was adjusted according to the latest weight measurement. The mice were administrated via tail vein injection at an administration volume of 10 1.11/g and twice a week for Date Recue/Date Received 2021-07-27 consecutive 3 weeks. After grouping and administration, the effects of tumor growth and treatment on noinial behaviors of animals, specifically including the activity of experimental animals, food intake and drinking status, weight gain or loss status, and other abnoinial conditions in eyes, hair, etc., were monitored. After grouping and administration, the weight of mice was measured twice a week to calculate the rate of weight change; at the same time, the long diameter and short diameter of the tumor were measured with a vernier caliper, and the tumor volume, relative tumor proliferation rate, tumor volume inhibition rate and other indicators were calculated.
The foimula of tumor volume is TV = 0.5 a x b2, wherein a is the long diameter of a tumor and b is the short diameter of a tumor.
Table 25. Inhibitory Effects of CR19428 in Lung Cancer PDX models Model TGI (%) Administration dosage/time CALU-3 51 50 mg/kg q2/w*3 SCLC-21H 66 50 mg/kg q2/w*3 SPC -A1 75 50 mg/kg q2/w*3 SPC -A1 91 100 mg/kg q2/w*3 CR-19428 is a drug conjugate of ligands FOLR1 and FOLH1 and a payload Dxd. It can be seen from the above table that the drug conjugate has an obvious anti-tumor growth effect in subcutaneous xenograft models of humanized CALU-3, SCLC-21H and SPC-Al cell lines.
9. Study on effectiveness of conjugate compound CBP-1018 in lung cancer LU2505 model (PDX model) The 3rd generation of lung cancer PDX model LU2505 (from Asian female patients), which is a rapid-growing tumor model, was used in this experiment.
BALB/c nude mice bore tumors subcutaneously and, when the tumor volume reached about 150 mm3, were grouped into: low-dose, medium-dose and high-dose test sample groups, a small molecule MMAE control group, a targeting polypeptide 20BK-SMO9 control group, and a blank control group (a total of 6 groups, and 8 mice per group); the mice were administered on day 1, day 8 and day 15 after the grouping; and the observation was continued for 14 days after the last Date Recue/Date Received 2021-07-27 administration. The active substance of test sample CBP-1018 is CB-20BK, which is obtained by mixing CB-20BK with auxiliary materials and then lyophilizing same.
Table 26. Phannacodynamic Results of CBP-1018 for Injection in Lung Cancer LU2505 models (first experiment) Tumor Test sample Tumor growth Dosage Weight Tumor weight /Control volume inhibition (mg/kg) (g) (mg) sample (mm3) rate TGI%
Blank control / 24.0 2219.62 / 2031.4 (D22) group 4.5 22.5 0.00 100.00 0.0 (D29) CBP-1018 1.5 22.1 386.16 82.60 400.7 (D29) 0.5 23.6 1634.50 26.36 2084.7 (D26) 20BK-SMO9 10 23.8 2024.75 8.78 1811.5 (D22) MMAE 0.375 23.5 1132.61 48.97 1597.1 (D29) Notes: 1) Due to requirements of animal welfare, animals in a group must be euthanized when the mean tumor volume in the group reaches 2000 mm3, leading to different execution time for each group. 2) The data of weight, tumor volume, and tumor growth inhibition rate is the data obtained on day 22 (D22).
As shown in Table 26 and Fig. 8A:
animals in all groups show no abnoinial clinical manifestations and no deaths after administration; and the weight of the animals in each group increases slowly.
The animals in the blank control group, 20BK-SMO9 group, and low-dose CBP-1018 group were euthanized on day 22, day 22, and day 26 (D26) because of the mean tumor volume exceeding 2000 mm3. Therefore, the data of weight, tumor volume, and tumor growth inhibition rate in Table 26 is the data obtained on D22.
The effectiveness of CBP-1018 for injection has an obvious dose correlation, wherein CBP-1018 for injection was ineffective in the low-dose group, and Date Recue/Date Received 2021-07-27 effective in the medium-dose group and the high-dose group. The tumor in the high-dose group was completely cured on day 19 (D19), and no signs of tumor growth were seen on day 29 (D29).
The polypeptide 20BK-SM09 group showed no obvious tumor growth inhibition effect, suggesting that a single targeting polypeptide is not enough to produce a clear anti-tumor effect.
The MMAE group showed a clear tumor growth inhibition effect. Equimolar MMAE is contained in MMAE at 0.375 mg/kg and in CBP-1018 at 1.5 mg/kg. It can be seen from the comparison that the tumor growth inhibition effect of CBP-1018 at 1.5 mg/kg is significantly better than that in the MMAE group, suggesting the advantage of ligand targeting (tumor growth inhibition rate:
82.60%
vs 48.97%).
10. Study on effectiveness of conjugate compound CBP-1018 in lung cancer LU1206 model (PDX model) The 5th generation of lung cancer PDX model LU1206 (from Asian female patients), which is a rapid-growing tumor model, was used in this experiment.
BALB/c nude mice bore tumors subcutaneously and, when the tumor volume reached about 150 mm3, were grouped into: low-dose, medium-dose and high-dose test sample groups, a small molecule MMAE control group, a targeting polypeptide 20BK-SMO9 control group, and a blank control group (a total of 6 groups, and 8 mice per group); the mice were administered on day 1, day 8 and day 15 after the grouping; and the observation was continued for 14 days after the last administration.
Table 27. Phannacodynamic Results of CBP-1018 for Injection in Lung Cancer LU1206 models (first experiment) Tumor Tumor Tumor volume Tumor weight Dosage Weight Groups volume Tumor growth weight Inhibition (mg/kg) (g) (mm3) inhibition rate (mg) ratio (%) (%) Blank control / 22.7 1216.67 / 856.14 /
Date Recue/Date Received 2021-07-27 group 4.5 23.4 31.44 97.42 13.55 98.42 CBP-1018 1.5 23.7 301.64 75.21 185.10 78.38 0.5 22.7 1118.30 8.08 852.19 0.46 20BK-SMO9 10 23.3 1158.80 4.76 914.36 -6.80 MMAE 0.375 23.6 778.32 36.03 437.73 54.53 As shown in Table 27 and Fig. 8B:
animals in all groups show no abnoinial clinical manifestations and no deaths after administration, and all animals were euthanized on day 29. The weight of the animals in each group remained basically unchanged, and is slightly higher than that on the first day of administration.
The effectiveness of CBP-1018 for injection has an obvious dose correlation, wherein CBP-1018 for injection was ineffective in the low-dose group (with a tumor growth inhibition rate of 8.08%), and effective in the medium-dose group and 113 the high-dose group, with a tumor growth inhibition rate of 75.21% and 97.42%, respectively. The difference in effectiveness between the low-dose group and the medium-dose group is relatively large.
The polypeptide 20BK-SM09 group showed no obvious tumor growth inhibition effect, suggesting that a single targeting polypeptide is not enough to produce a clear anti-tumor effect in this model.
The MMAE group showed a clear tumor growth inhibition effect. Equimolar MMAE is contained in MMAE at 0.375 mg/kg and in CBP-1018 at 1.5 mg/kg. It can be seen from the comparison that the tumor growth inhibition effect of CBP-1018 at 1.5 mg/kg is significantly better than that in the MMAE group, suggesting the advantage of ligand targeting (tumor volume inhibition rate:
75.21%
vs 36.03%; tumor weight inhibition rate: 78.38% vs 54.53%).
11. Tissue distribution of tumor-bearing mice (PDX model LU2505) 12 female tumor-bearing mice inoculated with tumor mass of an HuPrime lung cancer LU2505 model and 12 healthy male BALB/c nude mice were given 1.5 mg/140 fiCi/kg of [3fl]CBP-1018 (isotope labeling on MMAE) via single tail vein Date Recue/Date Received 2021-07-27 injection. At 0.5 hours, 2 hours, 6 hours and 24 hours respectively, 3 male mice and 3 female mice were placed in an induction box and anesthetized by inhaling an appropriate amount of carbon dioxide; and blood was collected by cardiac puncture, and then euthanasia was carried out immediately to collect samples.
Table 28. Total Radioactivity in Various Tissues at Different Time Points after Single Intravenous Administration of [3I-1]CBP-1018 Radioactivity concentration (ng Eq./g) Tissue 0.5 hours 2 hours 6 hours 24 hours (% C.) Tumor 763 / 643 / 566 / 413 Esophagus 915 784 628 508 521 172 286 91.7 Body fat 341 479 288 276 231 177 67.2 43.7 Skeletal muscle 321 311 207 137 188 95.5 90.8 61.5 Spleen 744 693 701 524 671 421 253 163 Stomach wall 658 623 414 370 460 259 211 Whole brain 66.3 67.9 53.8 39.5 82.0 48.2 76.4 68.3 Testicular epididymis 350 494 279 298 183 248 92.3 64.8 Heart 785 795 415 326 219 143 114 69.2 Lung 1493 1807 997 920 416 392 109 93.7 Kidney 5682 6118 3300 4197 1227 1391 241 238 Liver 1423 1306 1257 865 1138 599 712 518 Small intestine wall 726 778 641 816 490 334 210 90.9 Large intestine wall 550 520 672 823 1074 490 215 Whole blood 2087 3194 684 691 211 132 86.5 64.6 Plasma 7372 7610 1844 1760 495 306 214 140 It can be seen from the above table that the distribution in animal tissues shows no difference between male and female; after administration, the drug is mainly distributed in kidney, whole blood (mainly distributed in plasma), liver and lung; in all tissues, the highest concentration appears at 0.5 hours, and then the drug is eliminated rapidly, wherein the slowest elimination appears in liver and tumor; and Date Recue/Date Received 2021-07-27 the slow elimination in tumor can explain the advantages of CBP-1018 in terms of effectiveness.
12. Excretion experiment in rats Six noinial SD rats, half male and half female, were given 0.75 mg/70 fiCi/kg of [31-1]CBP-1018 via single tail vein injection. At designated time intervals, samples such as urine, feces, cage flushing/cleaning solution and corpses of integral rats were collected before administration and 0-168 hours after administration, and samples such as bile, urine, feces and cage flushing/cleaning solution of BDC
rats were collected before administration and 0-72 hours after administration. The above-mentioned samples were cryopreserved in a low-temperature refrigerator (-10 C to -30 C).
To each sample, an appropriate amount of scintillation fluid was added and uniformly mixed, and then the amount of radioactivity was measured using a liquid scintillation counter. The amount of radioactivity measured in samples such as bile, urine, feces, cage flushing/cleaning solution and corpses was used to calculate the percentage in the given dose. The amount of radioactivity in the plasma sample was used to calculate the total radioactivity in each gram of the sample. The main pharmacokinetic parameters of the total plasma radioactivity were calculated using WinNonLin software (version 7.0, Pharsight) according to a non-compartmental model.
Table 29. Results of Mass Balance Study on Integral Rats After Administration Cage Total Sex (number of Urine Feces Corpse flushing/cleaning recovery rats) (%) (%) (%) solution (%) (%) 50.26 23.93 4.51 84.40 Female (n = 3) 5.70 2.63 6.11 0.98 1.19 4.02 53.60 20.61 4.83 83.28 Male (n = 3) 4.24 2.16 3.68 1.74 0.42 0.71 Female and male 51.93 22.27 4.67 83.84 (n = 3 female and 4.97 2.30 4.87 2.21 0.82 2.65 3 male) Date Recue/Date Received 2021-07-27 It can be seen from the above table that CBP-1018 is mainly excreted in urine, which accounts for about 57% of the total given dose, and the amount excreted in feces accounts for less than 25%. The result of mainly excretion in urine through the kidney is consistent with the finding of large distribution in the kidney in the tissue distribution of nude mice.
13. Plasma stability CBP-1008 (the active substance thereof is LDC1OB described in WO
2017025047 Al, and CBP-1008 is obtained by mixing LDC1OB with auxiliary materials and lyophilizing same; WO 2017025047A1 is incorporated by reference in its entirety) and CBP-1018 at a concentration of 1 [tg/mL, 10 [tg/mL and [tg/mL were incubated with plasma of different species at 37 C for 2 hours to observe the stability of the two compounds. The results show (in Table 30) that CBP-1018 is stable in plasma of various species, and the remaining percentage thereof after 2 hours of incubation is above 90% relative to the concentration at 0 hours. However, CBP-1008 is only stable in plasma of rats (with 87.92%-91.82%
remaining), and is unstable in plasma of other species, with only 0.751%-24.52%
remaining.
The experimental results suggest that the plasma stability of CBP-1018 is better than that of CBP-1008.
Table 30. Summary of Plasma Stability Data of CBP-1008 and CBP-1018 at Different Concentrations in Various Species (% relative to 0 hours) Cynomolgus Compound Concentration Mouse Rat Human monkey 1 [tg/mL 0.805 87.92 0.961 0.751 CBP-1008 10 [tg/mL 1.27 91.47 0.988 0.807 100 [tg/mL 24.52 91.82 1.10 0.953 1 [tg/mL 94.4 92.2 97.3 99.1 CBP-1018 10 [tg/mL 96.5 95.0 95.8 102 100 [tg/mL 98.1 94.8 101 103 Date Recue/Date Received 2021-07-27 Notes:
1) The data in the table is the percentage of the compound concentration in plasma after plasma incubation for 2 hours relative to 0 hours.
2) Since the data in the table is obtained from two experiments, the effective digits after the decimal point are inconsistent. For the traceability and authenticity, the data in the report is directly referred without uniformity.
14. Half-life in PK
Consistent with the comparison of plasma stability, the pharmacokinetic half-life of CBP-1018 (about 1 hour) is significantly longer than that of CBP-(about 20 minutes) in rats and cynomolgus monkeys, suggesting that CBP-1018 is more stable than CBP-1008.
Table 31. Comparison of Half-life (in hour) of CBP-1008 and CBP-1018 in PK of Rats 0.15 mg/kg 0.5 mg/kg 1.5 mg/kg Male Female Male Female Male Female CBP-1008 0.245 0.215 0.249 0.202 0.222 0.368 CBP-1018 0.928 0.833 1.37 0.952 1.49 1.51 Date Recue/Date Received 2021-07-27
Consistent with the comparison of plasma stability, the pharmacokinetic half-life of CBP-1018 (about 1 hour) is significantly longer than that of CBP-(about 20 minutes) in rats and cynomolgus monkeys, suggesting that CBP-1018 is more stable than CBP-1008.
Table 31. Comparison of Half-life (in hour) of CBP-1008 and CBP-1018 in PK of Rats 0.15 mg/kg 0.5 mg/kg 1.5 mg/kg Male Female Male Female Male Female CBP-1008 0.245 0.215 0.249 0.202 0.222 0.368 CBP-1018 0.928 0.833 1.37 0.952 1.49 1.51 Date Recue/Date Received 2021-07-27
Claims (46)
1. A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a prostate-specific membrane antigen ligand moiety, respectively.
2. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the prostate-specific membrane antigen ligand has the following structure:
3. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the prostate-specific membrane antigen ligand has the following structure:
4. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the prostate-specific membrane antigen ligand has the following structure:
5. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the prostate-specific membrane antigen ligand has the following structure:
6. A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and a ligand moiety represented by foimula (I), respectively:
7. A conjugate compound or a pharmaceutically acceptable salt thereof, comprising a payload and two targeting molecules, wherein the two targeting molecules are a synergistic molecule moiety and P10, respectively, and the payload is camptothecin or any derivative thereof
8. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-7, wherein the two targeting molecules are different.
9. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein the synergistic molecule is a cell-interacting molecule.
10. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-9, wherein the two targeting molecules are cell-interacting molecules that interact with different cell molecules.
11. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-10, wherein the synergistic molecule is an endocytosis molecule capable of mediating endocytosis.
12. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-11, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), GNRHR, Her2, Trop2, Her3, NECTIN4, LRP1, GLUT1, EGFR1, AXL, CA9, CD44, Claudin18.2, APN, DLL3, CEACAM5, FZD10, TFRC, MET, IGFR1, SSTR2, CCKBR, LFA1, ICAM, GPR87, GM-CSF, GM-CSFR, TIM3, TLR family, CD40, CD4OL, 0X40, OX4OL, GITRL, GITR, 4-BBL, 4-1BB, CD70, CD27, ICOSL, ICOS, HHLA2, CD28, CD86/80, CD28, MHCII antigen, TCR, CTLA-4, CD155, CD122, CD113, IGIT, PD-L1, PD1, Galectin-9, TIM-3, HVEM, BTLA, CD160, VISTA, B7-H4, B7-H3, phosphatidylserine, HHLA2, LAG3, Galectin-3, LILRB4, SIGLEC15, NKG2A, NKG2D, SLAMF7, MR2DL1, KIR2DL2, KIR2DL3, FGFR1, FGFR2, FGFR4, NeuGcGM3 and CXCR4.
13. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-12, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA), SSTR2 and GNRHR.
14. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, SSTR2 and GNRHR.
15. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein the synergistic molecule binds to a molecule selected from the group consisting of FOLR1, TRPV6, FOLH1 (PMSA) and GNRHR.
16. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-15, wherein the synergistic molecule is a folate or an analog thereof
17. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 16, wherein the analog of a folate is selected from the group consisting of 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, methotrexate, and 5,10-methyl enetetrahydro folate .
18. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1 or 6, comprising one, two, three, four or more payloads.
19. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1 or 6, wherein the payload is selected from the group consisting of a small molecule compound, a nucleotide, a peptide, and a protein.
20. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 19, wherein the payload is a small molecule compound.
21. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 20, wherein the small molecule compound is selected from the group consisting of camptothecin and any derivative thereof, auristatin and any derivative thereof, maytansine and any derivative thereof, a radionuclide complex, a cyclooxygenase-2 inhibitor, paclitaxel and any derivative thereof, epothilone and any derivative thereof, bleomycin and any derivative thereof, dactinomycin and any derivative thereof, plicamycin and any derivative thereof, and mitomycin C.
22. The conjugate compound or the phannaceutically acceptable salt thereof according to claim 21, wherein the small molecule compound is camptothecin or any derivative thereof, auristatin or any derivative thereof, a radionuclide complex or a cyclooxygenase-2 inhibitor.
23. The conjugate compound or the phannaceutically acceptable salt thereof according to any one of claims 1-22, wherein the payload is linked to at least one of the targeting molecules via a linker.
24. The conjugate compound or the phannaceutically acceptable salt thereof according to claim 23, wherein the linker is a peptide linker, a disulfide linker, a pH-dependent linker or a combination of the above-mentioned linkers.
25. The conjugate compound or the phannaceutically acceptable salt thereof according to claim 24, wherein the peptide linker is cleavable under a certain physiological environment by protease cleavage or reduction.
26. The conjugate compound or the phannaceutically acceptable salt thereof according to claim 24 or 25, wherein the peptide linker is selected from the group consisting of cysteine, lysine, lysine-lysine, valine-citrulline, phenylalanine-lysine, valine-lysine, cysteine-lysine, cysteine-glutamic acid, aspartic acid-aspartic acid, and aspartic acid-aspartic acid-lysine, and optionally, the carboxylic acid in the above-mentioned amino acids is amidated.
27 The conjugate compound or the phannaceutically acceptable salt thereof according to claim 24, wherein the disulfide linker is selected from the group consisting of DMDS, MDS, DSDM and NDMDS.
28. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 24, wherein the pH-dependent linker is a cis-aconitic anhydride.
29. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 23, wherein the linker has the following structures:
or the linker is a combination of the above-mentioned structures and a peptide linker.
or the linker is a combination of the above-mentioned structures and a peptide linker.
30. The conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-29, wherein the two targeting molecules are linked to each other via a spacer.
31. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 30, wherein the spacer comprises the amino acid sequence selected from the group consisting of SEQ ID NOs: 1-14, Arg-Arg, Ala-Ser-Asn, Ala-Ala-Ala, Ser-Ser-Arg, Pro-Arg and Pro-Leu-Gly.
32. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 1, wherein the conjugate compound is selected from the group consisting of the following compounds:
(CB-20R), wherein M is a radionuclide.
(CB-20R), wherein M is a radionuclide.
33. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 6, wherein the conjugate compound is:
34. The conjugate compound or the pharmaceutically acceptable salt thereof according to claim 7, wherein the conjugate compound is
35. A phannaceutical composition comprising the conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-34, and a pharmaceutically acceptable carrier.
36. The phannaceutical composition according to claim 35, wherein the composition is administered intravenously, subcutaneously, orally, intramuscularly or intraventricularly.
37. A method for delivering a payload to a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-34, or the phannaceutical composition according to claim 35 or 36.
38. A method for treating a disease in a subject, comprising administering to the subject a therapeutically effective amount of the conjugate compound or the pharmaceutically acceptable salt thereof according to any one of claims 1-34, or the pharmaceutical composition according to claim 35 or 36.
39. The method according to claim 38, wherein the disease is selected from the group consisting of a cancer, an immunological disease, a cardiovascular disease, a metabolic disease, and a neurological disease.
40. The method according to claim 39, wherein the cancer is selected from the group consisting of prostatic cancer, breast cancer, lung cancer, renal cancer, leukemia, ovarian cancer, gastric cancer, uterine cancer, endometrial carcinoma, liver cancer, colon cancer, thyroid cancer, pancreatic cancer, colorectal cancer, esophageal cancer, testicular cancer, skin cancer, lymphoma, and multiple myeloma.
41. The method according to claim 39, wherein the immunological disease is an autoimmune disease.
42. The method according to claim 41, wherein the autoimmune disease is selected from the group consisting of connective tissue disease, systemic sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.
43. The method according to claim 39, wherein the cardiovascular disease is selected from the group consisting of angina, myocardial infarction, stroke, heart attack, hypertensive heart disease, rheumatic heart disease, cardiomyopathy, arrhythmia, and congenital heart disease.
44. The method according to claim 39, wherein the metabolic disease is selected from the group consisting of diabetes, gout, obesity, hypoglycemia, hyperglycemia, and dyslipidemia.
45. The method according to claim 39, wherein the neurological disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, head injury, multiple sclerosis, vertigo, coma, and epilepsy.
46. The method according to any one of claims 38-45, wherein the method further comprises administering one or more therapeutic agents in combination with the conjugate compound or the phannaceutically acceptable salt thereof, or the pharmaceutical composition.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CNPCT/CN2019/073962 | 2019-01-30 | ||
CN202010048593.4 | 2020-01-16 | ||
CN202010048593 | 2020-01-16 | ||
PCT/CN2020/074117 WO2020156513A1 (en) | 2019-01-30 | 2020-01-31 | Bi-ligand drug conjugate and use thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CA3127903A1 true CA3127903A1 (en) | 2020-08-06 |
Family
ID=77369515
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA3127903A Pending CA3127903A1 (en) | 2019-01-30 | 2020-01-31 | Bi-ligand drug conjugate and use thereof |
Country Status (1)
Country | Link |
---|---|
CA (1) | CA3127903A1 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113122543A (en) * | 2021-04-01 | 2021-07-16 | 厦门大学 | Aptamer of sialic acid binding immunoglobulin-like lectin-15 protein and application thereof |
-
2020
- 2020-01-31 CA CA3127903A patent/CA3127903A1/en active Pending
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113122543A (en) * | 2021-04-01 | 2021-07-16 | 厦门大学 | Aptamer of sialic acid binding immunoglobulin-like lectin-15 protein and application thereof |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP3903825A1 (en) | Bi-ligand drug conjugate and use thereof | |
CN109316605B (en) | Folate receptor binding ligand-drug conjugates | |
KR101413955B1 (en) | Aziridinyl-epothilone compounds | |
KR102301596B1 (en) | Multi-ligand drug conjugates and uses thereof | |
US20150320799A1 (en) | Chimeric antigen receptor-expressing t cells as anti-cancer therapeutics | |
US20180078656A1 (en) | Cryptophycin-based antibody-drug conjugates with novel self-immolative linkers | |
US20220194983A1 (en) | Peptide ligands for binding to psma | |
Zhou et al. | A designed cyclic peptide based on Trastuzumab used to construct peptide-drug conjugates for its HER2-targeting ability | |
US20210122804A1 (en) | Peptide ligands for binding to cd38 | |
JP2019524871A (en) | α (V) β (6) integrin-binding peptide and method of use thereof | |
CN114053426A (en) | Double-drug linked assembly unit and double-drug targeting joint-drug conjugate | |
CA3127903A1 (en) | Bi-ligand drug conjugate and use thereof | |
CA2529555A1 (en) | Leukocyte internalized peptide-drug conjugates | |
EP4265275A1 (en) | Trop2 targeting antibody-drug conjugate, and preparation method and use therefor | |
CA3226897A1 (en) | Tumor-associated calcium signal transducer 2 (tacstd2) antibody-maytansine conjugates and methods of use thereof | |
CA3198788A1 (en) | Camptothecine antibody-drug conjugates and methods of use thereof | |
CA3144951A1 (en) | Cd38-binding agents and uses thereof | |
US20240058463A1 (en) | B7-h3 targeting antibody-drug conjugate, and preparation method therefor and use thereof | |
IL309543A (en) | Ligand-drug conjugate and use thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |
Effective date: 20240126 |