WO2015050957A2 - Treatments for systemic lupus erythematosus - Google Patents
Treatments for systemic lupus erythematosus Download PDFInfo
- Publication number
- WO2015050957A2 WO2015050957A2 PCT/US2014/058576 US2014058576W WO2015050957A2 WO 2015050957 A2 WO2015050957 A2 WO 2015050957A2 US 2014058576 W US2014058576 W US 2014058576W WO 2015050957 A2 WO2015050957 A2 WO 2015050957A2
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- inhibitor
- eif4e
- cells
- sle
- bankl
- Prior art date
Links
- 201000000596 systemic lupus erythematosus Diseases 0.000 title claims abstract description 101
- 238000011282 treatment Methods 0.000 title description 32
- 238000000034 method Methods 0.000 claims abstract description 60
- 230000014509 gene expression Effects 0.000 claims abstract description 35
- 239000003112 inhibitor Substances 0.000 claims description 84
- 101001082110 Acanthamoeba polyphaga mimivirus Eukaryotic translation initiation factor 4E homolog Proteins 0.000 claims description 73
- 101001082109 Danio rerio Eukaryotic translation initiation factor 4E-1B Proteins 0.000 claims description 71
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical group N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 claims description 38
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 claims description 36
- 229960000329 ribavirin Drugs 0.000 claims description 36
- 102100026299 MAP kinase-interacting serine/threonine-protein kinase 1 Human genes 0.000 claims description 26
- 101710139011 MAP kinase-interacting serine/threonine-protein kinase 1 Proteins 0.000 claims description 26
- 101100232687 Drosophila melanogaster eIF4A gene Proteins 0.000 claims description 18
- 230000027455 binding Effects 0.000 claims description 16
- 230000000692 anti-sense effect Effects 0.000 claims description 12
- 238000002560 therapeutic procedure Methods 0.000 claims description 11
- 101710091919 Eukaryotic translation initiation factor 4G Proteins 0.000 claims description 9
- 102100023085 Serine/threonine-protein kinase mTOR Human genes 0.000 claims description 9
- 108010065917 TOR Serine-Threonine Kinases Proteins 0.000 claims description 9
- 239000003246 corticosteroid Substances 0.000 claims description 9
- 239000003018 immunosuppressive agent Substances 0.000 claims description 9
- 230000001684 chronic effect Effects 0.000 claims description 7
- 244000005700 microbiome Species 0.000 claims description 7
- 229940021182 non-steroidal anti-inflammatory drug Drugs 0.000 claims description 7
- 239000003430 antimalarial agent Substances 0.000 claims description 6
- 229960003444 immunosuppressant agent Drugs 0.000 claims description 6
- 238000011301 standard therapy Methods 0.000 claims description 6
- 108010033276 Peptide Fragments Proteins 0.000 claims description 5
- 102000007079 Peptide Fragments Human genes 0.000 claims description 5
- 238000001361 intraarterial administration Methods 0.000 claims description 5
- 238000007918 intramuscular administration Methods 0.000 claims description 5
- 238000001990 intravenous administration Methods 0.000 claims description 5
- 239000000041 non-steroidal anti-inflammatory agent Substances 0.000 claims description 5
- 230000001861 immunosuppressant effect Effects 0.000 claims description 4
- 230000002452 interceptive effect Effects 0.000 claims description 4
- 108090001005 Interleukin-6 Proteins 0.000 abstract description 108
- 102000004889 Interleukin-6 Human genes 0.000 abstract description 104
- 230000001404 mediated effect Effects 0.000 abstract description 9
- 208000023275 Autoimmune disease Diseases 0.000 abstract description 7
- 230000008685 targeting Effects 0.000 abstract description 6
- 239000012636 effector Substances 0.000 abstract 1
- 229940100601 interleukin-6 Drugs 0.000 description 98
- 210000003719 b-lymphocyte Anatomy 0.000 description 81
- 210000004027 cell Anatomy 0.000 description 70
- 108090000623 proteins and genes Proteins 0.000 description 56
- 230000000638 stimulation Effects 0.000 description 37
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 36
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 36
- 206010025135 lupus erythematosus Diseases 0.000 description 36
- 230000000694 effects Effects 0.000 description 35
- 239000003814 drug Substances 0.000 description 34
- 239000000203 mixture Substances 0.000 description 34
- 108090000765 processed proteins & peptides Proteins 0.000 description 34
- 102100021568 B-cell scaffold protein with ankyrin repeats Human genes 0.000 description 33
- 101000971155 Homo sapiens B-cell scaffold protein with ankyrin repeats Proteins 0.000 description 33
- 241000699670 Mus sp. Species 0.000 description 33
- 230000003393 splenic effect Effects 0.000 description 32
- 201000010099 disease Diseases 0.000 description 30
- 229940079593 drug Drugs 0.000 description 29
- 108020004999 messenger RNA Proteins 0.000 description 29
- 102000004169 proteins and genes Human genes 0.000 description 29
- 230000007812 deficiency Effects 0.000 description 28
- 230000026731 phosphorylation Effects 0.000 description 27
- 238000006366 phosphorylation reaction Methods 0.000 description 27
- 235000018102 proteins Nutrition 0.000 description 26
- 230000028327 secretion Effects 0.000 description 26
- 235000001014 amino acid Nutrition 0.000 description 24
- 150000001413 amino acids Chemical class 0.000 description 24
- 102000002574 p38 Mitogen-Activated Protein Kinases Human genes 0.000 description 24
- 108010068338 p38 Mitogen-Activated Protein Kinases Proteins 0.000 description 24
- 230000004913 activation Effects 0.000 description 23
- 238000004519 manufacturing process Methods 0.000 description 20
- 230000002829 reductive effect Effects 0.000 description 20
- -1 ΓΝΚ Proteins 0.000 description 20
- 230000011664 signaling Effects 0.000 description 19
- 241000699666 Mus <mouse, genus> Species 0.000 description 18
- 108020004459 Small interfering RNA Proteins 0.000 description 18
- 230000006870 function Effects 0.000 description 18
- 230000037361 pathway Effects 0.000 description 18
- 102000004196 processed proteins & peptides Human genes 0.000 description 18
- 238000002474 experimental method Methods 0.000 description 17
- 108091008611 Protein Kinase B Proteins 0.000 description 16
- 208000024891 symptom Diseases 0.000 description 16
- 230000014621 translational initiation Effects 0.000 description 16
- 102000004127 Cytokines Human genes 0.000 description 15
- 108090000695 Cytokines Proteins 0.000 description 15
- 239000003795 chemical substances by application Substances 0.000 description 15
- 230000000295 complement effect Effects 0.000 description 15
- 238000000338 in vitro Methods 0.000 description 15
- 230000014616 translation Effects 0.000 description 15
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 14
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 14
- 108020004414 DNA Proteins 0.000 description 13
- 230000015572 biosynthetic process Effects 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 238000013519 translation Methods 0.000 description 13
- 108090000994 Catalytic RNA Proteins 0.000 description 12
- 102000053642 Catalytic RNA Human genes 0.000 description 12
- 230000009266 disease activity Effects 0.000 description 12
- 239000002158 endotoxin Substances 0.000 description 12
- 230000017306 interleukin-6 production Effects 0.000 description 12
- DSPNTLCJTJBXTD-IRNRRZNASA-N pateamine Chemical compound C[C@H]1C[C@@H](N)CC(=O)O[C@@H](C)C\C(C)=C\C=C/C(=O)O[C@H](\C=C(/C)\C=C\C(\C)=C\CN(C)C)CC2=NC1=CS2 DSPNTLCJTJBXTD-IRNRRZNASA-N 0.000 description 12
- DSPNTLCJTJBXTD-UHFFFAOYSA-N pateamine A Natural products CC1CC(N)CC(=O)OC(C)CC(C)=CC=CC(=O)OC(C=C(C)C=CC(C)=CCN(C)C)CC2=NC1=CS2 DSPNTLCJTJBXTD-UHFFFAOYSA-N 0.000 description 12
- 108091092562 ribozyme Proteins 0.000 description 12
- 239000000243 solution Substances 0.000 description 12
- 210000001519 tissue Anatomy 0.000 description 12
- 239000002585 base Substances 0.000 description 11
- 239000002773 nucleotide Substances 0.000 description 11
- 230000004044 response Effects 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 238000003786 synthesis reaction Methods 0.000 description 11
- 238000002965 ELISA Methods 0.000 description 10
- 102000007338 Fragile X Mental Retardation Protein Human genes 0.000 description 10
- 108010032606 Fragile X Mental Retardation Protein Proteins 0.000 description 10
- 230000019491 signal transduction Effects 0.000 description 10
- 101150013553 CD40 gene Proteins 0.000 description 9
- 102100022466 Eukaryotic translation initiation factor 4E-binding protein 1 Human genes 0.000 description 9
- 108050000946 Eukaryotic translation initiation factor 4E-binding protein 1 Proteins 0.000 description 9
- 241001465754 Metazoa Species 0.000 description 9
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 description 9
- 239000004480 active ingredient Substances 0.000 description 9
- 230000003833 cell viability Effects 0.000 description 9
- 239000003431 cross linking reagent Substances 0.000 description 9
- 230000002950 deficient Effects 0.000 description 9
- 210000003743 erythrocyte Anatomy 0.000 description 9
- 238000001727 in vivo Methods 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 9
- 238000012986 modification Methods 0.000 description 9
- 125000003729 nucleotide group Chemical group 0.000 description 9
- 125000006239 protecting group Chemical group 0.000 description 9
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 8
- 108090000174 Interleukin-10 Proteins 0.000 description 8
- 230000005784 autoimmunity Effects 0.000 description 8
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 230000009368 gene silencing by RNA Effects 0.000 description 8
- 230000003993 interaction Effects 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 229920001184 polypeptide Polymers 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- NHKZSTHOYNWEEZ-AFCXAGJDSA-N taribavirin Chemical compound N1=C(C(=N)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 NHKZSTHOYNWEEZ-AFCXAGJDSA-N 0.000 description 8
- 229950006081 taribavirin Drugs 0.000 description 8
- 238000013518 transcription Methods 0.000 description 8
- 230000035897 transcription Effects 0.000 description 8
- 206010061218 Inflammation Diseases 0.000 description 7
- 102100024193 Mitogen-activated protein kinase 1 Human genes 0.000 description 7
- UQPMANVRZYYQMD-UHFFFAOYSA-N N3-(4-fluorophenyl)-2H-pyrazolo[3,4-d]pyrimidine-3,4-diamine Chemical compound C=12C(N)=NC=NC2=NNC=1NC1=CC=C(F)C=C1 UQPMANVRZYYQMD-UHFFFAOYSA-N 0.000 description 7
- 208000002193 Pain Diseases 0.000 description 7
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 7
- 102000002689 Toll-like receptor Human genes 0.000 description 7
- 108020000411 Toll-like receptor Proteins 0.000 description 7
- PYMYPHUHKUWMLA-LMVFSUKVSA-N aldehydo-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 description 7
- 230000000840 anti-viral effect Effects 0.000 description 7
- 210000004369 blood Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- 210000004556 brain Anatomy 0.000 description 7
- 229910052799 carbon Inorganic materials 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 230000006378 damage Effects 0.000 description 7
- 238000005516 engineering process Methods 0.000 description 7
- XXSMGPRMXLTPCZ-UHFFFAOYSA-N hydroxychloroquine Chemical compound ClC1=CC=C2C(NC(C)CCCN(CCO)CC)=CC=NC2=C1 XXSMGPRMXLTPCZ-UHFFFAOYSA-N 0.000 description 7
- 229960004171 hydroxychloroquine Drugs 0.000 description 7
- RAXXELZNTBOGNW-UHFFFAOYSA-N imidazole Natural products C1=CNC=N1 RAXXELZNTBOGNW-UHFFFAOYSA-N 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 150000007523 nucleic acids Chemical class 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 230000001105 regulatory effect Effects 0.000 description 7
- 230000001225 therapeutic effect Effects 0.000 description 7
- 238000001262 western blot Methods 0.000 description 7
- KDCGOANMDULRCW-UHFFFAOYSA-N 7H-purine Chemical compound N1=CNC2=NC=NC2=C1 KDCGOANMDULRCW-UHFFFAOYSA-N 0.000 description 6
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 6
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 6
- 101100276464 Drosophila melanogaster Sra-1 gene Proteins 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 6
- 101000669402 Homo sapiens Toll-like receptor 7 Proteins 0.000 description 6
- 108060003951 Immunoglobulin Proteins 0.000 description 6
- 108091000080 Phosphotransferase Proteins 0.000 description 6
- 108010060818 Toll-Like Receptor 9 Proteins 0.000 description 6
- 102000008235 Toll-Like Receptor 9 Human genes 0.000 description 6
- 102100039390 Toll-like receptor 7 Human genes 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 238000004113 cell culture Methods 0.000 description 6
- 230000003247 decreasing effect Effects 0.000 description 6
- 238000000684 flow cytometry Methods 0.000 description 6
- 210000000987 immune system Anatomy 0.000 description 6
- 102000018358 immunoglobulin Human genes 0.000 description 6
- 230000004054 inflammatory process Effects 0.000 description 6
- 230000000977 initiatory effect Effects 0.000 description 6
- 210000003734 kidney Anatomy 0.000 description 6
- 102000020233 phosphotransferase Human genes 0.000 description 6
- 102000040430 polynucleotide Human genes 0.000 description 6
- 108091033319 polynucleotide Proteins 0.000 description 6
- 239000002157 polynucleotide Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- XJMOSONTPMZWPB-UHFFFAOYSA-M propidium iodide Chemical compound [I-].[I-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CCC[N+](C)(CC)CC)=C1C1=CC=CC=C1 XJMOSONTPMZWPB-UHFFFAOYSA-M 0.000 description 6
- 102000005962 receptors Human genes 0.000 description 6
- 108020003175 receptors Proteins 0.000 description 6
- 150000003839 salts Chemical class 0.000 description 6
- 239000013598 vector Substances 0.000 description 6
- 230000009385 viral infection Effects 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 102100026008 Breakpoint cluster region protein Human genes 0.000 description 5
- GEWLYFZWVLXQME-UHFFFAOYSA-N Cercosporamide Natural products CC12C(=O)C(C(=O)C)=C(O)C=C1OC1=C2C(O)=CC(O)=C1C(N)=O GEWLYFZWVLXQME-UHFFFAOYSA-N 0.000 description 5
- 208000005777 Lupus Nephritis Diseases 0.000 description 5
- 102000043136 MAP kinase family Human genes 0.000 description 5
- 108091054455 MAP kinase family Proteins 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 208000007536 Thrombosis Diseases 0.000 description 5
- 230000005856 abnormality Effects 0.000 description 5
- 239000000556 agonist Substances 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- GEWLYFZWVLXQME-MRXNPFEDSA-N cercosporamide Chemical compound O=C([C@]12C)C(C(=O)C)=C(O)C=C1OC1=C2C(O)=CC(O)=C1C(N)=O GEWLYFZWVLXQME-MRXNPFEDSA-N 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 150000001875 compounds Chemical class 0.000 description 5
- 229960001334 corticosteroids Drugs 0.000 description 5
- 238000011161 development Methods 0.000 description 5
- 230000018109 developmental process Effects 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- 239000006185 dispersion Substances 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 125000000524 functional group Chemical group 0.000 description 5
- 230000002401 inhibitory effect Effects 0.000 description 5
- 230000007246 mechanism Effects 0.000 description 5
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 150000003384 small molecules Chemical class 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 230000004083 survival effect Effects 0.000 description 5
- 230000009261 transgenic effect Effects 0.000 description 5
- CMWRPDHVGMHLSZ-GFCCVEGCSA-N (10r)-10-methyl-3-(6-methylpyridin-3-yl)-9,10,11,12-tetrahydro-8h-[1,4]diazepino[5',6':4,5]thieno[3,2-f]quinolin-8-one Chemical compound C([C@H](NC1=O)C)NC(C2=C3C=C4)=C1SC2=CC=C3N=C4C1=CC=C(C)N=C1 CMWRPDHVGMHLSZ-GFCCVEGCSA-N 0.000 description 4
- 208000006820 Arthralgia Diseases 0.000 description 4
- 208000000094 Chronic Pain Diseases 0.000 description 4
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 206010019233 Headaches Diseases 0.000 description 4
- 241000282412 Homo Species 0.000 description 4
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 4
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 4
- 229940124789 MK2 inhibitor Drugs 0.000 description 4
- 108091030071 RNAI Proteins 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 208000036142 Viral infection Diseases 0.000 description 4
- RJURFGZVJUQBHK-UHFFFAOYSA-N actinomycin D Natural products CC1OC(=O)C(C(C)C)N(C)C(=O)CN(C)C(=O)C2CCCN2C(=O)C(C(C)C)NC(=O)C1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)NC4C(=O)NC(C(N5CCCC5C(=O)N(C)CC(=O)N(C)C(C(C)C)C(=O)OC4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-UHFFFAOYSA-N 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- HMFHBZSHGGEWLO-UHFFFAOYSA-N alpha-D-Furanose-Ribose Natural products OCC1OC(O)C(O)C1O HMFHBZSHGGEWLO-UHFFFAOYSA-N 0.000 description 4
- 125000000539 amino acid group Chemical group 0.000 description 4
- 125000003277 amino group Chemical group 0.000 description 4
- 238000004458 analytical method Methods 0.000 description 4
- 208000007502 anemia Diseases 0.000 description 4
- 230000003172 anti-dna Effects 0.000 description 4
- 206010003246 arthritis Diseases 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 229960003270 belimumab Drugs 0.000 description 4
- 230000015556 catabolic process Effects 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 239000002612 dispersion medium Substances 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 231100000869 headache Toxicity 0.000 description 4
- 210000002216 heart Anatomy 0.000 description 4
- 239000001257 hydrogen Substances 0.000 description 4
- 229910052739 hydrogen Inorganic materials 0.000 description 4
- 210000001503 joint Anatomy 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 230000001537 neural effect Effects 0.000 description 4
- 210000002569 neuron Anatomy 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 229940005483 opioid analgesics Drugs 0.000 description 4
- 230000036407 pain Effects 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000012545 processing Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000000770 proinflammatory effect Effects 0.000 description 4
- 238000011002 quantification Methods 0.000 description 4
- 238000003757 reverse transcription PCR Methods 0.000 description 4
- 206010039073 rheumatoid arthritis Diseases 0.000 description 4
- 239000011780 sodium chloride Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 235000000346 sugar Nutrition 0.000 description 4
- 210000002700 urine Anatomy 0.000 description 4
- 230000035899 viability Effects 0.000 description 4
- 229930024421 Adenine Natural products 0.000 description 3
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 3
- 208000003343 Antiphospholipid Syndrome Diseases 0.000 description 3
- 241000212384 Bifora Species 0.000 description 3
- 102100028892 Cardiotrophin-1 Human genes 0.000 description 3
- 108020004705 Codon Proteins 0.000 description 3
- CMSMOCZEIVJLDB-UHFFFAOYSA-N Cyclophosphamide Chemical compound ClCCN(CCCl)P1(=O)NCCCO1 CMSMOCZEIVJLDB-UHFFFAOYSA-N 0.000 description 3
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 3
- 101000678286 Danio rerio Eukaryotic translation initiation factor 4E-binding protein 3-like Proteins 0.000 description 3
- 101000800913 Dictyostelium discoideum Eukaryotic translation initiation factor 4E-1A-binding protein homolog Proteins 0.000 description 3
- 241000255581 Drosophila <fruit fly, genus> Species 0.000 description 3
- 101000800906 Drosophila melanogaster Eukaryotic translation initiation factor 4E-binding protein Proteins 0.000 description 3
- 238000008157 ELISA kit Methods 0.000 description 3
- 101000669447 Homo sapiens Toll-like receptor 4 Proteins 0.000 description 3
- 102000000589 Interleukin-1 Human genes 0.000 description 3
- 108010002352 Interleukin-1 Proteins 0.000 description 3
- 241000186660 Lactobacillus Species 0.000 description 3
- 102100033610 MAP kinase-interacting serine/threonine-protein kinase 2 Human genes 0.000 description 3
- 101710138999 MAP kinase-interacting serine/threonine-protein kinase 2 Proteins 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 206010071323 Neuropsychiatric syndrome Diseases 0.000 description 3
- 108090000630 Oncostatin M Proteins 0.000 description 3
- 102000004140 Oncostatin M Human genes 0.000 description 3
- 229910019142 PO4 Inorganic materials 0.000 description 3
- 108010044843 Peptide Initiation Factors Proteins 0.000 description 3
- 102000005877 Peptide Initiation Factors Human genes 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 238000011529 RT qPCR Methods 0.000 description 3
- 102100039360 Toll-like receptor 4 Human genes 0.000 description 3
- 241000700605 Viruses Species 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229960000643 adenine Drugs 0.000 description 3
- 230000000844 anti-bacterial effect Effects 0.000 description 3
- 230000003460 anti-nuclear Effects 0.000 description 3
- 229940121375 antifungal agent Drugs 0.000 description 3
- 239000003429 antifungal agent Substances 0.000 description 3
- 239000003435 antirheumatic agent Substances 0.000 description 3
- 239000002981 blocking agent Substances 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 3
- 108010041776 cardiotrophin 1 Proteins 0.000 description 3
- 210000000170 cell membrane Anatomy 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 210000001175 cerebrospinal fluid Anatomy 0.000 description 3
- 208000020832 chronic kidney disease Diseases 0.000 description 3
- 238000000576 coating method Methods 0.000 description 3
- 239000000306 component Substances 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 229960004397 cyclophosphamide Drugs 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000006731 degradation reaction Methods 0.000 description 3
- 239000000551 dentifrice Substances 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 239000002988 disease modifying antirheumatic drug Substances 0.000 description 3
- 239000003937 drug carrier Substances 0.000 description 3
- 208000028208 end stage renal disease Diseases 0.000 description 3
- 201000000523 end stage renal failure Diseases 0.000 description 3
- 239000000284 extract Substances 0.000 description 3
- 206010016256 fatigue Diseases 0.000 description 3
- 239000012634 fragment Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical group O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 3
- 230000008348 humoral response Effects 0.000 description 3
- 150000002431 hydrogen Chemical group 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000010166 immunofluorescence Methods 0.000 description 3
- 229940124589 immunosuppressive drug Drugs 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 239000004615 ingredient Substances 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- 239000000543 intermediate Substances 0.000 description 3
- 230000003834 intracellular effect Effects 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 239000007951 isotonicity adjuster Substances 0.000 description 3
- 229940039696 lactobacillus Drugs 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 210000004072 lung Anatomy 0.000 description 3
- 229920002521 macromolecule Polymers 0.000 description 3
- 210000002540 macrophage Anatomy 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 210000004789 organ system Anatomy 0.000 description 3
- 244000052769 pathogen Species 0.000 description 3
- 230000001717 pathogenic effect Effects 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 235000021317 phosphate Nutrition 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 201000001474 proteinuria Diseases 0.000 description 3
- 230000009712 regulation of translation Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 150000003431 steroids Chemical class 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 229940124597 therapeutic agent Drugs 0.000 description 3
- 230000000451 tissue damage Effects 0.000 description 3
- 231100000827 tissue damage Toxicity 0.000 description 3
- 229940044616 toll-like receptor 7 agonist Drugs 0.000 description 3
- 150000003852 triazoles Chemical class 0.000 description 3
- 235000002374 tyrosine Nutrition 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- VHRUMKCAEVRUBK-GODQJPCRSA-N 15-deoxy-Delta(12,14)-prostaglandin J2 Chemical group CCCCC\C=C\C=C1/[C@@H](C\C=C/CCCC(O)=O)C=CC1=O VHRUMKCAEVRUBK-GODQJPCRSA-N 0.000 description 2
- YEAHTLOYHVWAKW-UHFFFAOYSA-N 8-(1-hydroxyethyl)-2-methoxy-3-[(4-methoxyphenyl)methoxy]benzo[c]chromen-6-one Chemical compound C1=CC(OC)=CC=C1COC(C(=C1)OC)=CC2=C1C1=CC=C(C(C)O)C=C1C(=O)O2 YEAHTLOYHVWAKW-UHFFFAOYSA-N 0.000 description 2
- 206010000234 Abortion spontaneous Diseases 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- 201000001320 Atherosclerosis Diseases 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 206010067982 Butterfly rash Diseases 0.000 description 2
- 125000001433 C-terminal amino-acid group Chemical group 0.000 description 2
- 101710167800 Capsid assembly scaffolding protein Proteins 0.000 description 2
- 208000006547 Central Nervous System Lupus Vasculitis Diseases 0.000 description 2
- 108010005939 Ciliary Neurotrophic Factor Proteins 0.000 description 2
- 102100031614 Ciliary neurotrophic factor Human genes 0.000 description 2
- 208000035473 Communicable disease Diseases 0.000 description 2
- 102100027587 Copper-transporting ATPase 1 Human genes 0.000 description 2
- 150000008574 D-amino acids Chemical class 0.000 description 2
- MNQZXJOMYWMBOU-VKHMYHEASA-N D-glyceraldehyde Chemical compound OC[C@@H](O)C=O MNQZXJOMYWMBOU-VKHMYHEASA-N 0.000 description 2
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 2
- 102000053602 DNA Human genes 0.000 description 2
- 108010092160 Dactinomycin Proteins 0.000 description 2
- 108010042407 Endonucleases Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 206010018364 Glomerulonephritis Diseases 0.000 description 2
- NYHBQMYGNKIUIF-UUOKFMHZSA-N Guanosine Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O NYHBQMYGNKIUIF-UUOKFMHZSA-N 0.000 description 2
- 208000005176 Hepatitis C Diseases 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000936277 Homo sapiens Copper-transporting ATPase 1 Proteins 0.000 description 2
- 206010020751 Hypersensitivity Diseases 0.000 description 2
- 102000008379 I-kappa B Proteins Human genes 0.000 description 2
- 108010021699 I-kappa B Proteins Proteins 0.000 description 2
- 102000007640 Inositol 1,4,5-Trisphosphate Receptors Human genes 0.000 description 2
- 108010066979 Interleukin-27 Proteins 0.000 description 2
- 102100036678 Interleukin-27 subunit alpha Human genes 0.000 description 2
- 102100037792 Interleukin-6 receptor subunit alpha Human genes 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 102000015617 Janus Kinases Human genes 0.000 description 2
- 108010024121 Janus Kinases Proteins 0.000 description 2
- 150000008575 L-amino acids Chemical class 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- 241001490312 Lithops pseudotruncatella Species 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 240000002769 Morchella esculenta Species 0.000 description 2
- 235000002779 Morchella esculenta Nutrition 0.000 description 2
- 101001033265 Mus musculus Interleukin-10 Proteins 0.000 description 2
- 101001076414 Mus musculus Interleukin-6 Proteins 0.000 description 2
- 208000000112 Myalgia Diseases 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 102000038030 PI3Ks Human genes 0.000 description 2
- 108091007960 PI3Ks Proteins 0.000 description 2
- 229930012538 Paclitaxel Natural products 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 101710130420 Probable capsid assembly scaffolding protein Proteins 0.000 description 2
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 2
- 108010029485 Protein Isoforms Proteins 0.000 description 2
- 102000001708 Protein Isoforms Human genes 0.000 description 2
- XESARGFCSKSFID-UHFFFAOYSA-N Pyrazofurin Natural products OC1=C(C(=O)N)NN=C1C1C(O)C(O)C(CO)O1 XESARGFCSKSFID-UHFFFAOYSA-N 0.000 description 2
- 206010037660 Pyrexia Diseases 0.000 description 2
- 101710204410 Scaffold protein Proteins 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 108091027967 Small hairpin RNA Proteins 0.000 description 2
- 108091023040 Transcription factor Proteins 0.000 description 2
- 102000040945 Transcription factor Human genes 0.000 description 2
- 206010053614 Type III immune complex mediated reaction Diseases 0.000 description 2
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 2
- 206010047115 Vasculitis Diseases 0.000 description 2
- 208000028227 Viral hemorrhagic fever Diseases 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 239000003070 absorption delaying agent Substances 0.000 description 2
- RJURFGZVJUQBHK-IIXSONLDSA-N actinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=CC=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 RJURFGZVJUQBHK-IIXSONLDSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 108091005764 adaptor proteins Proteins 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000000443 aerosol Substances 0.000 description 2
- 208000026935 allergic disease Diseases 0.000 description 2
- 230000007815 allergy Effects 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000012736 aqueous medium Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- LMEKQMALGUDUQG-UHFFFAOYSA-N azathioprine Chemical compound CN1C=NC([N+]([O-])=O)=C1SC1=NC=NC2=C1NC=N2 LMEKQMALGUDUQG-UHFFFAOYSA-N 0.000 description 2
- 229960002170 azathioprine Drugs 0.000 description 2
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 2
- 230000001588 bifunctional effect Effects 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000008499 blood brain barrier function Effects 0.000 description 2
- 238000004820 blood count Methods 0.000 description 2
- 230000036770 blood supply Effects 0.000 description 2
- 210000001218 blood-brain barrier Anatomy 0.000 description 2
- 230000036760 body temperature Effects 0.000 description 2
- 229910021538 borax Inorganic materials 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- 201000011510 cancer Diseases 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 150000003857 carboxamides Chemical class 0.000 description 2
- 108010002871 cardiotrophin-like cytokine Proteins 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 238000001516 cell proliferation assay Methods 0.000 description 2
- 210000003169 central nervous system Anatomy 0.000 description 2
- 201000008191 cerebritis 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
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000002648 combination therapy Methods 0.000 description 2
- 230000002301 combined effect Effects 0.000 description 2
- 230000004154 complement system Effects 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 208000018631 connective tissue disease Diseases 0.000 description 2
- 239000012228 culture supernatant Substances 0.000 description 2
- OPTASPLRGRRNAP-UHFFFAOYSA-N cytosine Chemical compound NC=1C=CNC(=O)N=1 OPTASPLRGRRNAP-UHFFFAOYSA-N 0.000 description 2
- 229960000640 dactinomycin Drugs 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 231100000517 death Toxicity 0.000 description 2
- 238000000326 densiometry Methods 0.000 description 2
- 230000008021 deposition Effects 0.000 description 2
- 238000001212 derivatisation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 206010012601 diabetes mellitus Diseases 0.000 description 2
- 230000003828 downregulation Effects 0.000 description 2
- 241001493065 dsRNA viruses Species 0.000 description 2
- 206010014599 encephalitis Diseases 0.000 description 2
- 206010014665 endocarditis Diseases 0.000 description 2
- PJMPHNIQZUBGLI-UHFFFAOYSA-N fentanyl Chemical compound C=1C=CC=CC=1N(C(=O)CC)C(CC1)CCN1CCC1=CC=CC=C1 PJMPHNIQZUBGLI-UHFFFAOYSA-N 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 210000001280 germinal center Anatomy 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 230000002489 hematologic effect Effects 0.000 description 2
- FDGQSTZJBFJUBT-UHFFFAOYSA-N hypoxanthine Chemical compound O=C1NC=NC2=C1NC=N2 FDGQSTZJBFJUBT-UHFFFAOYSA-N 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000003053 immunization Effects 0.000 description 2
- 238000002649 immunization Methods 0.000 description 2
- 230000016784 immunoglobulin production Effects 0.000 description 2
- 230000001976 improved effect Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- CGIGDMFJXJATDK-UHFFFAOYSA-N indomethacin Chemical compound CC1=C(CC(O)=O)C2=CC(OC)=CC=C2N1C(=O)C1=CC=C(Cl)C=C1 CGIGDMFJXJATDK-UHFFFAOYSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 206010022000 influenza Diseases 0.000 description 2
- 238000001802 infusion Methods 0.000 description 2
- 210000005007 innate immune system Anatomy 0.000 description 2
- 108040006858 interleukin-6 receptor activity proteins Proteins 0.000 description 2
- 201000009941 intracranial hypertension Diseases 0.000 description 2
- 208000017169 kidney disease Diseases 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 210000001165 lymph node Anatomy 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 210000004962 mammalian cell Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000004379 membrane Anatomy 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 2
- HPNSFSBZBAHARI-UHFFFAOYSA-N micophenolic acid Natural products OC1=C(CC=C(C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-UHFFFAOYSA-N 0.000 description 2
- 201000005518 mononeuropathy Diseases 0.000 description 2
- 239000002324 mouth wash Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- HPNSFSBZBAHARI-RUDMXATFSA-N mycophenolic acid Chemical compound OC1=C(C\C=C(/C)CCC(O)=O)C(OC)=C(C)C2=C1C(=O)OC2 HPNSFSBZBAHARI-RUDMXATFSA-N 0.000 description 2
- 229930014626 natural product Natural products 0.000 description 2
- 239000002858 neurotransmitter agent Substances 0.000 description 2
- 230000000269 nucleophilic effect Effects 0.000 description 2
- 239000002777 nucleoside Substances 0.000 description 2
- 102000037831 nucleoside transporters Human genes 0.000 description 2
- 108091006527 nucleoside transporters Proteins 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 229960001592 paclitaxel Drugs 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 239000002245 particle Substances 0.000 description 2
- 239000006072 paste Substances 0.000 description 2
- 229930182977 pateamine Natural products 0.000 description 2
- 102000007863 pattern recognition receptors Human genes 0.000 description 2
- 108010089193 pattern recognition receptors Proteins 0.000 description 2
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- XESARGFCSKSFID-FLLFQEBCSA-N pirazofurin Chemical compound OC1=C(C(=O)N)NN=C1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 XESARGFCSKSFID-FLLFQEBCSA-N 0.000 description 2
- 210000004180 plasmocyte Anatomy 0.000 description 2
- 208000008423 pleurisy Diseases 0.000 description 2
- 229920001223 polyethylene glycol Polymers 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- 230000035935 pregnancy Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 229940002612 prodrug Drugs 0.000 description 2
- 239000000651 prodrug Substances 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000002510 pyrogen Substances 0.000 description 2
- 206010037844 rash Diseases 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 229960004641 rituximab Drugs 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- 239000004055 small Interfering RNA Substances 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 235000010339 sodium tetraborate Nutrition 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 210000004989 spleen cell Anatomy 0.000 description 2
- 208000000995 spontaneous abortion Diseases 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 238000003860 storage Methods 0.000 description 2
- 230000001629 suppression Effects 0.000 description 2
- 239000004094 surface-active agent Substances 0.000 description 2
- 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 description 2
- RWQNBRDOKXIBIV-UHFFFAOYSA-N thymine Chemical compound CC1=CNC(=O)NC1=O RWQNBRDOKXIBIV-UHFFFAOYSA-N 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- BSVBQGMMJUBVOD-UHFFFAOYSA-N trisodium borate Chemical compound [Na+].[Na+].[Na+].[O-]B([O-])[O-] BSVBQGMMJUBVOD-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 238000011870 unpaired t-test Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 description 1
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 1
- FDKWRPBBCBCIGA-REOHCLBHSA-N (2r)-2-azaniumyl-3-$l^{1}-selanylpropanoate Chemical compound [Se]C[C@H](N)C(O)=O FDKWRPBBCBCIGA-REOHCLBHSA-N 0.000 description 1
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- ZEWJFUNFEABPGL-UHFFFAOYSA-N 1,2,4-triazole-3-carboxamide Chemical compound NC(=O)C=1N=CNN=1 ZEWJFUNFEABPGL-UHFFFAOYSA-N 0.000 description 1
- QLHHRYZMBGPBJG-UHFFFAOYSA-N 1-[4-[1-(1,4-dioxaspiro[4.5]decan-8-yl)-4-(8-oxa-3-azabicyclo[3.2.1]octan-3-yl)-6-pyrazolo[3,4-d]pyrimidinyl]phenyl]-3-methylurea Chemical compound C1=CC(NC(=O)NC)=CC=C1C1=NC(N2CC3CCC(O3)C2)=C(C=NN2C3CCC4(CC3)OCCO4)C2=N1 QLHHRYZMBGPBJG-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 108020004463 18S ribosomal RNA Proteins 0.000 description 1
- LJVQHXICFCZRJN-UHFFFAOYSA-N 1h-1,2,4-triazole-5-carboxylic acid Chemical compound OC(=O)C1=NC=NN1 LJVQHXICFCZRJN-UHFFFAOYSA-N 0.000 description 1
- UPLPHRJJTCUQAY-WIRWPRASSA-N 2,3-thioepoxy madol Chemical compound C([C@@H]1CC2)[C@@H]3S[C@@H]3C[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@](C)(O)[C@@]2(C)CC1 UPLPHRJJTCUQAY-WIRWPRASSA-N 0.000 description 1
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 1
- OGHAROSJZRTIOK-KQYNXXCUSA-N 2-amino-9-[(2r,3r,4s,5r)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-7-methylpurin-9-ium-6-olate Chemical compound C12=NC(N)=NC([O-])=C2N(C)C=[N+]1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O OGHAROSJZRTIOK-KQYNXXCUSA-N 0.000 description 1
- ASJSAQIRZKANQN-CRCLSJGQSA-N 2-deoxy-D-ribose Chemical compound OC[C@@H](O)[C@@H](O)CC=O ASJSAQIRZKANQN-CRCLSJGQSA-N 0.000 description 1
- FLABMTVDNODXQV-UHFFFAOYSA-N 2h-tetrazole-5-carboxamide Chemical compound NC(=O)C=1N=NNN=1 FLABMTVDNODXQV-UHFFFAOYSA-N 0.000 description 1
- JUSFANSTBFGBAF-IRXDYDNUSA-N 3-[2,4-bis[(3s)-3-methylmorpholin-4-yl]pyrido[2,3-d]pyrimidin-7-yl]-n-methylbenzamide Chemical compound CNC(=O)C1=CC=CC(C=2N=C3N=C(N=C(C3=CC=2)N2[C@H](COCC2)C)N2[C@H](COCC2)C)=C1 JUSFANSTBFGBAF-IRXDYDNUSA-N 0.000 description 1
- YRNWIFYIFSBPAU-UHFFFAOYSA-N 4-[4-(dimethylamino)phenyl]-n,n-dimethylaniline Chemical compound C1=CC(N(C)C)=CC=C1C1=CC=C(N(C)C)C=C1 YRNWIFYIFSBPAU-UHFFFAOYSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- LRSASMSXMSNRBT-UHFFFAOYSA-N 5-methylcytosine Chemical compound CC1=CNC(=O)N=C1N LRSASMSXMSNRBT-UHFFFAOYSA-N 0.000 description 1
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 1
- USSIQXCVUWKGNF-UHFFFAOYSA-N 6-(dimethylamino)-4,4-diphenylheptan-3-one Chemical compound C=1C=CC=CC=1C(CC(C)N(C)C)(C(=O)CC)C1=CC=CC=C1 USSIQXCVUWKGNF-UHFFFAOYSA-N 0.000 description 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 1
- 108091006112 ATPases Proteins 0.000 description 1
- KVLFRAWTRWDEDF-IRXDYDNUSA-N AZD-8055 Chemical compound C1=C(CO)C(OC)=CC=C1C1=CC=C(C(=NC(=N2)N3[C@H](COCC3)C)N3[C@H](COCC3)C)C2=N1 KVLFRAWTRWDEDF-IRXDYDNUSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 206010048998 Acute phase reaction Diseases 0.000 description 1
- 102000011767 Acute-Phase Proteins Human genes 0.000 description 1
- 108010062271 Acute-Phase Proteins Proteins 0.000 description 1
- 102000057290 Adenosine Triphosphatases Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 201000004384 Alopecia Diseases 0.000 description 1
- 208000024827 Alzheimer disease Diseases 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 241000272522 Anas Species 0.000 description 1
- 102000008102 Ankyrins Human genes 0.000 description 1
- 108010049777 Ankyrins Proteins 0.000 description 1
- 241001523209 Antissa Species 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000203069 Archaea Species 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 208000006740 Aseptic Meningitis Diseases 0.000 description 1
- BSYNRYMUTXBXSQ-UHFFFAOYSA-N Aspirin Chemical compound CC(=O)OC1=CC=CC=C1C(O)=O BSYNRYMUTXBXSQ-UHFFFAOYSA-N 0.000 description 1
- 206010003671 Atrioventricular Block Diseases 0.000 description 1
- 206010003673 Atrioventricular block complete Diseases 0.000 description 1
- 208000030016 Avascular necrosis Diseases 0.000 description 1
- 108091008875 B cell receptors Proteins 0.000 description 1
- 101710135413 B-cell scaffold protein with ankyrin repeats Proteins 0.000 description 1
- 208000009137 Behcet syndrome Diseases 0.000 description 1
- 208000010392 Bone Fractures Diseases 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 208000020446 Cardiac disease Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 235000014653 Carica parviflora Nutrition 0.000 description 1
- 208000002177 Cataract Diseases 0.000 description 1
- 206010065941 Central obesity Diseases 0.000 description 1
- 229930186147 Cephalosporin Natural products 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 206010008748 Chorea Diseases 0.000 description 1
- 241000243321 Cnidaria Species 0.000 description 1
- 241001638933 Cochlicella barbara Species 0.000 description 1
- 102000000989 Complement System Proteins Human genes 0.000 description 1
- 108010069112 Complement System Proteins Proteins 0.000 description 1
- 108020004635 Complementary DNA Proteins 0.000 description 1
- 206010010305 Confusional state Diseases 0.000 description 1
- 206010010904 Convulsion Diseases 0.000 description 1
- 208000019736 Cranial nerve disease Diseases 0.000 description 1
- 208000000307 Crimean Hemorrhagic Fever Diseases 0.000 description 1
- 201000003075 Crimean-Congo hemorrhagic fever Diseases 0.000 description 1
- MIKUYHXYGGJMLM-GIMIYPNGSA-N Crotonoside Natural products C1=NC2=C(N)NC(=O)N=C2N1[C@H]1O[C@@H](CO)[C@H](O)[C@@H]1O MIKUYHXYGGJMLM-GIMIYPNGSA-N 0.000 description 1
- 102100031051 Cysteine and glycine-rich protein 1 Human genes 0.000 description 1
- FDKWRPBBCBCIGA-UWTATZPHSA-N D-Selenocysteine Natural products [Se]C[C@@H](N)C(O)=O FDKWRPBBCBCIGA-UWTATZPHSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UWTATZPHSA-N D-Serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 description 1
- 229930195711 D-Serine Natural products 0.000 description 1
- ODKSFYDXXFIFQN-SCSAIBSYSA-N D-arginine Chemical compound OC(=O)[C@H](N)CCCNC(N)=N ODKSFYDXXFIFQN-SCSAIBSYSA-N 0.000 description 1
- NYHBQMYGNKIUIF-UHFFFAOYSA-N D-guanosine Natural products C1=2NC(N)=NC(=O)C=2N=CN1C1OC(CO)C(O)C1O NYHBQMYGNKIUIF-UHFFFAOYSA-N 0.000 description 1
- KDXKERNSBIXSRK-RXMQYKEDSA-N D-lysine group Chemical group N[C@H](CCCCN)C(=O)O KDXKERNSBIXSRK-RXMQYKEDSA-N 0.000 description 1
- 108010076804 DNA Restriction Enzymes Proteins 0.000 description 1
- 241000450599 DNA viruses Species 0.000 description 1
- UQBOJOOOTLPNST-UHFFFAOYSA-N Dehydroalanine Chemical compound NC(=C)C(O)=O UQBOJOOOTLPNST-UHFFFAOYSA-N 0.000 description 1
- 206010012335 Dependence Diseases 0.000 description 1
- 208000006926 Discoid Lupus Erythematosus Diseases 0.000 description 1
- 206010052804 Drug tolerance Diseases 0.000 description 1
- 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 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102100031780 Endonuclease Human genes 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241001529459 Enterovirus A71 Species 0.000 description 1
- 108090000371 Esterases Proteins 0.000 description 1
- 108010008165 Etanercept Proteins 0.000 description 1
- 108010057192 Eukaryotic Initiation Factor-4G Proteins 0.000 description 1
- 102000012858 Eukaryotic Initiation Factor-4G Human genes 0.000 description 1
- 108010014863 Eukaryotic Initiation Factors Proteins 0.000 description 1
- 102000002241 Eukaryotic Initiation Factors Human genes 0.000 description 1
- 102100022462 Eukaryotic initiation factor 4A-II Human genes 0.000 description 1
- 102100027304 Eukaryotic translation initiation factor 4E Human genes 0.000 description 1
- 101710091918 Eukaryotic translation initiation factor 4E Proteins 0.000 description 1
- 102100022447 Eukaryotic translation initiation factor 4E-binding protein 2 Human genes 0.000 description 1
- 102100022457 Eukaryotic translation initiation factor 4E-binding protein 3 Human genes 0.000 description 1
- HKVAMNSJSFKALM-GKUWKFKPSA-N Everolimus Chemical compound C1C[C@@H](OCCO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 HKVAMNSJSFKALM-GKUWKFKPSA-N 0.000 description 1
- 208000010201 Exanthema Diseases 0.000 description 1
- 108700024394 Exon Proteins 0.000 description 1
- 241000710831 Flavivirus Species 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 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 1
- 208000002705 Glucose Intolerance Diseases 0.000 description 1
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 208000035895 Guillain-Barré syndrome Diseases 0.000 description 1
- 208000020061 Hand, Foot and Mouth Disease Diseases 0.000 description 1
- 208000025713 Hand-foot-and-mouth disease Diseases 0.000 description 1
- 208000008913 Hantavirus Infections Diseases 0.000 description 1
- 208000010271 Heart Block Diseases 0.000 description 1
- 206010019280 Heart failures Diseases 0.000 description 1
- 102000002812 Heat-Shock Proteins Human genes 0.000 description 1
- 108010004889 Heat-Shock Proteins Proteins 0.000 description 1
- 206010019799 Hepatitis viral Diseases 0.000 description 1
- 241001530201 Hippuris Species 0.000 description 1
- 101000959666 Homo sapiens Eukaryotic initiation factor 4A-I Proteins 0.000 description 1
- 101001044475 Homo sapiens Eukaryotic initiation factor 4A-II Proteins 0.000 description 1
- 101000678283 Homo sapiens Eukaryotic translation initiation factor 4E-binding protein 2 Proteins 0.000 description 1
- 101000678288 Homo sapiens Eukaryotic translation initiation factor 4E-binding protein 3 Proteins 0.000 description 1
- 101000933607 Homo sapiens Protein BTG3 Proteins 0.000 description 1
- 101000800483 Homo sapiens Toll-like receptor 8 Proteins 0.000 description 1
- 101000637726 Homo sapiens Toll/interleukin-1 receptor domain-containing adapter protein Proteins 0.000 description 1
- 241001502974 Human gammaherpesvirus 8 Species 0.000 description 1
- 108700003968 Human immunodeficiency virus 1 tat peptide (49-57) Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-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
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 1
- UGQMRVRMYYASKQ-UHFFFAOYSA-N Hypoxanthine nucleoside Natural products OC1C(O)C(CO)OC1N1C(NC=NC2=O)=C2N=C1 UGQMRVRMYYASKQ-UHFFFAOYSA-N 0.000 description 1
- 108091008585 IP3 receptors Proteins 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- SIKJAQJRHWYJAI-UHFFFAOYSA-N Indole Chemical compound C1=CC=C2NC=CC2=C1 SIKJAQJRHWYJAI-UHFFFAOYSA-N 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- 108010032354 Inositol 1,4,5-Trisphosphate Receptors Proteins 0.000 description 1
- 108010050904 Interferons Proteins 0.000 description 1
- 102000014150 Interferons Human genes 0.000 description 1
- 108050006617 Interleukin-1 receptor Proteins 0.000 description 1
- 102000019223 Interleukin-1 receptor Human genes 0.000 description 1
- 102000003815 Interleukin-11 Human genes 0.000 description 1
- 108090000177 Interleukin-11 Proteins 0.000 description 1
- 208000029523 Interstitial Lung disease Diseases 0.000 description 1
- 206010022773 Intracranial pressure increased Diseases 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- 102000019145 JUN kinase activity proteins Human genes 0.000 description 1
- 206010023232 Joint swelling Diseases 0.000 description 1
- YQEZLKZALYSWHR-UHFFFAOYSA-N Ketamine Chemical compound C=1C=CC=C(Cl)C=1C1(NC)CCCCC1=O YQEZLKZALYSWHR-UHFFFAOYSA-N 0.000 description 1
- RFSMUFRPPYDYRD-CALCHBBNSA-N Ku-0063794 Chemical compound C1=C(CO)C(OC)=CC=C1C1=CC=C(C(=NC(=N2)N3C[C@@H](C)O[C@@H](C)C3)N3CCOCC3)C2=N1 RFSMUFRPPYDYRD-CALCHBBNSA-N 0.000 description 1
- MNQZXJOMYWMBOU-GSVOUGTGSA-N L-(-)-glyceraldehyde Chemical compound OC[C@H](O)C=O MNQZXJOMYWMBOU-GSVOUGTGSA-N 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
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 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
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 1
- DWPCPZJAHOETAG-IMJSIDKUSA-N L-lanthionine Chemical compound OC(=O)[C@@H](N)CSC[C@H](N)C(O)=O DWPCPZJAHOETAG-IMJSIDKUSA-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
- FBOZXECLQNJBKD-ZDUSSCGKSA-N L-methotrexate Chemical compound C=1N=C2N=C(N)N=C(N)C2=NC=1CN(C)C1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 FBOZXECLQNJBKD-ZDUSSCGKSA-N 0.000 description 1
- ZFOMKMMPBOQKMC-KXUCPTDWSA-N L-pyrrolysine Chemical compound C[C@@H]1CC=N[C@H]1C(=O)NCCCC[C@H]([NH3+])C([O-])=O ZFOMKMMPBOQKMC-KXUCPTDWSA-N 0.000 description 1
- 240000001046 Lactobacillus acidophilus Species 0.000 description 1
- 241000218588 Lactobacillus rhamnosus Species 0.000 description 1
- 206010023927 Lassa fever Diseases 0.000 description 1
- 208000024369 Libman-Sacks endocarditis Diseases 0.000 description 1
- 206010062315 Lipohypertrophy Diseases 0.000 description 1
- 208000035752 Live birth Diseases 0.000 description 1
- 206010058225 Lupus endocarditis Diseases 0.000 description 1
- 206010057481 Lupus pneumonitis Diseases 0.000 description 1
- 102100034069 MAP kinase-activated protein kinase 2 Human genes 0.000 description 1
- 101710141394 MAP kinase-activated protein kinase 2 Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010027201 Meningitis aseptic Diseases 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 108060004795 Methyltransferase Proteins 0.000 description 1
- 206010049567 Miller Fisher syndrome Diseases 0.000 description 1
- 206010027918 Mononeuropathy multiplex Diseases 0.000 description 1
- 208000019022 Mood disease Diseases 0.000 description 1
- 208000016285 Movement disease Diseases 0.000 description 1
- 208000034578 Multiple myelomas Diseases 0.000 description 1
- MSFSPUZXLOGKHJ-UHFFFAOYSA-N Muraminsaeure Natural products OC(=O)C(C)OC1C(N)C(O)OC(CO)C1O MSFSPUZXLOGKHJ-UHFFFAOYSA-N 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- 101100260758 Mus musculus Tlr9 gene Proteins 0.000 description 1
- 241001668559 Mycale Species 0.000 description 1
- 102000010168 Myeloid Differentiation Factor 88 Human genes 0.000 description 1
- 108010077432 Myeloid Differentiation Factor 88 Proteins 0.000 description 1
- 206010028570 Myelopathy Diseases 0.000 description 1
- 208000009525 Myocarditis Diseases 0.000 description 1
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 1
- 206010028780 Nasal ulcer Diseases 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 102100032139 Neuroguidin Human genes 0.000 description 1
- 101710203741 Neuroguidin Proteins 0.000 description 1
- 102000019040 Nuclear Antigens Human genes 0.000 description 1
- 108010051791 Nuclear Antigens Proteins 0.000 description 1
- 108020003217 Nuclear RNA Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010073310 Occupational exposures Diseases 0.000 description 1
- 108700020796 Oncogene Proteins 0.000 description 1
- 102000043276 Oncogene Human genes 0.000 description 1
- 208000026251 Opioid-Related disease Diseases 0.000 description 1
- 208000007117 Oral Ulcer Diseases 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
- 206010031264 Osteonecrosis Diseases 0.000 description 1
- BRUQQQPBMZOVGD-XFKAJCMBSA-N Oxycodone Chemical compound O=C([C@@H]1O2)CC[C@@]3(O)[C@H]4CC5=CC=C(OC)C2=C5[C@@]13CCN4C BRUQQQPBMZOVGD-XFKAJCMBSA-N 0.000 description 1
- 102100024894 PR domain zinc finger protein 1 Human genes 0.000 description 1
- 201000010183 Papilledema Diseases 0.000 description 1
- 206010033712 Papilloedema Diseases 0.000 description 1
- 208000007542 Paresis Diseases 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108010013639 Peptidoglycan Proteins 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 206010035226 Plasma cell myeloma Diseases 0.000 description 1
- 208000002151 Pleural effusion Diseases 0.000 description 1
- 206010035664 Pneumonia Diseases 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 206010036105 Polyneuropathy Diseases 0.000 description 1
- 206010062519 Poor quality sleep Diseases 0.000 description 1
- 241000243142 Porifera Species 0.000 description 1
- 108010009975 Positive Regulatory Domain I-Binding Factor 1 Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 206010060862 Prostate cancer Diseases 0.000 description 1
- 208000000236 Prostatic Neoplasms Diseases 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 102000001253 Protein Kinase Human genes 0.000 description 1
- 102000004022 Protein-Tyrosine Kinases Human genes 0.000 description 1
- 108090000412 Protein-Tyrosine Kinases Proteins 0.000 description 1
- 108010071563 Proto-Oncogene Proteins c-fos Proteins 0.000 description 1
- 102000007568 Proto-Oncogene Proteins c-fos Human genes 0.000 description 1
- 208000028017 Psychotic disease Diseases 0.000 description 1
- 206010037394 Pulmonary haemorrhage Diseases 0.000 description 1
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 description 1
- 108090000944 RNA Helicases Proteins 0.000 description 1
- 102000004409 RNA Helicases Human genes 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 206010037742 Rabies Diseases 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 208000001647 Renal Insufficiency Diseases 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 206010062237 Renal impairment Diseases 0.000 description 1
- 241000725643 Respiratory syncytial virus Species 0.000 description 1
- 206010061603 Respiratory syncytial virus infection Diseases 0.000 description 1
- 241000219061 Rheum Species 0.000 description 1
- 208000025747 Rheumatic disease Diseases 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 1
- 101001034819 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) Eukaryotic initiation factor 4F subunit p130 Proteins 0.000 description 1
- 206010058556 Serositis Diseases 0.000 description 1
- 206010067739 Shrinking lung syndrome Diseases 0.000 description 1
- 208000031709 Skin Manifestations Diseases 0.000 description 1
- 206010040925 Skin striae Diseases 0.000 description 1
- 208000013738 Sleep Initiation and Maintenance disease Diseases 0.000 description 1
- 241000193998 Streptococcus pneumoniae Species 0.000 description 1
- 201000009594 Systemic Scleroderma Diseases 0.000 description 1
- 208000018359 Systemic autoimmune disease Diseases 0.000 description 1
- 206010042953 Systemic sclerosis Diseases 0.000 description 1
- 101150038509 TLR9 gene Proteins 0.000 description 1
- 108700012920 TNF Proteins 0.000 description 1
- CBPNZQVSJQDFBE-FUXHJELOSA-N Temsirolimus Chemical compound C1C[C@@H](OC(=O)C(C)(CO)CO)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 CBPNZQVSJQDFBE-FUXHJELOSA-N 0.000 description 1
- 108020005038 Terminator Codon Proteins 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical class O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 1
- 102100030859 Tissue factor Human genes 0.000 description 1
- 102100032120 Toll/interleukin-1 receptor domain-containing adapter protein Human genes 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- 102000014384 Type C Phospholipases Human genes 0.000 description 1
- 108010079194 Type C Phospholipases Proteins 0.000 description 1
- 201000009693 Venezuelan hemorrhagic fever Diseases 0.000 description 1
- 210000001766 X chromosome Anatomy 0.000 description 1
- 210000002593 Y chromosome Anatomy 0.000 description 1
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 1
- 229960003697 abatacept Drugs 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 210000003208 abducens nerve Anatomy 0.000 description 1
- 238000002679 ablation Methods 0.000 description 1
- 239000003082 abrasive agent Substances 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- 229960001138 acetylsalicylic acid Drugs 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 230000004658 acute-phase response Effects 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 210000005006 adaptive immune system Anatomy 0.000 description 1
- 239000012082 adaptor molecule Substances 0.000 description 1
- 102000035181 adaptor proteins Human genes 0.000 description 1
- 229960001570 ademetionine Drugs 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 150000001299 aldehydes Chemical class 0.000 description 1
- 125000002723 alicyclic group Chemical group 0.000 description 1
- 125000005907 alkyl ester group Chemical group 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 230000000172 allergic effect Effects 0.000 description 1
- 231100000360 alopecia Toxicity 0.000 description 1
- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 1
- 229960003805 amantadine Drugs 0.000 description 1
- 230000001668 ameliorated effect Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 125000000320 amidine group Chemical group 0.000 description 1
- 150000001409 amidines Chemical class 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 229940035676 analgesics Drugs 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000003042 antagnostic effect Effects 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 230000001745 anti-biotin effect Effects 0.000 description 1
- 230000002583 anti-histone Effects 0.000 description 1
- 230000000078 anti-malarial effect Effects 0.000 description 1
- 230000000340 anti-metabolite Effects 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 230000002421 anti-septic effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 229940100197 antimetabolite Drugs 0.000 description 1
- 239000002256 antimetabolite Substances 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 208000010668 atopic eczema Diseases 0.000 description 1
- 230000003305 autocrine Effects 0.000 description 1
- 230000001363 autoimmune Effects 0.000 description 1
- 230000002567 autonomic effect Effects 0.000 description 1
- HNYOPLTXPVRDBG-UHFFFAOYSA-N barbituric acid Chemical group O=C1CC(=O)NC(=O)N1 HNYOPLTXPVRDBG-UHFFFAOYSA-N 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 229940022836 benlysta Drugs 0.000 description 1
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 1
- 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 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 239000012503 blood component Substances 0.000 description 1
- 230000036772 blood pressure Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000001275 ca(2+)-mobilization Effects 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 108020001778 catalytic domains Proteins 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 230000011712 cell development Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 239000008004 cell lysis buffer Substances 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 108091092328 cellular RNA Proteins 0.000 description 1
- 230000005754 cellular signaling Effects 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 208000011235 central nervous system lupus Diseases 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 229940124587 cephalosporin Drugs 0.000 description 1
- 150000001780 cephalosporins Chemical class 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 208000012601 choreatic disease Diseases 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 230000001886 ciliary effect Effects 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- ZCZQDTUCMRSEAS-CCLYOLAMSA-N co-codamol Chemical compound OP(O)(O)=O.CC(=O)NC1=CC=C(O)C=C1.C([C@H]1[C@H](N(CC[C@@]112)C)C3)=C[C@H](O)[C@@H]1OC1=C2C3=CC=C1OC ZCZQDTUCMRSEAS-CCLYOLAMSA-N 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 230000003930 cognitive ability Effects 0.000 description 1
- 208000010877 cognitive disease Diseases 0.000 description 1
- 230000024203 complement activation Effects 0.000 description 1
- 230000001268 conjugating effect Effects 0.000 description 1
- 238000007596 consolidation process Methods 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 208000014826 cranial nerve neuropathy Diseases 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 238000009109 curative therapy Methods 0.000 description 1
- 208000004921 cutaneous lupus erythematosus Diseases 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- 230000016396 cytokine production Effects 0.000 description 1
- 108091092330 cytoplasmic RNA Proteins 0.000 description 1
- 229940104302 cytosine Drugs 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 231100000433 cytotoxic Toxicity 0.000 description 1
- 230000001472 cytotoxic effect Effects 0.000 description 1
- 229950006418 dactolisib Drugs 0.000 description 1
- JOGKUKXHTYWRGZ-UHFFFAOYSA-N dactolisib Chemical compound O=C1N(C)C2=CN=C3C=CC(C=4C=C5C=CC=CC5=NC=4)=CC3=C2N1C1=CC=C(C(C)(C)C#N)C=C1 JOGKUKXHTYWRGZ-UHFFFAOYSA-N 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000003413 degradative effect Effects 0.000 description 1
- 230000017858 demethylation Effects 0.000 description 1
- 238000010520 demethylation reaction Methods 0.000 description 1
- 230000003210 demyelinating effect Effects 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- NXQWWWGBTPUXMZ-YBLPYSLLSA-N dermaseptin s4 Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)N)=CNC2=C1 NXQWWWGBTPUXMZ-YBLPYSLLSA-N 0.000 description 1
- 229960004193 dextropropoxyphene Drugs 0.000 description 1
- XLMALTXPSGQGBX-GCJKJVERSA-N dextropropoxyphene Chemical compound C([C@](OC(=O)CC)([C@H](C)CN(C)C)C=1C=CC=CC=1)C1=CC=CC=C1 XLMALTXPSGQGBX-GCJKJVERSA-N 0.000 description 1
- 229960001259 diclofenac Drugs 0.000 description 1
- DCOPUUMXTXDBNB-UHFFFAOYSA-N diclofenac Chemical compound OC(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl DCOPUUMXTXDBNB-UHFFFAOYSA-N 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- XYYVYLMBEZUESM-UHFFFAOYSA-N dihydrocodeine Natural products C1C(N(CCC234)C)C2C=CC(=O)C3OC2=C4C1=CC=C2OC XYYVYLMBEZUESM-UHFFFAOYSA-N 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- 238000010494 dissociation reaction Methods 0.000 description 1
- 230000005593 dissociations Effects 0.000 description 1
- 238000009826 distribution Methods 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
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 238000012377 drug delivery Methods 0.000 description 1
- 239000003596 drug target Substances 0.000 description 1
- 201000004997 drug-induced lupus erythematosus Diseases 0.000 description 1
- 229940099191 duragesic Drugs 0.000 description 1
- 108010093366 eIF-4B Proteins 0.000 description 1
- 101150114135 eIF4E gene Proteins 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 230000002996 emotional effect Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 229960000403 etanercept Drugs 0.000 description 1
- BEFDCLMNVWHSGT-UHFFFAOYSA-N ethenylcyclopentane Chemical compound C=CC1CCCC1 BEFDCLMNVWHSGT-UHFFFAOYSA-N 0.000 description 1
- 210000004265 eukaryotic small ribosome subunit Anatomy 0.000 description 1
- 229940125632 eukaryotic translation initiation factor 4A inhibitor Drugs 0.000 description 1
- 229960005167 everolimus Drugs 0.000 description 1
- 230000005713 exacerbation Effects 0.000 description 1
- 201000005884 exanthem Diseases 0.000 description 1
- 238000013213 extrapolation Methods 0.000 description 1
- 229960002428 fentanyl Drugs 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000006260 foam Substances 0.000 description 1
- 239000004088 foaming agent Substances 0.000 description 1
- 230000003325 follicular Effects 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- ZZUFCTLCJUWOSV-UHFFFAOYSA-N furosemide Chemical compound C1=C(Cl)C(S(=O)(=O)N)=CC(C(O)=O)=C1NCC1=CC=CO1 ZZUFCTLCJUWOSV-UHFFFAOYSA-N 0.000 description 1
- 229960003692 gamma aminobutyric acid Drugs 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 238000003633 gene expression assay Methods 0.000 description 1
- 210000000585 glomerular basement membrane Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229940029575 guanosine Drugs 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 125000001475 halogen functional group Chemical group 0.000 description 1
- 210000004247 hand Anatomy 0.000 description 1
- 208000029629 hantavirus infectious disease Diseases 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 208000019622 heart disease Diseases 0.000 description 1
- 210000002443 helper t lymphocyte Anatomy 0.000 description 1
- 208000006750 hematuria Diseases 0.000 description 1
- 208000007475 hemolytic anemia Diseases 0.000 description 1
- 208000031169 hemorrhagic disease Diseases 0.000 description 1
- 208000010710 hepatitis C virus infection Diseases 0.000 description 1
- 206010019847 hepatosplenomegaly Diseases 0.000 description 1
- 125000001072 heteroaryl group Chemical group 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 102000050432 human BANK1 Human genes 0.000 description 1
- 239000003906 humectant Substances 0.000 description 1
- 210000004408 hybridoma Anatomy 0.000 description 1
- 235000011167 hydrochloric acid Nutrition 0.000 description 1
- LLPOLZWFYMWNKH-CMKMFDCUSA-N hydrocodone Chemical compound C([C@H]1[C@H](N(CC[C@@]112)C)C3)CC(=O)[C@@H]1OC1=C2C3=CC=C1OC LLPOLZWFYMWNKH-CMKMFDCUSA-N 0.000 description 1
- OROGSEYTTFOCAN-UHFFFAOYSA-N hydrocodone Natural products C1C(N(CCC234)C)C2C=CC(O)C3OC2=C4C1=CC=C2OC OROGSEYTTFOCAN-UHFFFAOYSA-N 0.000 description 1
- 229960000240 hydrocodone Drugs 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
- 150000004679 hydroxides Chemical class 0.000 description 1
- 229960002591 hydroxyproline Drugs 0.000 description 1
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 1
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000002989 hypothyroidism Effects 0.000 description 1
- 208000003532 hypothyroidism Diseases 0.000 description 1
- 210000002865 immune cell Anatomy 0.000 description 1
- 230000016178 immune complex formation Effects 0.000 description 1
- 230000036039 immunity Effects 0.000 description 1
- 238000003119 immunoblot Methods 0.000 description 1
- 229940072221 immunoglobulins Drugs 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 229940125721 immunosuppressive agent Drugs 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229960000905 indomethacin Drugs 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 230000006749 inflammatory damage Effects 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 239000003999 initiator Substances 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 206010022437 insomnia Diseases 0.000 description 1
- 230000031261 interleukin-10 production Effects 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 229940008228 intravenous immunoglobulins Drugs 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 229960003299 ketamine Drugs 0.000 description 1
- 201000006370 kidney failure Diseases 0.000 description 1
- 230000003907 kidney function Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000011545 laboratory measurement Methods 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 230000033001 locomotion Effects 0.000 description 1
- 230000012976 mRNA stabilization Effects 0.000 description 1
- 238000002826 magnetic-activated cell sorting Methods 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 206010025482 malaise Diseases 0.000 description 1
- 125000005439 maleimidyl group Chemical group C1(C=CC(N1*)=O)=O 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 238000007726 management method Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000008774 maternal effect Effects 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 238000002483 medication Methods 0.000 description 1
- 201000008350 membranous glomerulonephritis Diseases 0.000 description 1
- 230000015654 memory Effects 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 229910052753 mercury Inorganic materials 0.000 description 1
- DWPCPZJAHOETAG-UHFFFAOYSA-N meso-lanthionine Natural products OC(=O)C(N)CSCC(N)C(O)=O DWPCPZJAHOETAG-UHFFFAOYSA-N 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 208000037819 metastatic cancer Diseases 0.000 description 1
- 208000011575 metastatic malignant neoplasm Diseases 0.000 description 1
- 229960001797 methadone Drugs 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
- 230000000696 methanogenic effect Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 229960000485 methotrexate Drugs 0.000 description 1
- 239000011325 microbead Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- DDLIGBOFAVUZHB-UHFFFAOYSA-N midazolam Chemical compound C12=CC(Cl)=CC=C2N2C(C)=NC=C2CN=C1C1=CC=CC=C1F DDLIGBOFAVUZHB-UHFFFAOYSA-N 0.000 description 1
- 229960003793 midazolam Drugs 0.000 description 1
- 150000007522 mineralic acids Chemical class 0.000 description 1
- 208000015994 miscarriage Diseases 0.000 description 1
- 210000004115 mitral valve Anatomy 0.000 description 1
- 201000002003 mononeuritis multiplex Diseases 0.000 description 1
- 229940051866 mouthwash Drugs 0.000 description 1
- 230000004118 muscle contraction Effects 0.000 description 1
- 208000013465 muscle pain Diseases 0.000 description 1
- 206010028417 myasthenia gravis Diseases 0.000 description 1
- 229940014456 mycophenolate Drugs 0.000 description 1
- 229960000951 mycophenolic acid Drugs 0.000 description 1
- 210000000066 myeloid cell Anatomy 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 125000001624 naphthyl group Chemical group 0.000 description 1
- MRWXACSTFXYYMV-UHFFFAOYSA-N nebularine Chemical class OC1C(O)C(CO)OC1N1C2=NC=NC=C2N=C1 MRWXACSTFXYYMV-UHFFFAOYSA-N 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 208000020469 nerve plexus disease Diseases 0.000 description 1
- 210000000653 nervous system Anatomy 0.000 description 1
- 210000002241 neurite Anatomy 0.000 description 1
- 239000003076 neurotropic agent Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 239000000820 nonprescription drug Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 229940127073 nucleoside analogue Drugs 0.000 description 1
- 150000003833 nucleoside derivatives Chemical class 0.000 description 1
- 125000003835 nucleoside group Chemical group 0.000 description 1
- 231100000675 occupational exposure Toxicity 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 229940127240 opiate Drugs 0.000 description 1
- 201000005040 opiate dependence Diseases 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000008816 organ damage Effects 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 235000005985 organic acids Nutrition 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 210000002997 osteoclast Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000004792 oxidative damage Effects 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 229960002085 oxycodone Drugs 0.000 description 1
- 230000000803 paradoxical effect Effects 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 208000008494 pericarditis Diseases 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 210000004976 peripheral blood cell Anatomy 0.000 description 1
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 1
- 210000001428 peripheral nervous system Anatomy 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000000575 pesticide Substances 0.000 description 1
- 239000008177 pharmaceutical agent Substances 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 238000011458 pharmacological treatment Methods 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 125000002467 phosphate group Chemical group [H]OP(=O)(O[H])O[*] 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 201000006380 plexopathy Diseases 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 125000003367 polycyclic group Chemical group 0.000 description 1
- 230000007824 polyneuropathy Effects 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920002981 polyvinylidene fluoride Polymers 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000034190 positive regulation of NF-kappaB transcription factor activity Effects 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 208000001685 postmenopausal osteoporosis Diseases 0.000 description 1
- 235000007686 potassium Nutrition 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 235000015497 potassium bicarbonate Nutrition 0.000 description 1
- 229910000028 potassium bicarbonate Inorganic materials 0.000 description 1
- 239000011736 potassium bicarbonate Substances 0.000 description 1
- TYJJADVDDVDEDZ-UHFFFAOYSA-M potassium hydrogencarbonate Chemical compound [K+].OC([O-])=O TYJJADVDDVDEDZ-UHFFFAOYSA-M 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 229960004618 prednisone Drugs 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 230000002335 preservative effect Effects 0.000 description 1
- 230000007112 pro inflammatory response Effects 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
- 201000008171 proliferative glomerulonephritis Diseases 0.000 description 1
- 238000012342 propidium iodide staining Methods 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 108060006633 protein kinase Proteins 0.000 description 1
- 230000002685 pulmonary effect Effects 0.000 description 1
- 208000002815 pulmonary hypertension Diseases 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 150000003230 pyrimidines Chemical class 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- ZAHRKKWIAAJSAO-UHFFFAOYSA-N rapamycin Natural products COCC(O)C(=C/C(C)C(=O)CC(OC(=O)C1CCCCN1C(=O)C(=O)C2(O)OC(CC(OC)C(=CC=CC=CC(C)CC(C)C(=O)C)C)CCC2C)C(C)CC3CCC(O)C(C3)OC)C ZAHRKKWIAAJSAO-UHFFFAOYSA-N 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 230000009257 reactivity Effects 0.000 description 1
- 230000007115 recruitment Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 210000003289 regulatory T cell Anatomy 0.000 description 1
- 208000012263 renal involvement Diseases 0.000 description 1
- 230000001850 reproductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 230000006965 reversible inhibition Effects 0.000 description 1
- 230000000552 rheumatic effect Effects 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 239000000523 sample Substances 0.000 description 1
- 239000012723 sample buffer Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 229940055619 selenocysteine Drugs 0.000 description 1
- ZKZBPNGNEQAJSX-UHFFFAOYSA-N selenocysteine Natural products [SeH]CC(N)C(O)=O ZKZBPNGNEQAJSX-UHFFFAOYSA-N 0.000 description 1
- 235000016491 selenocysteine Nutrition 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000009528 severe injury Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 229960002930 sirolimus Drugs 0.000 description 1
- QFJCIRLUMZQUOT-HPLJOQBZSA-N sirolimus Chemical compound C1C[C@@H](O)[C@H](OC)C[C@@H]1C[C@@H](C)[C@H]1OC(=O)[C@@H]2CCCCN2C(=O)C(=O)[C@](O)(O2)[C@H](C)CC[C@H]2C[C@H](OC)/C(C)=C/C=C/C=C/[C@@H](C)C[C@@H](C)C(=O)[C@H](OC)[C@H](O)/C(C)=C/[C@@H](C)C(=O)C1 QFJCIRLUMZQUOT-HPLJOQBZSA-N 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 230000003860 sleep quality Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- CMZUMMUJMWNLFH-UHFFFAOYSA-N sodium metavanadate Chemical compound [Na+].[O-][V](=O)=O CMZUMMUJMWNLFH-UHFFFAOYSA-N 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 235000010199 sorbic acid Nutrition 0.000 description 1
- 229940075582 sorbic acid Drugs 0.000 description 1
- 239000004334 sorbic acid Substances 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 210000004988 splenocyte Anatomy 0.000 description 1
- 102000009076 src-Family Kinases Human genes 0.000 description 1
- 108010087686 src-Family Kinases Proteins 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 230000000153 supplemental effect Effects 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 210000000225 synapse Anatomy 0.000 description 1
- 230000000946 synaptic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000010189 synthetic method Methods 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 229960000235 temsirolimus Drugs 0.000 description 1
- 231100000462 teratogen Toxicity 0.000 description 1
- 239000003439 teratogenic agent Substances 0.000 description 1
- HNDGEYCCZGRMTN-UHFFFAOYSA-N thieno[3,2-f:4,5-f]bis[1]benzothiophene Chemical compound S1C2=CC=3SC=CC=3C=C2C2=C1C=C(SC=C1)C1=C2 HNDGEYCCZGRMTN-UHFFFAOYSA-N 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
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 150000003573 thiols Chemical class 0.000 description 1
- 201000002931 third-degree atrioventricular block Diseases 0.000 description 1
- 229940113082 thymine Drugs 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 229960003989 tocilizumab Drugs 0.000 description 1
- 230000000699 topical effect Effects 0.000 description 1
- MFAQYJIYDMLAIM-UHFFFAOYSA-N torkinib Chemical compound C12=C(N)N=CN=C2N(C(C)C)N=C1C1=CC2=CC(O)=CC=C2N1 MFAQYJIYDMLAIM-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 230000002110 toxicologic effect Effects 0.000 description 1
- 231100000723 toxicological property Toxicity 0.000 description 1
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 description 1
- LLPOLZWFYMWNKH-UHFFFAOYSA-N trans-dihydrocodeinone Natural products C1C(N(CCC234)C)C2CCC(=O)C3OC2=C4C1=CC=C2OC LLPOLZWFYMWNKH-UHFFFAOYSA-N 0.000 description 1
- 230000002463 transducing effect Effects 0.000 description 1
- 230000026683 transduction Effects 0.000 description 1
- 238000010361 transduction Methods 0.000 description 1
- 238000006276 transfer reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- 230000001052 transient effect Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 238000011277 treatment modality Methods 0.000 description 1
- 210000000591 tricuspid valve Anatomy 0.000 description 1
- 239000001226 triphosphate Substances 0.000 description 1
- 235000011178 triphosphate Nutrition 0.000 description 1
- UNXRWKVEANCORM-UHFFFAOYSA-N triphosphoric acid Chemical compound OP(O)(=O)OP(O)(=O)OP(O)(O)=O UNXRWKVEANCORM-UHFFFAOYSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 125000000430 tryptophan group Chemical group [H]N([H])C(C(=O)O*)C([H])([H])C1=C([H])N([H])C2=C([H])C([H])=C([H])C([H])=C12 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 210000004231 tunica media Anatomy 0.000 description 1
- 108091052247 type I cytokine receptor family Proteins 0.000 description 1
- 102000042286 type I cytokine receptor family Human genes 0.000 description 1
- 208000025883 type III hypersensitivity disease Diseases 0.000 description 1
- 150000003668 tyrosines Chemical class 0.000 description 1
- 231100000402 unacceptable toxicity Toxicity 0.000 description 1
- 241000712461 unidentified influenza virus Species 0.000 description 1
- 241001515965 unidentified phage Species 0.000 description 1
- 229940035893 uracil Drugs 0.000 description 1
- 230000004143 urea cycle Effects 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 235000015112 vegetable and seed oil Nutrition 0.000 description 1
- 239000008158 vegetable oil Substances 0.000 description 1
- 239000003981 vehicle Substances 0.000 description 1
- 230000002861 ventricular Effects 0.000 description 1
- 230000007444 viral RNA synthesis Effects 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
- 201000001862 viral hepatitis Diseases 0.000 description 1
- 210000001835 viscera Anatomy 0.000 description 1
- 229960005080 warfarin Drugs 0.000 description 1
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
- 229910000166 zirconium phosphate Inorganic materials 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/027—New or modified breeds of vertebrates
- A01K67/0275—Genetically modified vertebrates, e.g. transgenic
- A01K67/0276—Knock-out vertebrates
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
- A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2217/00—Genetically modified animals
- A01K2217/07—Animals genetically altered by homologous recombination
- A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K2227/00—Animals characterised by species
- A01K2227/10—Mammal
- A01K2227/105—Murine
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2500/00—Screening for compounds of potential therapeutic value
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/10—Musculoskeletal or connective tissue disorders
- G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
- G01N2800/104—Lupus erythematosus [SLE]
Definitions
- the present invention relates generally to the fields of medicine, autoimmune disease and molecular biology. More particularly, it concerns use of pharmaceutical agents to treat systemic lupus erythematosus.
- SLE Systemic lupus erythematosus
- SLE is associated with the production of antinuclear antibodies, circulating immune complexes, and activation of the complement system.
- Numerous autoantibodies of differing specificity are present in SLE.
- SLE patients often produce autoantibodies having anti-DNA, anti-Ro, and anti-platelet specificity and that are capable of initiating clinical features of the disease, such as glomerulonephritis, arthritis, serositis, complete heart block in newborns, and hematologic abnormalities. These autoantibodies are also possibly related to central nervous system disturbances.
- Untreated lupus can be fatal as it progresses from attack of skin and joints to internal organs, including lung, heart, and kidneys (with renal disease being the primary concern). Lupus mainly appears as a series of flare-ups, with intervening periods of little or no disease manifestation. Kidney damage, measured by the amount of proteinuria in the urine, is one of the most acute areas of damage associated with pathogenicity in SLE, and accounts for at least 50% of the mortality and morbidity of the disease.
- immunosuppressive drugs interfere with the person's ability to produce all antibodies, not just the self-reactive anti- DNA antibodies Immunosuppressants also weaken the body's defense against other potential pathogens, thereby making the patient extremely susceptible to infection and other potentially fatal diseases, such as cancer. In some of these instances, the side effects of current treatment modalities, combined with continued low-level manifestation of the disease, can cause serious impairment and premature death.
- IVIG intravenous immune globulin
- a method of treating a subject having systemic lupus erythematosus comprising administering to said subject an inhibitor of eIF4 function.
- Administering may comprise intravenous administration, intra-arterial administration, oral administration or intramuscular administration.
- Administering may also include providing an organism to the subject that produces the inhibitor, such as lactobacillus.
- the method may further comprise administering to said subject one or more standard SLE therapies, such as a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody (e.g., Rituximab, Belimumab).
- the one or more standard therapies are provided before or after said inhibitor, or at the same time as said inhibitor.
- the inhibitor may be an eIF4E inhibitor, such as ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E.
- the inhibitor may be an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor.
- the inhibitor may be given more than once, including chronic administration.
- Administering may comprise intravenous administration, intra-arterial administration, oral administration or intramuscular administration. Administering may also include providing an organism to the subject that produces the inhibitor, such as lactobacillus.
- the method may further comprise administering to said subject one or more standard SLE therapies, such as a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody (e.g., Rituximab, Belimumab).
- the one or more standard therapies are provided before or after said inhibitor, or at the same time as said inhibitor.
- the inhibitor may be an eIF4E inhibitor, such as ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E.
- the inhibitor may be an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor.
- the inhibitor may be given more than once, including chronic administration.
- compositions and kits of the disclosure can be used to achieve methods of the disclosure.
- the words “comprising” (and any form of comprising, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), "including” (and any form of including, such as “includes” and “include”) or “containing” (and any foam of containing, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
- FIGS. 1A-E CpG-induced p38 MAPK activation is down-regulated in Bankl ' ' B cells.
- FIG. 1A Proliferation assay of CFSE-labeled splenic B cells from Bankl '1' mice and its littermate control ( +/+ ) mice following 2 ⁇ CpG or 20 ⁇ g/ml LPS stimulation for 48, 72 and 96h. Propidium iodide (Pl)-negative viable cells were gated and analyzed by flow cytometry. Data shown is representative of 3 independent experiments.
- FIGS. 1A-E CpG-induced p38 MAPK activation is down-regulated in Bankl ' ' B cells.
- FIG. 1B-C Cell extracts from WT and Bankl KO splenic B cells stimulated with 10 ⁇ g/mL anti-mouse IgM F(ab') 2 (anti-IgM) (FIG. IB) or 2 ⁇ CpG (FIG. 1C) and analyzed by Western blot with specific antibodies for ERK, ⁇ , p38 and ⁇ ⁇ and the corresponding phospho-specific antibodies.
- FIG. ID Quantification of densitometry analysis of phospho-p38 Western blot data is shown as mean ⁇ SEM.
- FIGS. 2A-C CpG-induced IL-6 production is downregulated in Bankl ' ' B cells, but Tlr9 gene expression is not affected.
- FIG. 2A IL-6 and IL-10 were measured by ELISA of WT ( +/+ ) and Bankl KO ( _/ ⁇ ) B-cell supernatants collected at the indicated times following CpG, anti-IgM+ CpG or CpG+ anti-mouse CD40 (10 ⁇ g/ml). Data shown are mean ⁇ SD of 3 replicates, and is representative of 4 independent experiments.
- FIG. 2B Splenic B cells from Bankl +I+ and Bankl '1' mice were stimulated with 2 ⁇ CpG for the indicated times.
- TLR9 gene Relative expression of the TLR9 gene was analyzed by Taqman real-time PCR. Data shown is the mean ⁇ SEM of 3 replicates from a representative experiment out of 3 performed. (FIG. 2C) Bankl +/+ and Bankl ' ' ' splenic B cells were stimulated with LPS (20 ⁇ g/ml) or R848 (1 ⁇ g/mL), or left unstimulated for the times shown. Data are the representative of two independent experiments with 3 replicates each. Bars represent mean ⁇ SD.
- FIGS. 3A-D MK2 and MNK1/2 inhibitors suppress IL-6 cytokine secretion in mouse splenic B cells.
- Mouse C57BL/6J (WT) splenic B cells (1 * 10 6 cells/ml) were seeded in triplicate in RPMI 1640 complete medium in a 48-well tissue culture plate. The cells were pretreated with and without (FIG. 3 A) MK2 specific inhibitor, PF3644022 (1 and 10 ⁇ ) for an hour, (FIG. 3B) MNK1 inhibitor, CGP57380 (10 and 20 ⁇ ) for 30 min, and (FIG. 2C) MNKs inhibitor cercosporamide (20 and 50 ⁇ ) for an hour before stimulation of CpG (2 ⁇ ) for 24h.
- FIGS. 3A-D MK2 and MNK1/2 inhibitors suppress IL-6 cytokine secretion in mouse splenic B cells.
- FIGS. 3A-D MK2 and MNK1/2 inhibitors suppress IL-6 cytokine secret
- the control well contains CpG and cell culture grade DMSO ( ⁇ 0.2% v/v) only.
- the culture supernatants were collected after 24h of stimulation and the capture ELISA for IL-6 was performed.
- the culture supernatants were placed in triplicate in a 96-well ELISA plate and IL-6 ELISA was performed. Bars represent mean ⁇ SD from two independent experiments. Unpaired t-test was performed and all the treated groups were compared with control, **p ⁇ 0.001 and ***p ⁇ 0.0001.
- FIG. 3D Viability of cells after above treatment was tested by propidium iodide (PI) labeling, the whole cells were gated and analyzed by flow cytometry, the figure shows (Pl)-negative viable cells.
- PI propidium iodide
- FIGS. 4A-D Reduced IL-6 secretion can be attributed to downregulation of the phosphorylation of the p38-MNKl/2-eIF4E cascade in Bankl ' ' ' B cells following CpG stimulation and not to IL-6 gene transcription and IL-6 mRNA stability.
- FIG. 4A Splenic B cells from Bankl +/+ and Bankl '1' mice were stimulated with 2 ⁇ CpG for indicated time shown on the plot. Relative expression of the IL-6 gene was determined using Taqman RT-PCR. Data shown is the mean ⁇ SEM of 3 replicates from 1 out of 3 independent experiments. (FIG.
- FIG. 4C Bank +/+ or _/ ⁇ splenic B cells were stimulated with 2 ⁇ CpG for the indicated times and tested by Western blot using p-MNKl/2, MNK1/2, p-eIF4E, and eIF4E specific antibodies. The data are representative of three independent experiments.
- FIG. 4D Quantification analysis of phospho-MNKl/2 and phospho-eIF4E Western blot data by densitometry. The relative intensities of phospho-MNKl/2 and phospho-eIF4E were quantified using ImageJ software (freely available through the National Institutes of Health). The plots are from 3 independent experiments and data are expressed as mean ⁇ SD.
- FIGS. 5A-C Bankl deficiency does not affect 4E-BP1 phosphorylation controlled through the AKT-mTORCl signaling cascade.
- Splenic B cells from Bankl+/+ and Bankl-/- littermate mice were stimulated with (FIG. 5A) CpG (2 ⁇ ), (FIG. 5B) anti- mouse CD40 (10 ⁇ g/ml), and (FIG. 5C) CpG (2 ⁇ ) in combination with anti-mouse CD40 (10 ⁇ g/ml) at 37 °C in a water bath for the indicated time points.
- Cytoplasmic cell extracts were prepared by using B cell lysis buffer containing appropriate amount of protease inhibitor and sodium vanadate.
- Equal amount of cell lysate protein (approx. 2x l0 6 cell) was loaded in each lane and western blot was performed as written in the material and methods.
- the data are representative of two independent experiments.
- the relative intensities of the phospho-bands were quantified as described in FIGS. 4A-D.
- FIGS. 6A-C Cell viability and CFSE-labeled cell proliferation between Bankl +/+ and Bankl 'A splenic B cells.
- CFSE-labeled splenic B cells were treated with the indicated stimuli for the indicated time, harvested and analyzed by flow cytometry. Debris and doublet cells were discriminated and viable cells (propidium iodide-negative) were gated. % of viability was determined. Histograms represent overlaid data from un-stimulated CFSE- labeled B cells, stimulated Bankl +/+ (solid line) and Bankf B cells (dash line).
- FIGS. 7A-B Immunoglobulin isotype switch or expression of BLIMP1 (Prdml) is not significantly affected by BANK1 deficiency.
- FIG. 7A Splenic B cells (l x lO 6 cells/ml) from Bankl +/+ and BankP A littermate mice were seeded in RPMI 1640 complete medium in a 48 well tissue culture plate. The cells were either stimulated with CpG (2 ⁇ ) or left unstimulated for 6 days. The supernatants were analyzed for immunoglobulin isotype antibodies. The data are presented as O.D. except for IgG2a/2c where data are represented as ng/ml. Data are from 4 independent experiments. Bars represent mean ⁇ SD.
- FIGS. 8A-B Increased production of IL-6 by splenic purified B cells from BANKIFL-Tg with CpG or anti-CD40 + CpG stimulation.
- the cytokine IL-6 was measured with ELISA in supernatants of BANKIFL-Tg mice and compared to WT controls from purified B-cells collected at indicated times after CpG stimulation (FIG. 8A) or CpG and anti-CD40 stimulation (FIG. 8B). Data shown are mean ⁇ SD of 3 replicates, and representative of 3 independent experiments.
- FIGS. 9A-C Deficiency of BANK1 modifies several phenotypes present in
- FIGS. 9A-C B6.Slel z z .Yaa lupus prone mouse.
- FIGS. 9A-C Serum from 25 week old Be.Slel ⁇ Bankl ' ' .yaa mice and genotype littermates heterozygous and sufficient for Bankl was measured for presence of IgG anti-dsDNA antibodies using ELISA in serial dilutions. Black circles show positive control sera.
- FIG. 9B A pair of littermates of B6.Slel z z Bank ⁇ .yaa and B6.Slel z/z Bankl +M .yaa mice of 12 weeks of age was used in FIGS. 9B-C.
- FIG. 9C Picture of the inguinal lymph nodes of the same mice as in FIG. 9B.
- FIG. 10 Deficiency of Bankl restores IL-6 levels in serum of lupus-prone mice to normal. Serum from 25 week-old B6.Slel z/ ' Bankl '1' ' .yaa mice and genotype littermates sufficient for Bankl were measured for presence of IL-6 using ELISA (*p ⁇ 0.05).
- FIG. 11 Inhibitors of the translation initiation pathway are capable of inhibiting IL-6 production induced with CpG by BANK1 human transgenic B cells.
- Left panel - Twelve weeks old hBanklTg/wt.mBankl-/- female mice were used for total splenocyte isolation.
- BANK1 may participate in CpG-induced signaling. Their results established a function for BANK1 in CpG-induced responses that have important implications for the role of BANK1 in infections and autoimmunity, where BANK1 has been established as a susceptibility gene (Kozyrev et ah, 2008). In particular, the inventors have connected BANK1 function with IL-6 translation through the eIF4E pathway, and propose eIF4E as a target for treating SLE.
- SLE Systemic lupus erythematosus
- SLE can induce abnormalities in the adaptive and innate immune system, as well as mount Type III hypersensitivity reactions in which antibody-immune complexes precipitate and cause a further immune response. SLE most often damages the joints, skin, lungs, heart, blood components, blood vessels, kidneys, liver and nervous system.
- the course of the disease is unpredictable, often with periods of increased disease activity (called "flares") alternating with suppressed or decreased disease activity.
- a flare has been defined as a measurable increase in disease activity in one or more organ systems involving new or worse clinical signs and symptoms and/or laboratory measurements. It must be considered clinically significant by the assessor and usually there would be at least consideration of a change or an increase in treatment (Ruperto et ah, 2010).
- SLE has no cure, and leads to increased morbidity and early mortality in many patients.
- the most common causes of death in lupus patients include accelerated cardiovascular disease (likely associated with increased inflammation and perhaps additionally increased by select lupus therapies), complications from renal involvement and infections.
- Survival for people with SLE in the United States, Canada, and Europe has risen to approximately 95% at five years, 90% at 10 years, and 78% at 20 years in patients of European descent; however, similar improvements in mortality rates in non-Caucasian patients are not as evident.
- Childhood systemic lupus erythematosus generally presents between the ages of 3 and 15, with girls outnumbering boys 4: 1, and typical skin manifestations being butterfly eruption on the face and photosensitivity.
- SLE is one of several diseases known as "the great imitators" because it often mimics or is mistaken for other illnesses.
- SLE is a classical item in differential diagnosis, because SLE symptoms vary widely and come and go unpredictably. Diagnosis can thus be elusive, with some people suffering unexplained symptoms of untreated SLE for years. Common initial and chronic complaints include fever, malaise, joint pains, myalgias, fatigue, and temporary loss of cognitive abilities. Because they are so often seen with other diseases, these signs and symptoms are not part of the American College of Rheumatology SLE classification criteria. When occurring in conjunction with other signs and symptoms, however, they are suggestive.
- SLE sufferers Over half (65%) of SLE sufferers have some dermatological manifestations at some point in their disease, with approximately 30% to 50% suffering from the classic malar rash (or butterfly rash) associated with the name of the disorder. Some may exhibit chronic thick, annual scaly patches on the skin (referred to as discoid lupus). Alopecia, mouth ulcers, nasal ulcers, and photosensitive lesions on the skin are also possible manifestations. Anemia may develop in up to 50% of lupus cases. Low platelet and white blood cell counts may be due to the disease or as a side effect of pharmacological treatment. People with SLE may have an association with antiphospholipid antibody syndrome (a thrombotic disorder), wherein autoantibodies to phospholipids are present in their serum.
- antiphospholipid antibody syndrome a thrombotic disorder
- Abnormalities associated with antiphospholipid antibody syndrome include a paradoxical prolonged partial thromboplastin time (which usually occurs in hemorrhagic disorders) and a positive test for antiphospholipid antibodies; the combination of such findings has earned the term "lupus anticoagulant- positive.”
- SLE patients with anti-phospholipid autoantibodies have more ACR classification criteria of the disease and may suffer from a more severe lupus phenotype.
- a person with SLE may have inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis.
- the endocarditis of SLE is characteristically noninfective (Libman-Sacks endocarditis), and involves either the mitral valve or the tricuspid valve.
- Atherosclerosis also tends to occur more often and advances more rapidly than in the general population. Lung and pleura inflammation can cause pleuritis, pleural effusion, lupus pneumonitis, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome.
- Painless hematuria or proteinuria may often be the only presenting renal symptom.
- Acute or chronic renal impairment may develop with lupus nephritis, leading to acute or end- stage renal failure.
- end-stage renal failure occurs in less than 5% of cases.
- a histological hallmark of SLE is membranous glomerulonephritis with "wire loop" abnormalities. This finding is due to immune complex deposition along the glomerular basement membrane, leading to a typical granular appearance in immunofluorescence testing.
- Neuropsychiatric syndromes can result when SLE affects the central or peripheral nervous systems.
- the American College of Rheumatology defines 19 neuropsychiatric syndromes in systemic lupus erythematosus.
- the diagnosis of neuropsychiatric syndromes concurrent with SLE is one of the most difficult challenges in medicine, because it can involve so many different patterns of symptoms, some of which may be mistaken for signs of infectious disease or stroke.
- the most common neuropsychiatric disorder people with SLE have is headache, although the existence of a specific lupus headache and the optimal approach to headache in SLE cases remains controversial.
- CNS lupus can rarely present with intracranial hypertension syndrome, characterized by an elevated intracranial pressure, papilledema, and headache with occasional abducens nerve paresis, absence of a space-occupying lesion or ventricular enlargement, and normal cerebrospinal fluid chemical and hematological constituents.
- NPSLE neuropsychiatric systemic lupus erythematosus
- Neonatal lupus is the occurrence of SLE symptoms in an infant born from a mother with SLE, most commonly presenting with a rash resembling discoid lupus erythematosus, and sometimes with systemic abnormalities such as heart block or hepatosplenomegaly. Neonatal lupus is usually benign and self-limited.
- Fatigue in SLE is probably multifactorial and has been related to not only disease activity or complications such as anemia or hypothyroidism, but also to pain, depression, poor sleep quality, poor physical fitness and lack of social support.
- SLE patients Different clinical measurements have been used to determine whether a SLE patients is having a clinic flare.
- One of the most common measurements is the Systemic Lupus Erythematosus Disease Activity Index SELENA Modification (world-wide-web at rheumatology.org/Practice/Clinical/Indexes/Systemic_Lupus_Erythematosus_Disease_Activi ty_Index_SELENA_Modification/).
- This scale uses a point system to calculate when the accumulated significance of recent changes in various indicators translates into a mild/moderate (SLEDA Index of 3-1 1 point change) or a severe (12 of more point change) flare.
- Antinuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti-ENA) responses form the mainstay of SLE serologic testing.
- ANA Antinuclear antibody
- anti-ENA anti-extractable nuclear antigen
- Clinically the most widely used method is indirect immunofluorescence. The pattern of fluorescence suggests the type of antibody present in the patient's serum.
- Direct immunofluorescence can detect deposits of immunoglobulins and complement proteins in the patient's skin. When skin not exposed to the sun is tested, a positive direct IF (the so-called Lupus band test) is an evidence of systemic lupus erythematosus.
- ANA screening yields positive results in many connective tissue disorders and other autoimmune diseases, and may occur in healthy individuals.
- Subtypes of antinuclear antibodies include anti-Smith and anti-double-stranded DNA (dsDNA) antibodies (which are linked to SLE) and anti-histone antibodies (which are linked to drug-induced lupus).
- Anti- dsDNA antibodies are relatively specific for SLE; they are present in up to 50% of cases depending on ethnicity, whereas they appear in less than 2% of people without SLE.
- the anti-dsDNA antibody titers also tend to reflect disease activity, although not in all cases.
- anti-Ul RNP which also appears in systemic sclerosis
- anti-Ro or anti-SSA
- anti-La or anti-SSB; both of which are more common in Sj5gren's syndrome
- anti-Ro and anti-La when present in the maternal circulation, confer an increased risk for heart conduction block in neonatal lupus.
- Other tests routinely performed in suspected SLE are complement system levels (low levels suggest consumption by the immune system), electrolytes and renal function (disturbed if the kidneys are involved), liver enzymes, and complete blood count. II. IL-6, BANK1 and eIF4
- Interleukin 6 is an interleukin that acts as both a pro-inflammatory and an antiinflammatory cytokine. In humans, it is encoded by the IL-6 gene. IL-6 is secreted by T cells and macrophages to stimulate immune response, e.g., during infection and after trauma, especially burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against bacterium Streptococcus pneumoniae.
- IL-6 is also considered a myokine, a cytokine produced from muscle, and is elevated in response to muscle contraction. It is significantly elevated with exercise, and precedes the appearance of other cytokines in the circulation. During exercise, it is thought to act in a hormone-like manner to mobilize extracellular substrates and/or augment substrate delivery. In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation. Smooth muscle cells in the tunica media of many blood vessels also produce IL-6 as a pro-inflammatory cytokine. IL-6's role as an anti-inflammatory cytokine is mediated through its inhibitory effects on
- TNF-alpha and IL-1 TNF-alpha and IL-1
- activation of IL-lra and IL-10 TNF-alpha and IL-1
- IL-6 is one of the most important mediators of fever and of the acute phase response.
- IL-6 stimulates energy mobilization that leads to increased body temperature.
- IL-6 can be secreted by macrophages in response to specific microbial molecules, referred to as pathogen-associated molecular patterns (PAMPs).
- PAMPs pathogen-associated molecular patterns
- PRRs pattern recognition receptors
- TLRs Toll-like receptors
- IL-6 is also essential for hybridoma growth and is found in many supplemental cloning media such as briclone. Inhibitors of IL-6 (including estrogen) are used to treat postmenopausal osteoporosis. IL-6 is also produced by adipocytes and is thought to be a reason why obese individuals have higher endogeneous levels of CRP. Intranasally administered IL-6 has been shown to improve sleep-associated consolidation of emotional memories.
- IL-6 is responsible for stimulating acute phase protein synthesis, as well as the production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to regulatory T cells.
- CD130 is the common signal transducer for several cytokines including leukemia inhibitory factor (LIF), ciliary neurotropic factor, oncostatin M, IL-11 and cardiotrophin-1, and is almost ubiquitously expressed in most tissues. In contrast, the expression of CD 126 is restricted to certain tissues.
- LIF leukemia inhibitory factor
- ciliary neurotropic factor ciliary neurotropic factor
- oncostatin M IL-11
- cardiotrophin-1 the expression of CD 126 is restricted to certain tissues.
- IL-6 As IL-6 interacts with its receptor, it triggers the gpl30 and IL-6R proteins to form a complex, thus activating the receptor.
- These complexes bring together the intracellular regions of gpl30 to initiate a signal transduction cascade through
- IL-6 is probably the best-studied of the cytokines that use gpl30, also known as IL-6 signal transducer (IL6ST), in their signalling complexes.
- Other cytokines that signal through receptors containing gpl30 are Interleukin 1 1 (IL-1 1), Interleukin 27 (IL-27), ciliary neurotrophic factor (CNTF), cardiotrophin- 1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), oncostatin M (OSM), Kaposi's sarcoma-associated herpesvirus interleukin 6-like protein (KSHV-IL6).
- IL-1 1 Interleukin 1 1
- CNTF ciliary neurotrophic factor
- CT-1 cardiotrophin- 1
- CLC cardiotrophin-like cytokine
- LIF leukemia inhibitory factor
- OSM oncostatin M
- KSHV-IL6 Kaposi's sarcoma-associated
- sIL-6R soluble form of IL-6R
- Many neuronal cells are unresponsive to stimulation by IL-6 alone, but differentiation and survival of neuronal cells can be mediated through the action of sIL-6R.
- the sIL-6R/IL-6 complex can stimulate neurites outgrowth and promote survival of neurons and, hence, may be important in nerve regeneration through remyelination.
- IL-6 is relevant to many diseases such as diabetes, atherosclerosis, depression, Alzheimer's Disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, Behcet's disease, and rheumatoid arthritis.
- Advanced/metastatic cancer patients have higher levels of IL-6 in their blood.
- anti-IL-6 agents as therapy against many of these diseases.
- the first such is tocilizumab, which has been approved for rheumatoid arthritis.
- ALD518 is in clinical trials.
- BANK1 Human and mouse Bankl encode the B cell scaffold protein with ankyrin repeats ! (BANK1).
- BANK1 is a tyrosine kinase substrate that becomes extensively tyrosine phosphorylated and is capable of binding the Src family kinases Lyn and Blk (Castillejo- Lopez et ah, 2012; Yokoyama et ah, 2002) and promotes tyrosine phosphorylation of inositol 1,4,5-trisphosphate receptors. While apparently involved in BCR signaling, the function of BANK1 during signaling induced by CpG, an agonist of the major toll-like receptor, TLR9 expressed in B cells, is not known. It also is involved in B-cell receptor induced Ca 2+ mobilization from intracellular stores and promotes Lyn-mediated phosphorylation of IP3 receptors 1 and 2.
- the gene is located on the long arm of chromosome 4 (4q24) on the Watson (plus) strand and is 284,206 bases in length.
- the gene encodes a protein of 785 amino acids (molecular weight 89.335 kDa) and four isoforms are known.
- the gene is expressed in B-cell but not T-cell or myeloid cell lines. The greatest expression is in CD19 + B-cells with very low expression in other cell populations.
- BANK1 acts as an adaptor or scaffold protein in the same family as the B cell adapter for PI3K (BCAP) and the Drosophila homologue Dof (Yokoyama et al, 2002). Consistent with this hypothesis, the inventors' recent studies have shown that exon 2 of human BANK1 encodes a highly hydrophobic domain, which renders the protein susceptible to aggregation (Kozyrev et al, 2012); scaffold and adaptor proteins are known to form complex structures to facilitate intracellular signaling at the proper time and differentiation stage.
- exon 2 also encodes a predicted N-terminal toll/IL-1 receptor (TIR) domain that is shared by BCAP (Troutman et al, 2012) and used in the interaction of BCAP with the adaptors MyD88 and TIRAP.
- TIR N-terminal toll/IL-1 receptor
- TLR9 is the major endosomal TLR in B cells that recognizes viral nucleic acids, and TLR9 signaling is believed to have an important role in autoimmunity (Christensen et al, 2005). TLR9 signaling is stimulated by hypomethylated DNA oligonucleotides or CpG (Bernasconi et al, 2003), leading to a pro-inflammatory response (Sun et al, 2007). CpG- induced signaling activates mitogen activated protein kinase (MAPK) pathways, including p38, JNK and ERK.
- MAPK mitogen activated protein kinase
- Stimulation of p38 and ERK signaling by growth factors, stress or viral infections can induce transcriptional activation, but can also induce two pathways of post- transcriptional regulation of protein synthesis: (a) control of mRNA stabilization (Neininger et al, 2002 and Bollig et al, 2003) by the mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase) MK2, and (b) the transient formation of the heterotrimeric eIF4E/eIF4F/eIF4G translation initiation complex through phosphorylation of eIF4E (Banerjee et al, 2002; Morley, 1997).
- mice In mice, the only kinases known to phosphorylate eIF4E are MNK1 and MNK2.
- MNK2 is constitutively active, while MNK1 is regulated by the MAP kinases (Sonenberg, 2008).
- a second axis of control of eIF4E activation is through the AKT/mTORCl pathway. This pathway regulates the phosphorylation of 4E-BP1, the eIF4E binding protein. Under non-phosphorylated conditions, 4E-BP 1 retains eIF4E (Gingras et al, 1999 and Richter and Sonenberg, 2005). Once 4E-BP1 becomes phosphorylated by mTORCl, it releases eIF4E, which is in turn phosphorylated by MNK1/2 (Livingstone et al, 2009).
- Eukaryotic initiation factor (elF) complex 2 forms a ternary complex with GTP and the initiator Met-tRNA - this process is regulated by guanine nucleotide exchange and phosphorylation and serves as the main regulatory element of the bottleneck of protein expression.
- a number of initiation factors must facilitate the synergy of the ribosome and the mRNA and ensure that the 5' UTR of the mRNA is sufficiently devoid of secondary structure. Binding in this way is facilitated by group 4 eukaryotic initiation factors; eIF4 has implications in the normal regulation of translation as well as the transformation and progression of cancerous cells; as such, it represents an interesting field of research.
- Eukaryotic translation initiation factor 4E is a protein that in humans is encoded by the EIF4E gene.
- eIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of a multiprotein complex termed eIF4F.
- the eIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis.
- eIF4F The other subunits of eIF4F are (a) a 50-kD polypeptide, termed eIF4A, that possesses ATPase and RNA helicase activities, and (b) a 220-kD polypeptide, eIF4G.
- eIF4E has been shown to interact with eIF4Al, eIF4EBP3, eIF4EBP l, eIF4EBP2, eukaryotic translation initiation factor 4 ⁇ , eIF4G2 and eIF4ENIF l.
- eIF4E's function is to bind an mRNA cap and ultimately bring it to the ribosome.
- eIF4E is part of the eIF4F pre-initiation complex, which is made up of eIF4E, and eIF4G (eIF4F is sometimes considered to have additional protein components).
- eIF4E binds the first nucleotide on the 5' end of an mRNA molecule (known as the cap): a 7 methyl guanosine (m7G). It sandwiches m7G between 2 tryptophan residues, and other amino acids are involved in the binding.
- viruses cut eIF4G in such a way that the eIF4E binding site is removed and the virus is able to translate its proteins without eIF4E. Also, some cellular proteins (the most notable being heat shock proteins) do not require eIF4E in order to be translated. Both viruses and cellular proteins achieve this through an IRES structure in the RNA.
- Fragile X mental retardation protein acts to regulate translation of specific mRNAs through its binding of eIF4E.
- FMRP acts by binding CYFIP 1 , which directly binds eIF4e at a domain that is structurally similar to those found in 4E-BPs including EIF4EBP3, EIF4EBP 1, and EIF4EBP2.
- the FMRP/CYFIP 1 complex binds in such a way as to prevent the eIF4E-eIF4G interaction, which is necessary for translation (biology) to occur.
- the FMRP/CYFIP l/eIF4E interaction is strengthened by the presence of mRNA(s).
- BC1 RNA allows for an optimal interaction between FMRP and CYFIP 1.
- RNA-BC1 is a non-translatable, dendritic mRNA, which binds FMRP to allow for its association with a specific target mRNA.
- BC1 may function to regulate FMRP and mRNA interactions at synapse(s) through its recruitment of FMRP to the appropriate mRNA.
- FMRP may recruit CYFIP 1 to specific mRNAs in order to repress translation.
- the FMRP-CYFIP 1 translational inhibitor is regulated by stimulation of neuron(s). Increased synaptic stimulation resulted in the dissociation of eIF4E and CYFIP 1, allowing for the initiation of translation.
- inhibitors of eIF4 contemplates the use of inhibitors of eIF4 to treat SLE.
- inhihitors those that inhibit eIF4A/E/G expression, and those that do not inhibit expression but instead inhibit the activity of eIF4A/E/G.
- inhibitors may be biological - nucleic acid or peptides - or pharmaceutical (i.e., small molecules).
- Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences.
- complementary it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
- Antisense polynucleotides when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability.
- Antisense RNA constructs, or DNA encoding such antisense RNA's may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
- Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
- complementary or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
- ribozyme e.g., ribozyme; see below
- genomic DNA may be combined with cDNA or synthetic sequences to generate specific constructs.
- a genomic clone will need to be used.
- the cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
- Ribozymes Another general class of inhibitors is ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et ah, 1987; Forster and Symons, 1987).
- ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et ah, 1981 ; Michel and Westhof, 1990; Reinhold- Hurek and Shub, 1992).
- This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
- IGS internal guide sequence
- Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et ah, 1981).
- U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes.
- sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990).
- ribozymes can elicit genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that was cleaved by a specific ribozyme.
- RNA interference also referred to as "RNA-mediated interference” or RNAi
- RNAi Double- stranded RNA
- dsRNA double- stranded RNA
- dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp et al, 2000; Tabara et al, 1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction.
- RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp et al, 2000; Tabara et al, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma, Drosophila, and mammals (Grishok et al, 2000; Sharp, 1999; Sharp et al, 2000; Elbashir et al, 2001). It is generally accepted that RNAi acts post-transcriptionally, targeting RNA transcripts for degradation, and possibly by inhibiting translation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher et al, 2000).
- siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e. those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes. Typically, siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et al, 1998). Of particular interest are those siRNAs that span an exon-intron junction.
- siRNAs has been mainly through direct chemical synthesis; through processing of longer, double-stranded R As through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive.
- Chemical synthesis proceeds by making two single-stranded RNA-oligomers followed by the annealing of the two single-stranded oligomers into a double-stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889, 136, 4,415,732, and 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).
- RNA sequences having di-nucleotide overhangs may provide the greatest level of suppression.
- These protocols primarily use a sequence of two (2'-deoxy)thymidine nucleotides as the di- nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA.
- the literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight ( ⁇ 20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
- siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. This had been demonstrated by Elbashir et al. (2001) wherein concentrations of about 100 nM achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen et al, 2000; Elbashir et al, 2001).
- RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference.
- the enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. See U.S. Patent 5,795,715.
- the contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene.
- the length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
- RNA single-stranded RNA is enzymatically synthesized from the PCR® products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides.
- WO 01/36646 incorporated herein by reference, places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures.
- RNA polymerase e.g., T3, T7, SP6
- RNA interference no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
- U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized.
- the templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence.
- the templates can be attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.
- the inventors propose to inhibit eIF4A/E/G expression in adult tissues in vitro using siRNA or shRNA in an adenoviral vector.
- a GFP marker can be utilized to determine cells that take up the vector, and thus permit checking for appropriate inhibition of eIF4A/E/G production.
- the use of an inducible promoter allows for induction of the siRNA or shRNA only under specific growth conditions, thereby permitting reversible inhibition of eIF4A/E/G.
- the present invention contemplates the design, production and use of various eIF4A/E/G peptides.
- the peptides will be 50 residues or less, again, comprising no more than 20 consecutive residues of eIF4A/E/G.
- the overall length may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues.
- Ranges of peptide length of 4-50 residues, 5-50 residues, 6-50 residues, 7-50 residues, 7-25, residues, 4- 20 residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15 residues are contemplated.
- the number of consecutive eIF4A/E/G residues may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20.
- Ranges of consecutive residues of 4-20 residues, 5-20 residues, 6-20 residues, 7-20 residues and 4-15 residues, 5-15, residues, 6-15 residues or 7-15 residues are contemplated.
- the present invention may utilize L-configuration amino acids, D-configuration amino acids, or a mixture thereof. While L-amino acids represent the vast majority of amino acids found in proteins, D-amino acids are found in some proteins produced by exotic sea- dwelling organisms, such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain.
- L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L- glyceraldehyde is levorotary).
- Retro-inverso modification of naturally-occurring polypeptides involves the synthetic assemblage of amino acids with a- carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids in reverse order with respect to the native peptide sequence.
- a retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds ( H-CO rather than CO- NH) while approximately maintaining the topology of the side chains as in the native peptide sequence. See U.S. Patent 6,261,569, incorporated herein by reference.
- the present invention contemplates fusing or conjugating a cell delivery domain (also called a cell delivery vector, or cell transduction domain).
- a cell delivery domain also called a cell delivery vector, or cell transduction domain.
- Such domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007).
- poly-D-Arg and poly-D-Lys sequences e.g., dextrorotary residues, eight residues in length
- peptides modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake.
- blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino- and/or carboxyl-terminal residues.
- the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group.
- the protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support.
- the protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide.
- the peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids.
- Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
- non-standard amino acids Aside from the 20 standard amino acids can be used, there are a vast number of "non-standard” amino acids. Two of these can be specified by the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid, dehydroalanine and the neurotransmitter gamma-aminobutyric acid.
- Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism.
- Nonstandard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.
- Linkers or cross-linking agents may be used to fuse peptides to other proteinaceous sequences.
- Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies.
- Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites.
- Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially.
- the bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied.
- a majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.
- heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described in U.S. Patent 5,889, 155, specifically incorporated herein by reference in its entirety.
- the cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols.
- the cross-linking reagent can be modified to cross-link various functional groups and is thus useful for cross-linking polypeptides. In instances where a particular peptide does not contain a residue amenable for a given cross-linking reagent in its native sequence, conservative genetic or synthetic amino acid changes in the primary sequence can be utilized.
- Dominant negative mutants are mutant defective proteins with can negate the effects of normal, functional proteins when both are present in the same environment. In many cases, dominant negative proteins homo-multimerize and are thus able to "poison" a complext that contains one or more functional proteins.
- dominant negative molecules one may design molecules that interact with one binding partner but fail to interact with another, such as due to a conformational change or single site mutation or trunctation. Another option is to design a molecule that binds normally, but lacks a subsequent function such as an enzymatic activity (phosphorylation, cleavage, etc.). Yet another option is to remove a sequence from the molecule that would permit its translocation to another portion of the cell. Other possibilities also exist. 2.
- hippuristinol a small molecule found in the coral Ms hippuris. Its structure is shown below:
- PatA pateamine A
- Another eIF4A inhibitor is 15-deoxy-delta(12, 14)-prostaglandin J2 (15d-PGJ2).
- Ribavirin is a guanosine (ribonucleic) analog used to stop viral RNA synthesis and viral mRNA capping; simply put, it is a nucleoside inhibitor. Its brand names include CopegusTM, RebetolTM, RibasphereTM, VilonaTM, and VirazoleTM, and it is an anti-viral drug indicated for severe RSV infection (individually), (notably for persistent) hepatitis C infection (can be used in conjunction with peg-interferon a2b or peg-interferon a2a), and some other viral infections. Ribavirin is a prodrug, which when metabolized resembles purine RNA nucleotides. In this form it interferes with RNA metabolism required for viral replication.
- Ribavirin is active against a number of DNA and RNA viruses. It is a member of the nucleoside antimetabolite drugs that interfere with duplication of viral genetic material. Ribavirin is active against influenzas, flaviviruses, and agents of many viral hemorrhagic fevers. In Europe and the U.S. the oral (capsule or tablet) form of ribavirin is used in the treatment of hepatitis C, in combination with pegylated interferon drugs.
- Ribavirin is the only known treatment for a variety of viral hemorrhagic fevers, including Lassa fever, Crimean-Congo hemorrhagic fever, Venezuelan hemorrhagic fever, and Hantavirus infection, although data regarding these infections are scarce and the drug might be effective only in early stages.
- ribavirin The primary observed serious adverse side effect of ribavirin is hemolytic anemia, which may worsen preexisting cardiac disease.
- the mechanism for this effect is due to ribavirin's buildup inside erythrocytes. Oxidative damage to erythrocyte cell membrane is usually inhibited by glutathione; however, with reduced ATP levels caused by ribavirin, glutathione levels are impaired, permitting oxidative erythrocyte cell lysis. The gradual loss of erythrocytes leads to anemia. The anemia is dose-dependent and may sometimes be compensated by decreasing dose. Ribavirin is also a teratogen in some animal species and thus poses a theoretical reproductive risk in humans, remaining a hazard as long as the drug is present, which can be as long as 6 months after a course of the drug has ended.
- the aerosol form has been used in the past to treat respiratory syncytial virus-related diseases in children. However, its efficacy has been called into question by multiple studies, and most institutions no longer use it. It is still used in some cases.
- ribavirin (“ribavirina”) has been sold for use against influenza. Studies have been mixed, but the derivative viramidine may have more promise. It has been used (in combination with ketamine, midazolam, and amantadine) in treatment of rabies.
- This drug is also used to control the life span of enterovirus 71 which causes hand, foot, and mouth disease. Notably, for severe RSV in children, the drug is delivered as aerosol particles for 12-18 hours daily. The method is very expensive, inconvenient and used rarely.
- ribavirin is similar to the sugar D-ribose from which it is derived. It is freely soluble in water, and is re-crystallized as fine silvery needles from boiling methanol. The three free sugar hydroxyls make the pure drug hydrophilic enough that it is only sparingly soluble in anhydrous ethanol.
- ribavirin is prepared from natural D- ribose by blocking the 2', 3' and 5' OH groups with benzyl groups, then derivatizing the OH with an acetyl group which acts as a suitable leaving group upon nucleophilic attack.
- the ribose ⁇ carbon attack is accomplished with a 1,2,4 triazole-3 -carboxymethyl ester, which directly attaches the nitrogen of the triazole to the carbon of the ribose, in the proper 1- ⁇ - D isomeric position.
- the bulky benzyl groups hinder attack at the other sugar carbons.
- treatment with ammonia in methanolic conditions then simultaneously deblocks the ribose hydroxyls, and converts the triazole carboxymethyl ester to the carboxamide.
- ribavirin may be recovered in good quantity by cooling and crystallization.
- Ribavirin is possibly best viewed as a ribosyl purine analogue with an incomplete purine 6-membered ring. This structural resemblance historically prompted replacement of the 2' nitrogen of the triazole with a carbon (which becomes the 5' carbon in an imidazole), in an attempt to partly "fill out” the second ring, but to no great effect.
- Such 5' imidazole riboside derivatives show antiviral activity with 5' hydrogen or halide, but the larger the substituent, the smaller the activity, and all proved less active than ribavirin.
- Antiviral activity is retained for acetate and phosphate derivation of the ribose hydroxyls, including the triphosphate and 3',5' cyclic phosphates, but these compounds are no more active than the parent molecule, reflecting the high efficiency of esterase and kinase activity in the body.
- ribavirin derivative The most successful ribavirin derivative to date is the 3-carboxamidine derivative of the parent 3-carboxamide, first reported in 1973 and now called taribavirin (former names viramidine and ribamidine).
- This drug shows a similar spectrum of antiviral activity to ribavirin, which is not surprising as it is now known to be a pro-drug for ribavirin.
- Viramidine has useful properties of less erythrocyte-trapping and better liver- targeting than ribavirin.
- the first property is due to viramidine's basic amidine group which inhibits drug entry into RBCs, and the second property is probably due to increased concentration of the enzymes which convert amidine to amide, in liver tissue.
- Viramidine is in phase III human trials and may one day be used in place of ribavirin, at least against certain kinds of viral hepatitis. Viramidine's slightly superior toxicological properties may eventually cause it to replace ribavirin in all uses of ribavirin.
- Ribavirin is absorbed from the GI tract probably by nucleoside transporters. Absorption is about 45%, and this is modestly increased (to about 75%) by a fatty meal. Once in the plasma, ribavirin is transported through the cell membrane also by nucleoside transporters. Ribavirin is widely distributed in all tissues, including the CSF and brain. The pharmacokinetics of ribavirin is dominated by trapping of the phosphate form inside cells, particularly red blood cells (RBCs) which lack the enzyme to remove the phosphate once it has been added by kinases, and therefore attain high concentrations of the drug. Most of the kinase activity which converts the drug to active nucleotide form, is provided by adenine kinase. This enzyme is more active in virally infected cells.
- RBCs red blood cells
- the volume of distribution of ribavirin is large (2000 L/kg) and the length of time the drug is trapped varies greatly from tissue to tissue.
- the mean half-life for multiple doses in the body is about 12 days, but very long-term kinetics are dominated by the kinetics of RBCs (half-life 40 days).
- RBCs store ribavirin for the lifetime of the cells, releasing it into the body's systems when old cells are degraded in the spleen.
- About a third of absorbed ribavirin is excreted into the urine unchanged, and the rest is excreted into urine as the de-ribosylated base 1,2,4-triazole 3-carboxamide, and the hydrolysis product of this, 1,2,4-triazole 3- carboxylic acid.
- Ri is a hydrazone thiazole moiety of the structure:
- R2 is hydrogen, hydroxyl or a nitro group present in one, two or three locations on the ring to which it is attached;
- R 3 is a group individually present in one, two or three locations on the ring, wherein the group may be halo, hydrogen, conjugated or unconjugated aryl or heteroaryl, a alicyclic or polycyclic group, or R 3 , taken with the ring to which it is attached, forms a conjugated ring structure, e.g., a naphthalene ring;
- R4 is hydrogen, carboxyl, a lower alkyl ester, e.g. :
- R 5 is N (in which case the dotted bond is present), NH, or carbonyl
- R-6 is NH or carbonyl
- MNK1/2 inhibitors cercosporamide, S209, CGP57380, PF3644022
- mTOR ATP-active site inhibitors rapamycin, RAD001, CCI- 779, PP242, Torinl, WYE-132, Ku-0063794, Palomid 529, and AZD8055, ⁇ 128, AZD2014 Palomid 529 BEZ235, PF-04691502, XL765).
- the present invention also contemplates the treatment of SLE using standard therapeutic approaches where indicated in combination with the eIF4E therapies of the present disclosure.
- the treatment of SLE involves treating elevated disease activity and trying to minimize the organ damage that can be associated with this increased inflammation and increased immune complex formation/deposition/complement activation.
- the compositions would be provided in a combined amount effective to exert a combined effect on the damaged tissue. This process may involve contacting the cells with the eIF4E inhibitor, in combination with a second therapeutic agent or factor(s) at the same time.
- treatment with the eIF4E inhibitor may precede or follow the additional agent treatment by intervals ranging from minutes to weeks.
- the second agent is applied separately to the target, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent would still be able to exert an advantageously combined effect on the target.
- Foundational treatment for combination therapies as described above can include corticosteroids and anti-malarial drugs.
- Certain types of lupus nephritis such as diffuse proliferative glomerulonephritis require round of cytotoxic drugs. These drugs include, most commonly, cyclophosphamide and mycophenolate.
- Hydroxychloroquine (HCQ) was approved by the FDA for lupus in 1955. Some drugs approved for other diseases are used for SLE 'off-label' In November 2010, an FDA advisory panel recommended approving belimumab (Benlysta) as a treatment for elevated disease activity seen in autoantibody- positive lupus patients. The drug was approved by the FDA in March 201 1.
- Hydroxychloroquine is an FDA-approved antimalarial used for constitutional, cutaneous, and articular manifestations. Hydroxychloroquine has relatively few side effects, and there is evidence that it improves survival among people who have SLE and stopping HCQ in stable SLE patients led to increased disease flares in Canadian lupus patients.
- DMARDs Disease-modifying antirheumatic drugs
- SLE Disease-modifying antirheumatic drugs
- DMARDs commonly in use are methotrexate and azathioprine.
- medications that aggressively suppress the immune system primarily high-dose corticosteroids and major immunosuppressants
- Cyclophosphamide is used for severe glomerulonephritis, as well as other life-threatening or organ-damaging complications, such as vasculitis and lupus cerebritis.
- Mycophenolic acid is also used for treatment of lupus nephritis, but it is not FDA-approved for this indication.
- Belimumab or a humanized monoclonal antibody against B-lymphocyte stimulating factor (BlyS or BAFF), is FDA approved for lupus treatment and decreased SLE disease activity, especially in patients with baseline elevated disease activity and the presence of autoantibodies.
- Addition drugs such as abatacept, epratuzimab, etanercept and others, are actively being studied in SLE patients and some of these drugs are already FDA-approved for treatment of rheumatoid arthritis or other disorders.
- NSAIDs such as indomethacin and diclofenac are relatively contraindicated for patients with SLE because they increase the risk of kidney failure and heart failure.
- Moderate pain is typically treated with mild prescription opiates such as dextropropoxyphene and co-codamol.
- Moderate to severe chronic pain is treated with stronger opioids, such as hydrocodone or longer-acting continuous-release opioids, such as oxycodone, MS Contin, or methadone.
- the fentanyl duragesic transdermal patch is also a widely used treatment option for the chronic pain caused by complications because of its long-acting timed release and ease of use.
- opioids are used for prolonged periods, drug tolerance, chemical dependency, and addiction may occur.
- Opiate addiction is not typically a concern, since the condition is not likely to ever completely disappear.
- lifelong treatment with opioids is fairly common for chronic pain symptoms, accompanied by periodic titration that is typical of any long-term opioid regimen.
- Intravenous immunoglobulins may be used to control SLE with organ involvement, or vasculitis. It is believed that they reduce antibody production or promote the clearance of immune complexes from the body, even though their mechanism of action is not well- understood. Unlike immunosuppressives and corticosteroids, IVIGs do not suppress the immune system, so there is less risk of serious infections with these drugs. Avoiding sunlight is the primary change to the lifestyle of SLE sufferers, as sunlight is known to exacerbate the disease, as is the debilitating effect of intense fatigue. These two problems can lead to patients becoming housebound for long periods of time. Drugs unrelated to SLE should be prescribed only when known not to exacerbate the disease. Occupational exposure to silica, pesticides and mercury can also make the disease worsen.
- Renal transplants are the treatment of choice for end-stage renal disease, which is one of the complications of lupus nephritis, but the recurrence of the full disease in the transplanted kidney is common in up to 30% of patients.
- Antiphospholipid syndrome is also related to the onset of neural lupus symptoms in the brain.
- thromboses blood clots or "sticky blood" form in blood vessels, which prove to be fatal if they move within the blood stream. If the thromboses migrate to the brain, they can potentially cause a stroke by blocking the blood supply to the brain. If this disorder is suspected in patients, brain scans are usually required for early detection. These scans can show localized areas of the brain where blood supply has not been adequate. The treatment plan for these patients requires anticoagulation. Often, low-dose aspirin is prescribed for this purpose, although for cases involving thrombosis anticoagulants such as warfarin are used.
- compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
- compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula.
- pharmaceutically or pharmacologically acceptable refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
- compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Such routes include oral, nasal, buccal, rectal, vaginal or topical route. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.
- the active compounds may also be administered parenterally or intraperitoneally.
- Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose.
- Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
- the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions.
- the form must be sterile and must be fluid to the extent that easy syringability exists. It must 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 carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
- the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- a coating such as lecithin
- surfactants for example, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sulfate, sodium sorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars or sodium chloride.
- Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
- the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like.
- the use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
- the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices.
- a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
- the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate.
- the active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries.
- the active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
- compositions of the present invention may be formulated in a neutral or salt form.
- Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
- solutions Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
- the formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like.
- the solution For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
- sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure.
- one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences,” 15 th Ed., 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
- microbes can be engineered to deliver drugs according to the present disclosure.
- Lactobacillus strains have been used for the purpose (drug delivery of paclitaxel).
- L. acidophilus and L. rhamnosus can form minicells in modified MRS broth. Nanoparticle size of obtained minicells is ⁇ 400 nm in diameter.
- These minicells have been packaged paclitaxel (10 ⁇ g/ml) and cephalosporin (10 ⁇ g/ml).
- synthetic machinery can be embedded in microorganisms for the production of drugs. Delivery of the microorganism therefore constitutes administration of the drug itself.
- mice Bankl ' mice were kindly provided by Dr T. Kurosaki (Riken Research Centre for Allergy and Immunology, Kyoto, Japan) and were backcrossed nine generations onto the C57BL/6J background. C57BL/6J mice were purchased from Jackson Laboratory, Bar Harbor, Maine, USA. Mice were maintained under specific pathogen free (SPF) barrier conditions. This study was approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.
- SPF pathogen free
- Antibodies and reagents Phospho-specific antibodies to p38 (Thrl 80/Tyrl82, clonel2F8 #4631), JNK (Thrl83/Tyrl85, clone 81E11, #4668), ERK (Thr202/Tyr204, clone D13.14.4E, #4370), I /c B a (Ser32, clone 14D4, #2859), eIF4E (Ser209, #9741), M K1/2 (Thrl97/202, #211 1), 4E-BP1 (Thr37/46, clone 236B4, #2855), mTOR (Ser2448, clone D9C2, #5536), and AKT (Ser473, clone 193H12, #4058) and phosphorylation-state- independent antibodies to p38 (#9212), JNK, ERK, I /c B a , eIF4E (clone C46
- Splenic B cells were purified by negative selection using magnetic bead separation. Briefly, spleen cells from Bankl +/+ and Bankl "7" littermates were labeled with a cocktail of biotin-conjugated antibodies for 15 minutes. Cells were incubated an additional 15 minutes with anti-biotin micro beads (B-Cell Isolation Kit, mouse; Miltenyi Biotech, Auburn, CA) at 4° C. The labeled non-B-cells were depleted by magnetic retention in a MACS column while unlabeled B cells were recovered. The purity of the resulting cell population was typically more than 95% B220 + CD3 " as assessed by flow cytometry analysis.
- Cell Culture was performed with RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), 2-ME (50 ⁇ ) and antibiotics for all experiments, except for p38 analyses and the Akt axis where cells were serum-starved two hours prior to stimulation.
- Purified splenic B cells (10 6 cells/mL) were treated with 10 ⁇ g/ml F(ab')2 fragment of anti-mouse IgM (Jackson Immunoresearch, West Grove, PA), 2 ⁇ CpG (ODN Type B; 1668, Invivogen, San Diego, CA) or combination of anti-mouse IgM and CpG, 20 ⁇ g/mL TLR4 agonist LPS or 1 ⁇ g/mL of the TLR7 agonist R848 (Invivogen, San Diego, CA), or were left unstimulated.
- Cytokine secretion by in vitro stimulated B cells was measured using the ELISA kit for IL-6 and IL-10 purchased from BD Bioscience (San Jose, CA). For this, 96-well plates were coated with antibody against mouse IL-6 or mouse IL-10 overnight at 4°C. Plates were then blocked for 1 h at room temperature with 10% FBS in PBS. Supernatants were then added to the plate and left for 2 h at room temperature. Following washing, biotinylated anti-IL-6 or anti-IL-10 was added and plates incubated for further an hour at room temperature. Streptavidine alkaline phosphatase was then added following incubation at room temperature and washing with tetramethylbenzidine liquid substrate.
- Splenic B cells (0.5 l0 6 cells/well) were stimulated for 6 days and supernatants were analyzed for immunoglobulin isotype antibodies as per the instructions from BD PharmingenTM mouse immunoglobulin isotype ELISA kit (BD Biosciences, San Diego, CA, US).
- the inventors quantified IgG2a and IgG2c isotype antibodies using mouse IgG2a Ready-Set-Go ELISA kit (eBioscience Inc. San Diego CA, US) and mouse IgG2c ELISA Quantification Set (Bethyl Laboratories, Inc. TX, US) respectively and data are presented as ng/ml. For other antibodies, only OD's are shown.
- Cells were then stimulated with 10 ⁇ g/ml F(ab')2 fragment of anti-IgM, 2 ⁇ CpG, 10 ⁇ g/ml anti-CD40, 20 ⁇ g/ml LPS, and 1 ⁇ g/ml R848, respectively, or the shown combinations and cultured for 48, 72 and 96 hr. After harvest, cells were stained with propidium iodide (PI, final concentration of 1 ⁇ g/ml; eBioscience Inc. San Diego CA, US) and acquired by LSR II or FACScalibur (BD bioscience, San Jose, CA) 36,000 events in total. For flow cytometry, debris and doublet cells were discriminated and viable cells (PI- negative) were gated and % of cell viability was determined.
- PI propidium iodide
- the histograms shown with FL1 channel represent overlaid data from un-stimulated CFSE-labeled B cells, stimulated Bankl +/+ and Bankl 7" B cells, and the y-axis is presented as % viability of maximum.
- the analysis was conducted using FlowJo software (Tree Star Inc., Ashland, OR).
- CGP57380 [4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine] was obtained from Calbiochem®-Millipore (Billerica, MA), and MNKs inhibitor cercosporamide and MK2 inhibitor, PF-3644022 [(10R)-9,10,1 l,12-tetrahydro-10-methyl-3-(6-methyl-3-pyridinyl)-8H- [l,4]diazepino[5',6':4,5]thieno[3,2-f ]quinolin-8-one hydrate] were purchased from Sigma- Aldrich (St. Louis, MI).
- the purified splenic B cells (5 x 10 5 cells/ well) were seeded in complete RPMI1640 medium in a 48 well tissue culture plate. Then the cells were pretreated with or without inhibitor for 30 min or lh, respectively, before CpG stimulation for 24 hr time at which cells were harvested and subjected to determine % of viability by using propidium iodide staining (final concentration 1 ⁇ g/ml) and flow cytometry analysis. Supernatants were collected and stored at -80 °C until use and IL-6 was measured using a capture ELISA. The control well contained CpG and cell culture grade DMSO (Pierce, Rockford, IL) only (17).
- the qRT-PCR reactions were performed on a 7900 HT Fast Real-Time PCR system.
- the primers and probes for mouse TLR9 (Assay ID: Mm00446193_ml), Prdml (Mm00476128_ml) and IL-6 (Assay ID: Mm00446190_ml) and internal control gene 18S rRNA (4319413E) were purchased from Taqman Gene expression Assays (Life Technologies, Carlsbad, CA).
- IL-6 mRNA stability assay 5 x 10 5 splenic B cells were cultured in triplicate and treated with 2 ⁇ CpG for 20h followed by addition of 1 ⁇ actinomycin D (Sigma- Aldrich, St. Louis, MI) for 0, 50, 100, 200 min. RNA was isolated and IL-6 transcription was determined with Taqman qRT-PCR using the primers as described above.
- BANK1 Deficiency Reduces CpG-Induced p38 Activation in Splenic B Cells.
- the inventors tested if BANK1 deficiency altered B cell proliferation after stimulation with CpG. Using purified splenic B-cells from Bankr 7" and littermate control ( +/+ ) mice, they did not observe any differences at any of the time points tested in proliferation with CpG or LPS, a TLR4 agonist (FIG. 1A).
- the inventors also did not observe differences in cell proliferation when using anti-IgM, anti-IgM+CpG, anti-CD40, CpG + anti-CD40, R848, R848+anti-CD40, LPS, LPS + anti-CD40, or anti- IgM+LPS (FIGS. 6A-C). They then tested the effects of BANK 1 deficiency on IgM-induced activation of NF ⁇ B by analyzing phosphorylation and degradation of I ⁇ B a , and activation of the MAPK pathways by studying phosphorylation of ERK and p38. BANK1 deficiency did not affect phosphorylation of MAPKs, or I ⁇ B a following stimulation with anti-IgM alone (FIG. IB).
- MAPK p38-signaling pathway regulates the production of several cytokines including IL-6, TNFa, IFNy, and IL-10 (Enslen et al, 1998, Nagaleekar et al, 201 1 and Foey et al, 1998). Having shown that BANK1 deficiency reduced p38 phosphorylation after CpG stimulation, the inventors asked whether reduction in p38 phosphorylation leads to modulation of IL-6 or IL-10 secretion by Bankl 7" B cells.
- CpG is a known agonist of Tlr9.
- Tlr9 gene expression was no different between Bankl +/+ and Bankl "7" mice (FIG. 2B).
- LPS and the TLR7 agonist R848 induced subtle amounts of IL-6 (Vanden and Bishop, 2008 and Poudrier et al, 1998) as compared to CpG stimulation and BANKl deficiency had no effect (FIG. 2C).
- BANKl deficiency reduces secretion of IL-6 in response to CpG stimulation, overcomes the effect of anti-CD40 and is not due to changes in cell viability or to reduced Tlr9 expression.
- IL-6 gene expression was altered by the deficiency of BANK1. As shown in FIG. 4A, IL-6 gene expression showed no differences between BANK 1 -sufficient and BANK 1 -deficient B cells following CpG stimulation. Since the p38-MK2 signal pathway is responsible in maintaining mRNA stability, the inventors tested the effect of BANK1 deficiency on MK2- mediated IL-6 secretion by testing the stability oiIL-6 mRNA. CpG-stimulated Bankl +/+ and Bankr 7" splenic B-cells showed comparable IL-6 mRNA stability (FIG. 4B).
- MNK1/2 regulates the translation initiation factor eIF4E through phosphorylation (Raught and Gingras, 1999, Andersson and Sundler, 2006 and Shveygert et al, 2010).
- the inventors therefore analyzed phosphorylation of MNK1/2 and eIF4E. CpG- induced MNK1/2 and eIF4E phosphorylation were consistently reduced in Bankl 7" cells (FIGS. 4C-D).
- BANK1 influences IL-6 secretion by its effects on the p38-regulated MNKl/2/eIF4E/eIF4G pathway of translation initiation while BANK1 does not affect IL-6 secretion via the MK2 pathway.
- BANK1 has no effect on the AKT-mTORCl-4E-BPl Signaling Cascade. Activation of eIF4E is also controlled via the AKT-mTORCl-4E-BPl signaling cascade. Activation of this pathway leads to phosphorylation of 4E-BP1, which in turn releases eIF4E for it to be phosphorylated by MNK1/2 and initiate translation. In addition, stimulation through CD40 has been reported to lead to increased AKT phosphorylation in Bankl "7" purified B cells (Aiba et al, 2006). The cascade was induced with CpG alone (FIG.
- BANK1 acts only in the MNK1/2 and eIF4E arm of p38 signaling to induce IL-6 secretion following CpG stimulation, and that absence of BANK1 does not affect activation of AKT, mTORCl or 4E- BP1 of induction of translation initiation.
- BANKl deficiency leads to a tendency towards increased production of IgG2a/c subclass antibodies but does not affect expression of the Blimpl gene (Prdml).
- Prdml Blimpl gene
- IL-6 Production of IL-6 is increased following CpG stimulation in a transgenic for the human BANKl gene.
- the inventors have shown that deficiency of BANKl specifically affects the phosphorylation of p38 and the downstream signaling of the translation initiation complex molecule eIF4E leading to decreased production of IL-6 protein (Wu et al, 2013).
- the inventors produced a mouse transgenic for the human full-length isoform of BANKl .
- BANKl is expressed only in B cells thanks to the CD 19 promoter included in the construct.
- BANKl -deficient animals with B6.Slel z/z .yaa, a congenic mouse containing the NZW locus Slel that leads to several lupus-related B cell phenotypes (Morel et al, 2001; Shi et al, 2007; Sobel et al, 2002; Vuyyuru et al, 2009).
- B6.Slel z/z .yaa a congenic mouse containing the NZW locus Slel that leads to several lupus-related B cell phenotypes.
- the yaa region in male animals amplifies the phenotypic effect, leading to the development of kidney disease in male mice (Hwang et al, 2012; Subramanian et al, 2006).
- the yaa "mutation" is the result of a duplication of the genomic region in the X chromosome containing the TLR7 and TLR8 genes, which has naturally translocated onto the Y chromosome of the BXSB mouse (Subramanian et al, 2006), previously known as the BXSB.yaa strain Izui et al, 2000).
- the inventors performed experiments on the strain Be.Slel ⁇ Bankl ' .yaa and used as control the B6.Slelz/zBankl +/+ .yaa littermate. The results show that the deficiency of BANKl does suppress the development of lupus phenotypes (FIGS. 9A-C).
- Serum 11-6 is reduced in lupus prone mice deficient for Bankl.
- lupus phenotypes is the increase in serum IL-6, which promotes IgG antibody production and inflammation.
- Darise it has been shown that the KO of IL-6 normalizes all the lupus phenotypes of the B6.Slel.yaa mouse.
- the inventors now show that lack of Bankl in this mouse led to a reduction of the in vivo serum levels of IL-6 excessively produced due to the presence of the yaa genomic segment, as detected in serum of the B6.Slel z/z Bankl "A .yaa mice (FIG. 10).
- deficiency of Bankl reduces the effects induced by the presence of the yaa translocation (or TLR7 duplication) supporting that Bankl has an effect on TLR7 signaling.
- Translation inhibitors can reduce the production of IL-6 induced by CpG in B cells from the BANKl Tg mouse.
- Pateamine A PanA
- PatA alters the activity of the mRNA helicase EIF4A, needed for the assembly of the 40s ribosome subunit (Cencic et al, 2012).
- inhibitors such as Cercosporamide or CGP57380, inhibit MNK1/2 (Altman et al, 2013), the kinase that following p38 activation, phosphorylates eIF4E. Inhibition of assembly of the complex by PatA at low doses has been shown to inhibit the production of pro-inflammatory cytokines (Di Marco et al, 2012; Gingras et al, 1999), but the effects of M K1 inhibition on IL-6 production, have never been shown in mice.
- FIG. 11 shows how 0.01 ⁇ Pateamine is non-toxic (right panel) and is capable of inhibiting IL-6 secretion following CpG stimulation in purified B cells from the transgenic BANKIFL-Tg mice.
- the transgenic animals were crossed onto the Bankl-/- mice in order to have the human trans gene without the expression of the endogenous murine Bankl .
- BANKl influences signaling leading to the formation of the translation initiation eIF4E complex following stimulation with CpG, and that BANKl deficiency results in reduction of the translation of the proinflammatory cytokine IL-6. They have also shown that mRNA stability of IL-6 is not affected, nor the second axis of regulation of translation initiation via AKT, which is also induced by CpG and strongly amplified with anti-CD40 treatment. Clearly, the combination of CpG and anti-CD40 treatment leads to stronger signaling of the AKT axis followed by strong activation of mTOR and 4E-BP1. However, absence of BANKl has no effect on the signaling pathway through this axis, except weak activation of AKT.
- BANKl The involvement of BANKl in modulating the IL-6 response after CpG stimulation has important implications in the pathogenesis of autoimmunity and viral infection.
- Sera from autoimmunity -prone animals and cerebrospinal fluid from SLE patients have elevated levels of IL-6 (Alcocer-Varela et al, 1992 and Stuart et al, 1995) and peripheral blood cells from SLE patients spontaneously secrete increased levels of IL-6 (Linker-Israeli et al, 1991).
- Autoimmunity is ameliorated by IL-6 ablation (Barr et al, 2012). It is also important to note that IL-6, coordinated with IL-21, has been reported to control the differentiation towards plasma cells (Eto et al, 2011). More recently, B cell derived IL-6 was shown to function in an autocrine manner and trigger receptor revision through re-expression of RAG in the post germinal center response (Yan et al, 2012).
- IL-6 has also important roles in infectious diseases. It has been reported that B cells release IL-6 to promote a follicular helper T cell (T FH ) response to viral infection. B cell- derived IL-6 was necessary and sufficient to induce IL-21 from CD4+ T cells in vitro and to support TFH cell development in vivo upon acute influenza virus infection (Karnowski et al, 2012). IL-6 is also involved in the induction but not maintenance of plasma cells (Cassese et al, 2003). BANKl deficient mice have normal T cell-dependent humoral responses following immunization with NP-CGG (Aiba et al, 2006).
- BANKl has been genetically associated with SLE and other autoimmune diseases, and here the inventors observe that BANKl deficiency decreased IL-6 secretion by altering the translation initiation pathway upon CpG stimulation. These results therefore suggest that BANKl could be involved in controlling disease development through the control of IL-6 secretion. While there are several mechanisms through which IL-6 production is regulated, it is clear that the production of IL-6 is controlled through a multitude of pathways and through different genes.
- BANKl was found to contain a conformational modular TIR domain at the N- terminus, similar to its relative, the molecule BCAP.
- BCAP is required for TLR-mediated activation of PI3K and AKT in macrophages (Troutman et al, 2012).
- CpG- induced AKT activation is normal in Bankl-/- B cells (FIG. 5A).
- BCAP rather than BANKl, may play a critical role in TLR-mediated activation of AKT, and further, BCAP and CD19 have complementary roles in BCR-mediated-PI3K activation (Aiba et al, 2008).
- BANKl appears to mediate AKT activation upon CD40 ligation in B cells (Aiba et al, 2006; FIG. 5B), these results support a specific role for BANKl in transducing CpG-induced signals via p38-MNKl/2 and the translation initiation factor eIF4E in B cells. Thus, BANKl and BCAP rather than playing redundant roles, appear to have very different ones.
- BANKl with a putative TIR domain, would be prone to bind molecules containing TIR domains, and explain the very specific role of BANKl in p38 signaling and translation initiation.
- BANKl is an adaptor molecule with a modular structure. It has up to 23 tyrosines susceptible of phosphorylation, and their substrate could alternate among a variety of molecules forming specific complexes during CpG-induced activation, different from those occurring following BCR-induced ligation, for instance. One of those complexes induced by CpG could include p38. How BANKl controls the p38-MNKl/2 pathway downstream of TLR9 is at present not known, but is a subject of study in the inventors' laboratory.
- BANKl shows an interaction with the Src tyrosine kinase BLK (1), and that BLK serves to promote the interaction between BANKl and phospholipase C y2, a key molecule in signal transduction during BCR ligation (Bernal- Quiros et al, 2013).
- This phenomenon is apparently not linked to CpG-induced signaling, as the inventors do not observe phosphorylation of BANKl or changes in binding of BANKl to BLK following CpG stimulation (data not shown).
- the inventors provide here a role for BANK1 in CpG-induced signaling in the production of IL-6, which could be highly relevant in the study of autoimmunity and inflammation.
- compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Urology & Nephrology (AREA)
- Public Health (AREA)
- Environmental Sciences (AREA)
- Zoology (AREA)
- Pharmacology & Pharmacy (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Epidemiology (AREA)
- Cell Biology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Marine Sciences & Fisheries (AREA)
- Rheumatology (AREA)
- Gastroenterology & Hepatology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Rehabilitation Therapy (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Abstract
The present invention involves the identification of the eIF4 machinery as a downstream effector of Bankl -mediated regulation of IL-6 expression. Thus, methods for treating patients suffering from autoimmune diseases such as systemic lupus erythematosus by targeting eIF4 function are provided.
Description
DESCRIPTION
TREATMENTS FOR SYSTEMIC LUPUS ERYTHEMATOSUS
This application claims benefit of priority to U.S. Provisional Application Serial No. 61/885,859, filed October 2, 2013, the entire contents of which are hereby incorporated by reference.
GOVERNMENT SUPPORT CLAUSE
This invention was made with government support under grant no. GM 103456 awarded by the National Institutes Health. The government has certain rights in the invention BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention relates generally to the fields of medicine, autoimmune disease and molecular biology. More particularly, it concerns use of pharmaceutical agents to treat systemic lupus erythematosus.
2. Description of Related Art
Systemic lupus erythematosus (SLE) is an autoimmune disease that wreaks havoc through the body's own immune system. The disease is estimated to affect nearly 1 million Americans, primarily women between the ages of 20-40. SLE has an incidence of about 1- in-700 women between the ages of 20 and 60. SLE can affect any organ system and can cause severe tissue damage.
SLE is associated with the production of antinuclear antibodies, circulating immune complexes, and activation of the complement system. Numerous autoantibodies of differing specificity are present in SLE. SLE patients often produce autoantibodies having anti-DNA, anti-Ro, and anti-platelet specificity and that are capable of initiating clinical features of the disease, such as glomerulonephritis, arthritis, serositis, complete heart block in newborns, and hematologic abnormalities. These autoantibodies are also possibly related to central nervous system disturbances.
Untreated lupus can be fatal as it progresses from attack of skin and joints to internal organs, including lung, heart, and kidneys (with renal disease being the primary concern). Lupus mainly appears as a series of flare-ups, with intervening periods of little or no disease
manifestation. Kidney damage, measured by the amount of proteinuria in the urine, is one of the most acute areas of damage associated with pathogenicity in SLE, and accounts for at least 50% of the mortality and morbidity of the disease.
Currently, there are no really curative treatments for patients who have been diagnosed with SLE. From a practical standpoint, physicians generally employ a number of powerful immunosuppressive drugs such as high-dose corticosteroids, e.g., prednisone, or azathioprine or cyclophosphamide, which are given during periods of flare-ups, but may also be given persistently for those who have experienced frequent flare-ups. Even with effective treatment, which reduces symptoms and prolongs life, many of these drugs have potentially harmful side effects to the patients being treated. In addition, these immunosuppressive drugs interfere with the person's ability to produce all antibodies, not just the self-reactive anti- DNA antibodies Immunosuppressants also weaken the body's defense against other potential pathogens, thereby making the patient extremely susceptible to infection and other potentially fatal diseases, such as cancer. In some of these instances, the side effects of current treatment modalities, combined with continued low-level manifestation of the disease, can cause serious impairment and premature death.
Methods for treatment of SLE involving antibodies are also described. For example, monoclonal antibodies against anti-DNA antibodies (the monoclonal antibodies being referred to therein as anti-idiotypic antibodies) are used to remove the pathogenic anti-DNA antibodies from the patient's system. However, the removal of large quantities of blood for treatment can be a dangerous, complicated process. High-dose intravenous immune globulin (IVIG) infusions have also been used in treating certain autoimmune diseases. Up until the present time, treatment of SLE with IVIG has provided mixed results, including both resolution of lupus nephritis (Akashi et al, 1990), and in a few instances, exacerbation of proteinuria and kidney damage (Jordan et al, 1989). In short, improved methods of treating SLE are desperately needed.
SUMMARY OF THE INVENTION
Thus, in accordance with the present disclosure, there is provided a method of treating a subject having systemic lupus erythematosus (SLE) comprising administering to said subject an inhibitor of eIF4 function. Administering may comprise intravenous administration, intra-arterial administration, oral administration or intramuscular administration. Administering may also include providing an organism to the subject that produces the inhibitor, such as lactobacillus. The method may further comprise administering to said subject one or more standard SLE therapies, such as a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody (e.g., Rituximab, Belimumab). The one or more standard therapies are provided before or after said inhibitor, or at the same time as said inhibitor. The inhibitor may be an eIF4E inhibitor, such as ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E. The inhibitor may be an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor. The inhibitor may be given more than once, including chronic administration.
Also provided is a method of reducing flare duration or severity in a subject having systemic lupus erythematosus (SLE) comprising administering to said subject an inhibitor of eIF4 function. Administering may comprise intravenous administration, intra-arterial administration, oral administration or intramuscular administration. Administering may also include providing an organism to the subject that produces the inhibitor, such as lactobacillus. The method may further comprise administering to said subject one or more standard SLE therapies, such as a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody (e.g., Rituximab, Belimumab). The one or more standard therapies are provided before or after said inhibitor, or at the same time as said inhibitor. The inhibitor may be an eIF4E inhibitor, such as ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E. The inhibitor may be an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor. The inhibitor may be given more than once, including chronic administration.
Other embodiments of the disclosure are discussed throughout this application. Any embodiment discussed with respect to one aspect of the disclosure applies to other aspects of the disclosure as well and vice versa. The embodiments in the Examples section are
understood to be embodiments of the disclosure that are applicable to all aspects of the disclosure.
The terms "inhibiting," "reducing," or "prevention," or any variation of these terms, when used in the claims and/or the specification includes any measurable decrease or complete inhibition to achieve a desired result.
The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."
It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosure, and vice versa. Furthermore, compositions and kits of the disclosure can be used to achieve methods of the disclosure.
Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.
The use of the term "or" in the claims is used to mean "and/or" unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive, although the disclosure supports a definition that refers to only alternatives and "and/or." It is also contemplated that anything listed using the term "or" may also be specifically excluded.
As used in this specification and claim(s), the words "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include") or "containing" (and any foam of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.
Other objects, features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the examples, while indicating specific embodiments of the disclosure, are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.
BRIEF DESCRIPTION OF THE DRAWINGS
The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.
FIGS. 1A-E. CpG-induced p38 MAPK activation is down-regulated in Bankl '' B cells. (FIG. 1A) Proliferation assay of CFSE-labeled splenic B cells from Bankl'1' mice and its littermate control (+/+) mice following 2 μΜ CpG or 20 μg/ml LPS stimulation for 48, 72 and 96h. Propidium iodide (Pl)-negative viable cells were gated and analyzed by flow cytometry. Data shown is representative of 3 independent experiments. (FIGS. 1B-C) Cell extracts from WT and Bankl KO splenic B cells stimulated with 10 μg/mL anti-mouse IgM F(ab')2 (anti-IgM) (FIG. IB) or 2 μΜ CpG (FIG. 1C) and analyzed by Western blot with specific antibodies for ERK, ΓΝΚ, p38 and Ι Β and the corresponding phospho-specific antibodies. (FIG. ID) Quantification of densitometry analysis of phospho-p38 Western blot data is shown as mean ± SEM. (FIG. IE) Cell extracts from splenic B cells stimulated with 20 μg/mL LPS and 1 μg/mL of the TLR7 agonist (R848) were analyzed by Western blot with specific antibodies to p38. WB data shown is representative of 2 independent experiments.
FIGS. 2A-C. CpG-induced IL-6 production is downregulated in Bankl '' B cells, but Tlr9 gene expression is not affected. (FIG. 2A) IL-6 and IL-10 were measured by ELISA of WT (+/+) and Bankl KO (_/~) B-cell supernatants collected at the indicated times following CpG, anti-IgM+ CpG or CpG+ anti-mouse CD40 (10 μg/ml). Data shown are mean ± SD of 3 replicates, and is representative of 4 independent experiments. (FIG. 2B) Splenic B cells from Bankl+I+ and Bankl'1' mice were stimulated with 2 μΜ CpG for the indicated times. Relative expression of the TLR9 gene was analyzed by Taqman real-time PCR. Data shown is the mean ± SEM of 3 replicates from a representative experiment out of 3 performed. (FIG. 2C) Bankl+/+ and Bankl''' splenic B cells were stimulated with LPS (20 μg/ml) or R848 (1 μg/mL), or left unstimulated for the times shown. Data are the representative of two independent experiments with 3 replicates each. Bars represent mean ± SD.
FIGS. 3A-D. MK2 and MNK1/2 inhibitors suppress IL-6 cytokine secretion in mouse splenic B cells. Mouse C57BL/6J (WT) splenic B cells (1 * 106 cells/ml) were seeded in triplicate in RPMI 1640 complete medium in a 48-well tissue culture plate. The cells were
pretreated with and without (FIG. 3 A) MK2 specific inhibitor, PF3644022 (1 and 10 μΜ) for an hour, (FIG. 3B) MNK1 inhibitor, CGP57380 (10 and 20 μΜ) for 30 min, and (FIG. 2C) MNKs inhibitor cercosporamide (20 and 50 μΜ) for an hour before stimulation of CpG (2 μΜ) for 24h. The control well contains CpG and cell culture grade DMSO (<0.2% v/v) only. The culture supernatants were collected after 24h of stimulation and the capture ELISA for IL-6 was performed. The culture supernatants were placed in triplicate in a 96-well ELISA plate and IL-6 ELISA was performed. Bars represent mean ± SD from two independent experiments. Unpaired t-test was performed and all the treated groups were compared with control, **p<0.001 and ***p<0.0001. (FIG. 3D) Viability of cells after above treatment was tested by propidium iodide (PI) labeling, the whole cells were gated and analyzed by flow cytometry, the figure shows (Pl)-negative viable cells.
FIGS. 4A-D. Reduced IL-6 secretion can be attributed to downregulation of the phosphorylation of the p38-MNKl/2-eIF4E cascade in Bankl''' B cells following CpG stimulation and not to IL-6 gene transcription and IL-6 mRNA stability. (FIG. 4A) Splenic B cells from Bankl+/+ and Bankl'1' mice were stimulated with 2 μΜ CpG for indicated time shown on the plot. Relative expression of the IL-6 gene was determined using Taqman RT-PCR. Data shown is the mean ± SEM of 3 replicates from 1 out of 3 independent experiments. (FIG. 4B) Splenic B cells from Bankl+/+ and Bankl'1' littermate mice were stimulated with 2 μΜ CpG for 20h followed by the addition of 1 μg/mL of actinomycin D (Act D). Relative expression of the IL-6 gene was analyzed with RT-PCR, and % of IL-6 mRNA was determined. Data shown is the mean of 3 replicates from one representative experiment out of three performed. The thin dotted line indicates 50% degradation of IL-6 mRNA. (FIG. 4C) Bank +/+ or _/~ splenic B cells were stimulated with 2 μΜ CpG for the indicated times and tested by Western blot using p-MNKl/2, MNK1/2, p-eIF4E, and eIF4E specific antibodies. The data are representative of three independent experiments. (FIG. 4D) Quantification analysis of phospho-MNKl/2 and phospho-eIF4E Western blot data by densitometry. The relative intensities of phospho-MNKl/2 and phospho-eIF4E were quantified using ImageJ software (freely available through the National Institutes of Health). The plots are from 3 independent experiments and data are expressed as mean ± SD.
FIGS. 5A-C. Bankl deficiency does not affect 4E-BP1 phosphorylation controlled through the AKT-mTORCl signaling cascade. Splenic B cells from Bankl+/+ and Bankl-/- littermate mice were stimulated with (FIG. 5A) CpG (2 μΜ), (FIG. 5B) anti- mouse CD40 (10 μg/ml), and (FIG. 5C) CpG (2 μΜ) in combination with anti-mouse CD40
(10 μg/ml) at 37 °C in a water bath for the indicated time points. Cytoplasmic cell extracts were prepared by using B cell lysis buffer containing appropriate amount of protease inhibitor and sodium vanadate. Equal amount of cell lysate protein (approx. 2x l06cell) was loaded in each lane and western blot was performed as written in the material and methods. The data are representative of two independent experiments. The relative intensities of the phospho-bands were quantified as described in FIGS. 4A-D.
FIGS. 6A-C. Cell viability and CFSE-labeled cell proliferation between Bankl+/+ and Bankl'A splenic B cells. CFSE-labeled splenic B cells were treated with the indicated stimuli for the indicated time, harvested and analyzed by flow cytometry. Debris and doublet cells were discriminated and viable cells (propidium iodide-negative) were gated. % of viability was determined. Histograms represent overlaid data from un-stimulated CFSE- labeled B cells, stimulated Bankl+/+ (solid line) and Bankf B cells (dash line).
FIGS. 7A-B. Immunoglobulin isotype switch or expression of BLIMP1 (Prdml) is not significantly affected by BANK1 deficiency. (FIG. 7A) Splenic B cells (l x lO6 cells/ml) from Bankl+/+ and BankPA littermate mice were seeded in RPMI 1640 complete medium in a 48 well tissue culture plate. The cells were either stimulated with CpG (2 μΜ) or left unstimulated for 6 days. The supernatants were analyzed for immunoglobulin isotype antibodies. The data are presented as O.D. except for IgG2a/2c where data are represented as ng/ml. Data are from 4 independent experiments. Bars represent mean ± SD. Significance of IgG2a/2c between Bankl+/+ and BankT1' was tested using an unpaired t-test. p<0.05% was considered significant. (FIG. 7B) The relative expression of Prdml was determined with Taqman RT-PCR. Data shown is the mean ± SEM of 3 replicates from one representative out of three independent experiments.
FIGS. 8A-B. Increased production of IL-6 by splenic purified B cells from BANKIFL-Tg with CpG or anti-CD40 + CpG stimulation. The cytokine IL-6 was measured with ELISA in supernatants of BANKIFL-Tg mice and compared to WT controls from purified B-cells collected at indicated times after CpG stimulation (FIG. 8A) or CpG and anti-CD40 stimulation (FIG. 8B). Data shown are mean ± SD of 3 replicates, and representative of 3 independent experiments.
FIGS. 9A-C. Deficiency of BANK1 modifies several phenotypes present in
B6.Slelz z.Yaa lupus prone mouse. (FIG. 9A) Serum from 25 week old Be.Slel^Bankl '' .yaa mice and genotype littermates heterozygous and sufficient for Bankl was measured for presence of IgG anti-dsDNA antibodies using ELISA in serial dilutions. Black circles show
positive control sera. (FIG. 9B) A pair of littermates of B6.Slelz zBank ~ .yaa and B6.Slelz/zBankl+M .yaa mice of 12 weeks of age was used in FIGS. 9B-C. Stark differences in populations of B and T cells in the spleen and lymph nodes between B6.Slel.yaa Bankl- sufficient and 5aw 7-deficient mice were observed. (FIG. 9C) Picture of the inguinal lymph nodes of the same mice as in FIG. 9B.
FIG. 10. Deficiency of Bankl restores IL-6 levels in serum of lupus-prone mice to normal. Serum from 25 week-old B6.Slelz/ 'Bankl'1' ' .yaa mice and genotype littermates sufficient for Bankl were measured for presence of IL-6 using ELISA (*p<0.05).
FIG. 11. Inhibitors of the translation initiation pathway are capable of inhibiting IL-6 production induced with CpG by BANK1 human transgenic B cells. Left panel - Twelve weeks old hBanklTg/wt.mBankl-/- female mice were used for total splenocyte isolation. 0.5 x 106 Spleen cells/500 μΐ CRPMI pre-treated with and without the MNK1 inhibitor CGP57380 at 5, 10 and 20 μΜ; and PatA 0.1 to 0.005 μΜ concentrations for 30 min. Then these cells were stimulated with and without CpG and in combination with anti-CD40 ligand for 24h. After stimulation, supernatants were checked for inhibition of IL-6 using ELISA, and cells were processed for staining and cell viability (right panel) checked with FACSCalibur. P values: ** p <0.01 ; **** p <0.0001.
DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
As discussed above, there remains a critical need for improved therapies for SLE. Because of the role of CpG-induced signaling in autoimmunity, and the putative role of BANK1 as a TIR-containing adaptor, the inventors hypothesized that BANK1 may participate in CpG-induced signaling. Their results established a function for BANK1 in CpG-induced responses that have important implications for the role of BANK1 in infections and autoimmunity, where BANK1 has been established as a susceptibility gene (Kozyrev et ah, 2008). In particular, the inventors have connected BANK1 function with IL-6 translation through the eIF4E pathway, and propose eIF4E as a target for treating SLE. These and other aspects of the disclosure are described in greater detail below.
I. Systemic Lupus Erythematosus
A. Disease Manifestations
Systemic lupus erythematosus (SLE) is a systemic autoimmune disease (or autoimmune connective tissue disease) that can affect any part of the body. The disease occurs nine times more often in women than in men, especially in women in child-bearing years ages 15 to 35, and is also more common in those of non-European descent.
As occurs in other autoimmune diseases, the immune system attacks the body's cells and tissue, resulting in inflammation and tissue damage. SLE can induce abnormalities in the adaptive and innate immune system, as well as mount Type III hypersensitivity reactions in which antibody-immune complexes precipitate and cause a further immune response. SLE most often damages the joints, skin, lungs, heart, blood components, blood vessels, kidneys, liver and nervous system. The course of the disease is unpredictable, often with periods of increased disease activity (called "flares") alternating with suppressed or decreased disease activity. A flare has been defined as a measurable increase in disease activity in one or more organ systems involving new or worse clinical signs and symptoms and/or laboratory measurements. It must be considered clinically significant by the assessor and usually there would be at least consideration of a change or an increase in treatment (Ruperto et ah, 2010).
SLE has no cure, and leads to increased morbidity and early mortality in many patients. The most common causes of death in lupus patients include accelerated cardiovascular disease (likely associated with increased inflammation and perhaps additionally increased by select lupus therapies), complications from renal involvement and infections. Survival for people with SLE in the United States, Canada, and Europe has risen
to approximately 95% at five years, 90% at 10 years, and 78% at 20 years in patients of European descent; however, similar improvements in mortality rates in non-Caucasian patients are not as evident. Childhood systemic lupus erythematosus generally presents between the ages of 3 and 15, with girls outnumbering boys 4: 1, and typical skin manifestations being butterfly eruption on the face and photosensitivity.
SLE is one of several diseases known as "the great imitators" because it often mimics or is mistaken for other illnesses. SLE is a classical item in differential diagnosis, because SLE symptoms vary widely and come and go unpredictably. Diagnosis can thus be elusive, with some people suffering unexplained symptoms of untreated SLE for years. Common initial and chronic complaints include fever, malaise, joint pains, myalgias, fatigue, and temporary loss of cognitive abilities. Because they are so often seen with other diseases, these signs and symptoms are not part of the American College of Rheumatology SLE classification criteria. When occurring in conjunction with other signs and symptoms, however, they are suggestive.
The most common clinical symptom which brings a patient for medical attention is joint pain, with the small joints of the hand and wrist usually affected, although nearly all joints are at risk. Between 80 and 90% of those affected will experience joint and/or muscle pain at some time during the course of their illness. Unlike rheumatoid arthritis, many lupus arthritis paitents will have joint swelling and pain, but no X-ray changes and minimal loss of function. Fewer than 10% of people with lupus arthritis will develop deformities of the hands and feet. SLE patients are at particular risk of developing articular tuberculosis. An association between osteoporosis and SLE has been found, and SLE may be associated with an increased risk of bone fractures in relatively young women.
Over half (65%) of SLE sufferers have some dermatological manifestations at some point in their disease, with approximately 30% to 50% suffering from the classic malar rash (or butterfly rash) associated with the name of the disorder. Some may exhibit chronic thick, annual scaly patches on the skin (referred to as discoid lupus). Alopecia, mouth ulcers, nasal ulcers, and photosensitive lesions on the skin are also possible manifestations. Anemia may develop in up to 50% of lupus cases. Low platelet and white blood cell counts may be due to the disease or as a side effect of pharmacological treatment. People with SLE may have an association with antiphospholipid antibody syndrome (a thrombotic disorder), wherein autoantibodies to phospholipids are present in their serum. Abnormalities associated with antiphospholipid antibody syndrome include a paradoxical prolonged partial thromboplastin time (which usually occurs in hemorrhagic disorders) and a positive test for antiphospholipid
antibodies; the combination of such findings has earned the term "lupus anticoagulant- positive." SLE patients with anti-phospholipid autoantibodies have more ACR classification criteria of the disease and may suffer from a more severe lupus phenotype.
A person with SLE may have inflammation of various parts of the heart, such as pericarditis, myocarditis, and endocarditis. The endocarditis of SLE is characteristically noninfective (Libman-Sacks endocarditis), and involves either the mitral valve or the tricuspid valve. Atherosclerosis also tends to occur more often and advances more rapidly than in the general population. Lung and pleura inflammation can cause pleuritis, pleural effusion, lupus pneumonitis, chronic diffuse interstitial lung disease, pulmonary hypertension, pulmonary emboli, pulmonary hemorrhage, and shrinking lung syndrome.
Painless hematuria or proteinuria may often be the only presenting renal symptom. Acute or chronic renal impairment may develop with lupus nephritis, leading to acute or end- stage renal failure. Because of early recognition and management of SLE, end-stage renal failure occurs in less than 5% of cases. A histological hallmark of SLE is membranous glomerulonephritis with "wire loop" abnormalities. This finding is due to immune complex deposition along the glomerular basement membrane, leading to a typical granular appearance in immunofluorescence testing.
Neuropsychiatric syndromes can result when SLE affects the central or peripheral nervous systems. The American College of Rheumatology defines 19 neuropsychiatric syndromes in systemic lupus erythematosus. The diagnosis of neuropsychiatric syndromes concurrent with SLE is one of the most difficult challenges in medicine, because it can involve so many different patterns of symptoms, some of which may be mistaken for signs of infectious disease or stroke. The most common neuropsychiatric disorder people with SLE have is headache, although the existence of a specific lupus headache and the optimal approach to headache in SLE cases remains controversial. Other common neuropsychiatric manifestations of SLE include cognitive dysfunction, mood disorder (including depression), cerebrovascular disease, seizures, polyneuropathy, anxiety disorder, cerebritis, and psychosis. CNS lupus can rarely present with intracranial hypertension syndrome, characterized by an elevated intracranial pressure, papilledema, and headache with occasional abducens nerve paresis, absence of a space-occupying lesion or ventricular enlargement, and normal cerebrospinal fluid chemical and hematological constituents. More rare manifestations are acute confusional state, Guillain-Barre syndrome, aseptic meningitis, autonomic disorder, demyelinating syndrome, mononeuropathy (which might manifest as mononeuritis multiplex), movement disorder (more specifically, chorea), myasthenia gravis, myelopathy, cranial
neuropathy and plexopathy. Neural symptoms contribute to a significant percentage of morbidity and mortality in patients with lupus. As a result, the neural side of lupus is being studied in hopes of reducing morbidity and mortality rates. The neural manifestation of lupus is known as neuropsychiatric systemic lupus erythematosus (NPSLE). One aspect of this disease is severe damage to the epithelial cells of the blood-brain barrier.
SLE causes an increased rate of fetal death in utero and spontaneous abortion (miscarriage). The overall live-birth rate in SLE patients has been estimated to be 72%. Pregnancy outcome appears to be worse in SLE patients whose disease flares up during pregnancy. Neonatal lupus is the occurrence of SLE symptoms in an infant born from a mother with SLE, most commonly presenting with a rash resembling discoid lupus erythematosus, and sometimes with systemic abnormalities such as heart block or hepatosplenomegaly. Neonatal lupus is usually benign and self-limited.
Fatigue in SLE is probably multifactorial and has been related to not only disease activity or complications such as anemia or hypothyroidism, but also to pain, depression, poor sleep quality, poor physical fitness and lack of social support.
Different clinical measurements have been used to determine whether a SLE patients is having a clinic flare. One of the most common measurements is the Systemic Lupus Erythematosus Disease Activity Index SELENA Modification (world-wide-web at rheumatology.org/Practice/Clinical/Indexes/Systemic_Lupus_Erythematosus_Disease_Activi ty_Index_SELENA_Modification/). This scale uses a point system to calculate when the accumulated significance of recent changes in various indicators translates into a mild/moderate (SLEDA Index of 3-1 1 point change) or a severe (12 of more point change) flare. Although helpful in defining clinical flares in therapeutic and observational SLE clinical trials, this information only defines a flare state and does not help predict or identify patients who likely have an impending flare (an important clinical problem). In addition, no consensus, objective molecular test or tests are consistently associated individually with increased disease activity, nor with imminent SLE disease flare. Having such a molecular test would be greatly beneficial to SLE clinical care to help guide therapy, prevent damage, and minimize therapeutic toxicity.
B. Diagnosis
Antinuclear antibody (ANA) testing and anti-extractable nuclear antigen (anti-ENA) responses form the mainstay of SLE serologic testing. Several techniques are used to detect ANAs. Clinically the most widely used method is indirect immunofluorescence. The pattern
of fluorescence suggests the type of antibody present in the patient's serum. Direct immunofluorescence can detect deposits of immunoglobulins and complement proteins in the patient's skin. When skin not exposed to the sun is tested, a positive direct IF (the so-called Lupus band test) is an evidence of systemic lupus erythematosus.
ANA screening yields positive results in many connective tissue disorders and other autoimmune diseases, and may occur in healthy individuals. Subtypes of antinuclear antibodies include anti-Smith and anti-double-stranded DNA (dsDNA) antibodies (which are linked to SLE) and anti-histone antibodies (which are linked to drug-induced lupus). Anti- dsDNA antibodies are relatively specific for SLE; they are present in up to 50% of cases depending on ethnicity, whereas they appear in less than 2% of people without SLE. The anti-dsDNA antibody titers also tend to reflect disease activity, although not in all cases. Other ANA that may occur in SLE sufferers are anti-Ul RNP (which also appears in systemic sclerosis), anti-Ro (or anti-SSA) and anti-La (or anti-SSB; both of which are more common in Sj5gren's syndrome). anti-Ro and anti-La, when present in the maternal circulation, confer an increased risk for heart conduction block in neonatal lupus. Other tests routinely performed in suspected SLE are complement system levels (low levels suggest consumption by the immune system), electrolytes and renal function (disturbed if the kidneys are involved), liver enzymes, and complete blood count. II. IL-6, BANK1 and eIF4
A. IL-6
Interleukin 6 (IL-6) is an interleukin that acts as both a pro-inflammatory and an antiinflammatory cytokine. In humans, it is encoded by the IL-6 gene. IL-6 is secreted by T cells and macrophages to stimulate immune response, e.g., during infection and after trauma, especially burns or other tissue damage leading to inflammation. IL-6 also plays a role in fighting infection, as IL-6 has been shown in mice to be required for resistance against bacterium Streptococcus pneumoniae.
IL-6 is also considered a myokine, a cytokine produced from muscle, and is elevated in response to muscle contraction. It is significantly elevated with exercise, and precedes the appearance of other cytokines in the circulation. During exercise, it is thought to act in a hormone-like manner to mobilize extracellular substrates and/or augment substrate delivery. In addition, osteoblasts secrete IL-6 to stimulate osteoclast formation. Smooth muscle cells in the tunica media of many blood vessels also produce IL-6 as a pro-inflammatory cytokine.
IL-6's role as an anti-inflammatory cytokine is mediated through its inhibitory effects on
TNF-alpha and IL-1, and activation of IL-lra and IL-10.
IL-6 is one of the most important mediators of fever and of the acute phase response.
It is capable of crossing the blood-brain barrier and initiating synthesis of PGE2 in the hypothalamus, thereby changing the body's temperature setpoint. In muscle and fatty tissue,
IL-6 stimulates energy mobilization that leads to increased body temperature. IL-6 can be secreted by macrophages in response to specific microbial molecules, referred to as pathogen-associated molecular patterns (PAMPs). These PAMPs bind to a highly important group of detection molecules of the innate immune system, called pattern recognition receptors (PRRs), including Toll-like receptors (TLRs). These are present on the cell surface and intracellular compartments and induce intracellular signaling cascades that give rise to inflammatory cytokine production.
IL-6 is also essential for hybridoma growth and is found in many supplemental cloning media such as briclone. Inhibitors of IL-6 (including estrogen) are used to treat postmenopausal osteoporosis. IL-6 is also produced by adipocytes and is thought to be a reason why obese individuals have higher endogeneous levels of CRP. Intranasally administered IL-6 has been shown to improve sleep-associated consolidation of emotional memories.
IL-6 is responsible for stimulating acute phase protein synthesis, as well as the production of neutrophils in the bone marrow. It supports the growth of B cells and is antagonistic to regulatory T cells.
IL-6 signals through a cell-surface type I cytokine receptor complex consisting of the ligand-binding IL-6Ra chain (CD 126), and the signal-transducing component gpl30 (also called CD130). CD130 is the common signal transducer for several cytokines including leukemia inhibitory factor (LIF), ciliary neurotropic factor, oncostatin M, IL-11 and cardiotrophin-1, and is almost ubiquitously expressed in most tissues. In contrast, the expression of CD 126 is restricted to certain tissues. As IL-6 interacts with its receptor, it triggers the gpl30 and IL-6R proteins to form a complex, thus activating the receptor. These complexes bring together the intracellular regions of gpl30 to initiate a signal transduction cascade through certain transcription factors, Janus kinases (JAKs) and Signal Transducers and Activators of Transcription (STATs).
IL-6 is probably the best-studied of the cytokines that use gpl30, also known as IL-6 signal transducer (IL6ST), in their signalling complexes. Other cytokines that signal through receptors containing gpl30 are Interleukin 1 1 (IL-1 1), Interleukin 27 (IL-27), ciliary
neurotrophic factor (CNTF), cardiotrophin- 1 (CT-1), cardiotrophin-like cytokine (CLC), leukemia inhibitory factor (LIF), oncostatin M (OSM), Kaposi's sarcoma-associated herpesvirus interleukin 6-like protein (KSHV-IL6). These cytokines are commonly referred to as the IL-6 like or gpl30 utiliing cytokines.
In addition to the membrane-bound receptor, a soluble form of IL-6R (sIL-6R) has been purified from human serum and urine. Many neuronal cells are unresponsive to stimulation by IL-6 alone, but differentiation and survival of neuronal cells can be mediated through the action of sIL-6R. The sIL-6R/IL-6 complex can stimulate neurites outgrowth and promote survival of neurons and, hence, may be important in nerve regeneration through remyelination.
IL-6 is relevant to many diseases such as diabetes, atherosclerosis, depression, Alzheimer's Disease, systemic lupus erythematosus, multiple myeloma, prostate cancer, Behcet's disease, and rheumatoid arthritis. Advanced/metastatic cancer patients have higher levels of IL-6 in their blood. Hence there is an interest in developing anti-IL-6 agents as therapy against many of these diseases. The first such is tocilizumab, which has been approved for rheumatoid arthritis. Another, ALD518, is in clinical trials.
B. BANK1
Human and mouse Bankl encode the B cell scaffold protein with ankyrin repeats ! (BANK1). BANK1 is a tyrosine kinase substrate that becomes extensively tyrosine phosphorylated and is capable of binding the Src family kinases Lyn and Blk (Castillejo- Lopez et ah, 2012; Yokoyama et ah, 2002) and promotes tyrosine phosphorylation of inositol 1,4,5-trisphosphate receptors. While apparently involved in BCR signaling, the function of BANK1 during signaling induced by CpG, an agonist of the major toll-like receptor, TLR9 expressed in B cells, is not known. It also is involved in B-cell receptor induced Ca2+ mobilization from intracellular stores and promotes Lyn-mediated phosphorylation of IP3 receptors 1 and 2.
The gene is located on the long arm of chromosome 4 (4q24) on the Watson (plus) strand and is 284,206 bases in length. The gene encodes a protein of 785 amino acids (molecular weight 89.335 kDa) and four isoforms are known. The gene is expressed in B-cell but not T-cell or myeloid cell lines. The greatest expression is in CD19+ B-cells with very low expression in other cell populations.
It has been proposed that BANK1 acts as an adaptor or scaffold protein in the same family as the B cell adapter for PI3K (BCAP) and the Drosophila homologue Dof
(Yokoyama et al, 2002). Consistent with this hypothesis, the inventors' recent studies have shown that exon 2 of human BANK1 encodes a highly hydrophobic domain, which renders the protein susceptible to aggregation (Kozyrev et al, 2012); scaffold and adaptor proteins are known to form complex structures to facilitate intracellular signaling at the proper time and differentiation stage. Furthermore, exon 2 also encodes a predicted N-terminal toll/IL-1 receptor (TIR) domain that is shared by BCAP (Troutman et al, 2012) and used in the interaction of BCAP with the adaptors MyD88 and TIRAP.
TLR9 is the major endosomal TLR in B cells that recognizes viral nucleic acids, and TLR9 signaling is believed to have an important role in autoimmunity (Christensen et al, 2005). TLR9 signaling is stimulated by hypomethylated DNA oligonucleotides or CpG (Bernasconi et al, 2003), leading to a pro-inflammatory response (Sun et al, 2007). CpG- induced signaling activates mitogen activated protein kinase (MAPK) pathways, including p38, JNK and ERK. Stimulation of p38 and ERK signaling by growth factors, stress or viral infections can induce transcriptional activation, but can also induce two pathways of post- transcriptional regulation of protein synthesis: (a) control of mRNA stabilization (Neininger et al, 2002 and Bollig et al, 2003) by the mitogen activated protein kinase-activated protein kinase 2 (MAPKAP kinase) MK2, and (b) the transient formation of the heterotrimeric eIF4E/eIF4F/eIF4G translation initiation complex through phosphorylation of eIF4E (Banerjee et al, 2002; Morley, 1997).
In mice, the only kinases known to phosphorylate eIF4E are MNK1 and MNK2.
MNK2 is constitutively active, while MNK1 is regulated by the MAP kinases (Sonenberg, 2008). A second axis of control of eIF4E activation is through the AKT/mTORCl pathway. This pathway regulates the phosphorylation of 4E-BP1, the eIF4E binding protein. Under non-phosphorylated conditions, 4E-BP 1 retains eIF4E (Gingras et al, 1999 and Richter and Sonenberg, 2005). Once 4E-BP1 becomes phosphorylated by mTORCl, it releases eIF4E, which is in turn phosphorylated by MNK1/2 (Livingstone et al, 2009).
C. eIF4
Eukaryotic initiation factor (elF) complex 2 forms a ternary complex with GTP and the initiator Met-tRNA - this process is regulated by guanine nucleotide exchange and phosphorylation and serves as the main regulatory element of the bottleneck of protein expression. Before translation can progress to the elongation stage, a number of initiation factors must facilitate the synergy of the ribosome and the mRNA and ensure that the 5' UTR of the mRNA is sufficiently devoid of secondary structure. Binding in this way is facilitated
by group 4 eukaryotic initiation factors; eIF4 has implications in the normal regulation of translation as well as the transformation and progression of cancerous cells; as such, it represents an interesting field of research.
Eukaryotic translation initiation factor 4E, also known as eIF4E, is a protein that in humans is encoded by the EIF4E gene. eIF4E is a eukaryotic translation initiation factor involved in directing ribosomes to the cap structure of mRNAs. It is a 24-kD polypeptide that exists as both a free form and as part of a multiprotein complex termed eIF4F. The eIF4E polypeptide is the rate-limiting component of the eukaryotic translation apparatus and is involved in the mRNA-ribosome binding step of eukaryotic protein synthesis. The other subunits of eIF4F are (a) a 50-kD polypeptide, termed eIF4A, that possesses ATPase and RNA helicase activities, and (b) a 220-kD polypeptide, eIF4G. eIF4E has been shown to interact with eIF4Al, eIF4EBP3, eIF4EBP l, eIF4EBP2, eukaryotic translation initiation factor 4γ, eIF4G2 and eIF4ENIF l.
eIF4E's function is to bind an mRNA cap and ultimately bring it to the ribosome. eIF4E is part of the eIF4F pre-initiation complex, which is made up of eIF4E, and eIF4G (eIF4F is sometimes considered to have additional protein components). Almost all cellular proteins require eIF4E in order to be translated into protein. eIF4E binds the first nucleotide on the 5' end of an mRNA molecule (known as the cap): a 7 methyl guanosine (m7G). It sandwiches m7G between 2 tryptophan residues, and other amino acids are involved in the binding.
Some viruses cut eIF4G in such a way that the eIF4E binding site is removed and the virus is able to translate its proteins without eIF4E. Also, some cellular proteins (the most notable being heat shock proteins) do not require eIF4E in order to be translated. Both viruses and cellular proteins achieve this through an IRES structure in the RNA.
Fragile X mental retardation protein (FMR1) acts to regulate translation of specific mRNAs through its binding of eIF4E. FMRP acts by binding CYFIP 1 , which directly binds eIF4e at a domain that is structurally similar to those found in 4E-BPs including EIF4EBP3, EIF4EBP 1, and EIF4EBP2. The FMRP/CYFIP 1 complex binds in such a way as to prevent the eIF4E-eIF4G interaction, which is necessary for translation (biology) to occur. The FMRP/CYFIP l/eIF4E interaction is strengthened by the presence of mRNA(s). In particular, BC1 RNA allows for an optimal interaction between FMRP and CYFIP 1. RNA-BC1 is a non-translatable, dendritic mRNA, which binds FMRP to allow for its association with a specific target mRNA. BC1 may function to regulate FMRP and mRNA interactions at synapse(s) through its recruitment of FMRP to the appropriate mRNA. In addition, FMRP
may recruit CYFIP 1 to specific mRNAs in order to repress translation. The FMRP-CYFIP 1 translational inhibitor is regulated by stimulation of neuron(s). Increased synaptic stimulation resulted in the dissociation of eIF4E and CYFIP 1, allowing for the initiation of translation. III. Treating SLE
A. Treatments Targeting eIF4E
The present disclosure contemplates the use of inhibitors of eIF4 to treat SLE. In general, there are two categories of inhihitors - those that inhibit eIF4A/E/G expression, and those that do not inhibit expression but instead inhibit the activity of eIF4A/E/G. As discussed below, inhibitors may be biological - nucleic acid or peptides - or pharmaceutical (i.e., small molecules).
1. Biological Inhibitors
Antisense Constructs. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with "complementary" sequences. By complementary, it is meant that polynucleotides are those which are capable of base-pairing according to the standard Watson-Crick complementarity rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6- methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.
Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation; targeting RNA will lead to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense RNA constructs, or DNA encoding such antisense RNA's, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.
Antisense constructs may be designed to bind to the promoter and other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes an antisense construct with complementarity to regions within 50-200 bases of an intron-exon splice junction. It has
been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.
As stated above, "complementary" or "antisense" means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme; see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions.
It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.
Ribozymes. Another general class of inhibitors is ribozymes. Although proteins traditionally have been used for catalysis of nucleic acids, another class of macromolecules has emerged as useful in this endeavor. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987; Gerlach et ah, 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Cook et ah, 1981 ; Michel and Westhof, 1990; Reinhold- Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate bind via specific base-pairing interactions to the internal guide sequence ("IGS") of the ribozyme prior to chemical reaction.
Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989; Cook et ah, 1981). For
example, U.S. Patent 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al, 1991; Sarver et al, 1990). It has also been shown that ribozymes can elicit genetic changes in some cells lines to which they were applied; the altered genes included the oncogenes H-ras, c-fos and genes of HIV. Most of this work involved the modification of a target mRNA, based on a specific mutant codon that was cleaved by a specific ribozyme.
RNAi. RNA interference (also referred to as "RNA-mediated interference" or RNAi) is another mechanism by which protein expression can be reduced or eliminated. Double- stranded RNA (dsRNA) has been observed to mediate the reduction, which is a multi-step process. dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp et al, 2000; Tabara et al, 1999). Activation of these mechanisms targets mature, dsRNA-complementary mRNA for destruction. RNAi offers major experimental advantages for study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene (Fire et al, 1998; Grishok et al, 2000; Ketting et al, 1999; Lin et al, 1999; Montgomery et al, 1998; Sharp, 1999; Sharp et al, 2000; Tabara et al, 1999). Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma, Drosophila, and mammals (Grishok et al, 2000; Sharp, 1999; Sharp et al, 2000; Elbashir et al, 2001). It is generally accepted that RNAi acts post-transcriptionally, targeting RNA transcripts for degradation, and possibly by inhibiting translation. It appears that both nuclear and cytoplasmic RNA can be targeted (Bosher et al, 2000).
siRNAs must be designed so that they are specific and effective in suppressing the expression of the genes of interest. Methods of selecting the target sequences, i.e. those sequences present in the gene or genes of interest to which the siRNAs will guide the degradative machinery, are directed to avoiding sequences that may interfere with the siRNA's guide function while including sequences that are specific to the gene or genes. Typically, siRNA target sequences of about 21 to 23 nucleotides in length are most effective. This length reflects the lengths of digestion products resulting from the processing of much longer RNAs as described above (Montgomery et al, 1998). Of particular interest are those siRNAs that span an exon-intron junction.
The making of siRNAs has been mainly through direct chemical synthesis; through processing of longer, double-stranded R As through exposure to Drosophila embryo lysates; or through an in vitro system derived from S2 cells. Use of cell lysates or in vitro processing may further involve the subsequent isolation of the short, 21-23 nucleotide siRNAs from the lysate, etc., making the process somewhat cumbersome and expensive. Chemical synthesis proceeds by making two single-stranded RNA-oligomers followed by the annealing of the two single-stranded oligomers into a double-stranded RNA. Methods of chemical synthesis are diverse. Non-limiting examples are provided in U.S. Patents 5,889, 136, 4,415,732, and 4,458,066, expressly incorporated herein by reference, and in Wincott et al. (1995).
Several further modifications to siRNA sequences have been suggested in order to alter their stability or improve their effectiveness. It is suggested that synthetic complementary 21-mer RNAs having di-nucleotide overhangs (i.e., 19 complementary nucleotides + 3' non-complementary dimers) may provide the greatest level of suppression. These protocols primarily use a sequence of two (2'-deoxy)thymidine nucleotides as the di- nucleotide overhangs. These dinucleotide overhangs are often written as dTdT to distinguish them from the typical nucleotides incorporated into RNA. The literature has indicated that the use of dT overhangs is primarily motivated by the need to reduce the cost of the chemically synthesized RNAs. It is also suggested that the dTdT overhangs might be more stable than UU overhangs, though the data available shows only a slight (<20%) improvement of the dTdT overhang compared to an siRNA with a UU overhang.
Chemically synthesized siRNAs are found to work optimally when they are in cell culture at concentrations of 25-100 nM. This had been demonstrated by Elbashir et al. (2001) wherein concentrations of about 100 nM achieved effective suppression of expression in mammalian cells. siRNAs have been most effective in mammalian cell culture at about 100 nM. In several instances, however, lower concentrations of chemically synthesized siRNA have been used (Caplen et al, 2000; Elbashir et al, 2001).
WO 99/32619 and WO 01/68836 suggest that RNA for use in siRNA may be chemically or enzymatically synthesized. Both of these texts are incorporated herein in their entirety by reference. The enzymatic synthesis contemplated in these references is by a cellular RNA polymerase or a bacteriophage RNA polymerase (e.g., T3, T7, SP6) via the use and production of an expression construct as is known in the art. See U.S. Patent 5,795,715. The contemplated constructs provide templates that produce RNAs that contain nucleotide sequences identical to a portion of the target gene. The length of identical sequences provided by these references is at least 25 bases, and may be as many as 400 or more bases in length.
An important aspect of this reference is that the authors contemplate digesting longer dsRNAs to 21-25 mer lengths with the endogenous nuclease complex that converts long dsRNAs to siRNAs in vivo. They do not describe or present data for synthesizing and using in vitro transcribed 21-25 mer dsRNAs. No distinction is made between the expected properties of chemical or enzymatically synthesized dsRNA in its use in RNA interference.
Similarly, WO 00/44914, incorporated herein by reference, suggests that single strands of RNA can be produced enzymatically or by partial/total organic synthesis. Preferably, single-stranded RNA is enzymatically synthesized from the PCR® products of a DNA template, preferably a cloned cDNA template and the RNA product is a complete transcript of the cDNA, which may comprise hundreds of nucleotides. WO 01/36646, incorporated herein by reference, places no limitation upon the manner in which the siRNA is synthesized, providing that the RNA may be synthesized in vitro or in vivo, using manual and/or automated procedures. This reference also provides that in vitro synthesis may be chemical or enzymatic, for example using cloned RNA polymerase (e.g., T3, T7, SP6) for transcription of the endogenous DNA (or cDNA) template, or a mixture of both. Again, no distinction in the desirable properties for use in RNA interference is made between chemically or enzymatically synthesized siRNA.
U.S. Patent 5,795,715 reports the simultaneous transcription of two complementary DNA sequence strands in a single reaction mixture, wherein the two transcripts are immediately hybridized. The templates used are preferably of between 40 and 100 base pairs, and which is equipped at each end with a promoter sequence. The templates can be attached to a solid surface. After transcription with RNA polymerase, the resulting dsRNA fragments may be used for detecting and/or assaying nucleic acid target sequences.
In a specific embodiment, the inventors propose to inhibit eIF4A/E/G expression in adult tissues in vitro using siRNA or shRNA in an adenoviral vector. A GFP marker can be utilized to determine cells that take up the vector, and thus permit checking for appropriate inhibition of eIF4A/E/G production. The use of an inducible promoter allows for induction of the siRNA or shRNA only under specific growth conditions, thereby permitting reversible inhibition of eIF4A/E/G.
Peptide Fragments. The present invention contemplates the design, production and use of various eIF4A/E/G peptides. In general, the peptides will be 50 residues or less, again, comprising no more than 20 consecutive residues of eIF4A/E/G. The overall length may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues. Ranges of
peptide length of 4-50 residues, 5-50 residues, 6-50 residues, 7-50 residues, 7-25, residues, 4- 20 residues, 5-20 residues, 6-20 residues, 7-20 residues, and 7-15 residues are contemplated. The number of consecutive eIF4A/E/G residues may be 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20. Ranges of consecutive residues of 4-20 residues, 5-20 residues, 6-20 residues, 7-20 residues and 4-15 residues, 5-15, residues, 6-15 residues or 7-15 residues are contemplated.
The present invention may utilize L-configuration amino acids, D-configuration amino acids, or a mixture thereof. While L-amino acids represent the vast majority of amino acids found in proteins, D-amino acids are found in some proteins produced by exotic sea- dwelling organisms, such as cone snails. They are also abundant components of the peptidoglycan cell walls of bacteria. D-serine may act as a neurotransmitter in the brain. The L and D convention for amino acid configuration refers not to the optical activity of the amino acid itself, but rather to the optical activity of the isomer of glyceraldehyde from which that amino acid can theoretically be synthesized (D-glyceraldehyde is dextrorotary; L- glyceraldehyde is levorotary).
One form of an "all-D" peptide is a retro-inverso peptide. Retro-inverso modification of naturally-occurring polypeptides involves the synthetic assemblage of amino acids with a- carbon stereochemistry opposite to that of the corresponding L-amino acids, i.e., D-amino acids in reverse order with respect to the native peptide sequence. A retro-inverso analogue thus has reversed termini and reversed direction of peptide bonds ( H-CO rather than CO- NH) while approximately maintaining the topology of the side chains as in the native peptide sequence. See U.S. Patent 6,261,569, incorporated herein by reference.
As mentioned above, the present invention contemplates fusing or conjugating a cell delivery domain (also called a cell delivery vector, or cell transduction domain). Such domains are well known in the art and are generally characterized as short amphipathic or cationic peptides and peptide derivatives, often containing multiple lysine and arginine resides (Fischer, 2007). Of particular interest are poly-D-Arg and poly-D-Lys sequences (e.g., dextrorotary residues, eight residues in length), while others are shown in Table 1, below.
TABLE 1
CDD/CTD PEPTIDES SEQ ID NO
QAATATRG SAASRPTERPRAPARSASRPRRPVE 5
RQIKI WF QNRRMKWKK 6
RRMKWKK 7
RRWRRWWRRWWRRWRR 8
RGGRLSYSRRRFSTSTGR 9
YGRK RRQRRR 10
RKKRRQRRR 11
YARAAARQARA 12
RRRRRRRR 13
KKKKKKKK 14
GWTLNSAGYLLGKINLKALAALAKXIL 15
LLILLRRRIR Q ANAH SK 16
SRRHHCRSKAKRSRHH 17
NRARRNRRRVR 18
RQLRIAGRRLRGRSR 19
KLIKGRTPIKFGK 20
RRIPNRRPRR 21
KLALKLALKALKAALKLA 22
KLAKLAKKLAKLAK 23
GALFLGFLGAAGSTNGAWSQPKKKRKV 24
KETWWETWWTEWSQPKKKRKV 25
GALFLGWLGAAGSTMGAKKKRKV 26
MGLGLHLLVLAAALQGAKSKRKV 27
AAVALLPAVLLALLAPAAANYKKPKL 28
MANLGYWLLALFVTMWTDVGLCKKRPKP 29
LGTYTQDFNKFHTFPQTAIGVGAP 30
DP GDP GVTVTVTVTVTG GDPXPD 31
PPPPPPPPPPPPPP 32
VRLPPPVRLPPPVRLPPP 33
PRPLPPPRPG 34
SVRRRPRPPYLPRPRPPPFFPPRLPPRIPP 35
TRSSRAGLQFPVGRVHRLLR 36
GIGKFLHSAKKFGKAFVGEIMNS 37
KWKLFKKIEKVGQNIRDGIIKAGPAVAVVGQATQIAK 38
ALWMTLLKKVLKAAAKAALNAVLVGANA 39
GIGAVL VLTTGLPALISWI RKRQQ 40
INLKALAALAKKIL 41
GFFALIP IISSPLP TLLSAVGSALGGSGGQE 42
LAKWALKQGFAKLKS 43
SMAQDIISTIGDLVKWIIQTVNXFTKK 44
LLGDFFRKSKEKIGKEFKRIVQRIKQRIKDFLANLVPRTES 45
LKKLLKKLLKKLLKKLLKKL 46
KLKLKLKLKLKLKLKLKL 47
PAWRKAFRWAWRMLKKAA 48
Also as mentioned above, peptides modified for in vivo use by the addition, at the amino- and/or carboxyl-terminal ends, of a blocking agent to facilitate survival of the peptide in vivo are contemplated. This can be useful in those situations in which the peptide termini tend to be degraded by proteases prior to cellular uptake. Such blocking agents can include, without limitation, additional related or unrelated peptide sequences that can be attached to the amino and/or carboxyl terminal residues of the peptide to be administered. These agents
can be added either chemically during the synthesis of the peptide, or by recombinant DNA technology by methods familiar in the art. Alternatively, blocking agents such as pyroglutamic acid or other molecules known in the art can be attached to the amino- and/or carboxyl-terminal residues.
It will be advantageous to produce peptides using the solid-phase synthetic techniques
(Merrifield, 1963). Other peptide synthesis techniques are well known to those of skill in the art (Bodanszky et al, 1976; Peptide Synthesis, 1985; Solid Phase Peptide Synthelia, 1984). Appropriate protective groups for use in such syntheses will be found in the above texts, as well as in Protective Groups in Organic Chemistry, 1973. These synthetic methods involve the sequential addition of one or more amino acid residues or suitable protected amino acid residues to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid residue is protected by a suitable, selectively removable protecting group. A different, selectively removable protecting group is utilized for amino acids containing a reactive side group, such as lysine.
Using solid phase synthesis as an example, the protected or derivatized amino acid is attached to an inert solid support through its unprotected carboxyl or amino group. The protecting group of the amino or carboxyl group is then selectively removed and the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected is admixed and reacted with the residue already attached to the solid support. The protecting group of the amino or carboxyl group is then removed from this newly added amino acid residue, and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been linked in the proper sequence, any remaining terminal and side group protecting groups (and solid support) are removed sequentially or concurrently, to provide the final peptide. The peptides of the invention are preferably devoid of benzylated or methylbenzylated amino acids. Such protecting group moieties may be used in the course of synthesis, but they are removed before the peptides are used. Additional reactions may be necessary, as described elsewhere, to form intramolecular linkages to restrain conformation.
Aside from the 20 standard amino acids can can be used, there are a vast number of "non-standard" amino acids. Two of these can be specified by the genetic code, but are rather rare in proteins. Selenocysteine is incorporated into some proteins at a UGA codon, which is normally a stop codon. Pyrrolysine is used by some methanogenic archaea in enzymes that they use to produce methane. It is coded for with the codon UAG. Examples of non-standard amino acids that are not found in proteins include lanthionine, 2-aminoisobutyric acid,
dehydroalanine and the neurotransmitter gamma-aminobutyric acid. Non-standard amino acids often occur as intermediates in the metabolic pathways for standard amino acids - for example ornithine and citrulline occur in the urea cycle, part of amino acid catabolism. Nonstandard amino acids are usually formed through modifications to standard amino acids. For example, homocysteine is formed through the transsulfuration pathway or by the demethylation of methionine via the intermediate metabolite S-adenosyl methionine, while hydroxyproline is made by a posttranslational modification of proline.
Linkers or cross-linking agents may be used to fuse peptides to other proteinaceous sequences. Bifunctional cross-linking reagents have been extensively used for a variety of purposes including preparation of affinity matrices, modification and stabilization of diverse structures, identification of ligand and receptor binding sites, and structural studies. Homobifunctional reagents that carry two identical functional groups proved to be highly efficient in inducing cross-linking between identical and different macromolecules or subunits of a macromolecule, and linking of polypeptide ligands to their specific binding sites. Heterobifunctional reagents contain two different functional groups. By taking advantage of the differential reactivities of the two different functional groups, cross-linking can be controlled both selectively and sequentially. The bifunctional cross-linking reagents can be divided according to the specificity of their functional groups, e.g., amino-, sulfhydryl-, guanidino-, indole-, or carboxyl-specific groups. Of these, reagents directed to free amino groups have become especially popular because of their commercial availability, ease of synthesis and the mild reaction conditions under which they can be applied. A majority of heterobifunctional cross-linking reagents contains a primary amine-reactive group and a thiol-reactive group.
In another example, heterobifunctional cross-linking reagents and methods of using the cross-linking reagents are described in U.S. Patent 5,889, 155, specifically incorporated herein by reference in its entirety. The cross-linking reagents combine a nucleophilic hydrazide residue with an electrophilic maleimide residue, allowing coupling in one example, of aldehydes to free thiols. The cross-linking reagent can be modified to cross-link various functional groups and is thus useful for cross-linking polypeptides. In instances where a particular peptide does not contain a residue amenable for a given cross-linking reagent in its native sequence, conservative genetic or synthetic amino acid changes in the primary sequence can be utilized.
Dominant negative mutants. Dominant negative proteins are mutant defective proteins with can negate the effects of normal, functional proteins when both are present in
the same environment. In many cases, dominant negative proteins homo-multimerize and are thus able to "poison" a complext that contains one or more functional proteins. In designing dominant negative molecules, one may design molecules that interact with one binding partner but fail to interact with another, such as due to a conformational change or single site mutation or trunctation. Another option is to design a molecule that binds normally, but lacks a subsequent function such as an enzymatic activity (phosphorylation, cleavage, etc.). Yet another option is to remove a sequence from the molecule that would permit its translocation to another portion of the cell. Other possibilities also exist. 2. Pharmaceutical Inhibitors
An inhibitor of eIF4A function is hippuristinol, a small molecule found in the coral Ms hippuris. Its structure is shown below:
Another inhibitor of eIF4A is pateamine A (PatA), a natural product first isolated from marine sponges. This compound has attracted considerable attention as a potential anticancer agent. Recently, researchers have shown that PatA inhibits cap-dependent eukaryotic translation initiation. While PatA bound to and enhanced the intrinsic enzymatic activities of eIF4A, it inhibited eIF4A-eIF4G association and promoted the formation of a stable ternary complex between eIF4A and eIF4B. These changes in eIF4A affinity for its partner proteins upon binding to PatA caused the stalling of initiation complexes on mRNA in vitro and induced stress granule formation in vivo. Its structure is shown below:
Another eIF4A inhibitor is 15-deoxy-delta(12, 14)-prostaglandin J2 (15d-PGJ2).
An inhibitor of eIF4E function is ribavirin. Ribavirin is a guanosine (ribonucleic) analog used to stop viral RNA synthesis and viral mRNA capping; simply put, it is a nucleoside inhibitor. Its brand names include Copegus™, Rebetol™, Ribasphere™, Vilona™, and Virazole™, and it is an anti-viral drug indicated for severe RSV infection (individually), (notably for persistent) hepatitis C infection (can be used in conjunction with peg-interferon a2b or peg-interferon a2a), and some other viral infections. Ribavirin is a prodrug, which when metabolized resembles purine RNA nucleotides. In this form it interferes with RNA metabolism required for viral replication.
Ribavirin is active against a number of DNA and RNA viruses. It is a member of the nucleoside antimetabolite drugs that interfere with duplication of viral genetic material. Ribavirin is active against influenzas, flaviviruses, and agents of many viral hemorrhagic fevers. In Europe and the U.S. the oral (capsule or tablet) form of ribavirin is used in the treatment of hepatitis C, in combination with pegylated interferon drugs. Ribavirin is the only known treatment for a variety of viral hemorrhagic fevers, including Lassa fever, Crimean-Congo hemorrhagic fever, Venezuelan hemorrhagic fever, and Hantavirus infection, although data regarding these infections are scarce and the drug might be effective only in early stages.
The primary observed serious adverse side effect of ribavirin is hemolytic anemia, which may worsen preexisting cardiac disease. The mechanism for this effect is due to ribavirin's buildup inside erythrocytes. Oxidative damage to erythrocyte cell membrane is usually inhibited by glutathione; however, with reduced ATP levels caused by ribavirin, glutathione levels are impaired, permitting oxidative erythrocyte cell lysis. The gradual loss of erythrocytes leads to anemia. The anemia is dose-dependent and may sometimes be compensated by decreasing dose. Ribavirin is also a teratogen in some animal species and
thus poses a theoretical reproductive risk in humans, remaining a hazard as long as the drug is present, which can be as long as 6 months after a course of the drug has ended.
The aerosol form has been used in the past to treat respiratory syncytial virus-related diseases in children. However, its efficacy has been called into question by multiple studies, and most institutions no longer use it. It is still used in some cases. In Mexico, ribavirin ("ribavirina") has been sold for use against influenza. Studies have been mixed, but the derivative viramidine may have more promise. It has been used (in combination with ketamine, midazolam, and amantadine) in treatment of rabies. This drug is also used to control the life span of enterovirus 71 which causes hand, foot, and mouth disease. Notably, for severe RSV in children, the drug is delivered as aerosol particles for 12-18 hours daily. The method is very expensive, inconvenient and used rarely.
Physically ribavirin is similar to the sugar D-ribose from which it is derived. It is freely soluble in water, and is re-crystallized as fine silvery needles from boiling methanol. The three free sugar hydroxyls make the pure drug hydrophilic enough that it is only sparingly soluble in anhydrous ethanol. Classically, ribavirin is prepared from natural D- ribose by blocking the 2', 3' and 5' OH groups with benzyl groups, then derivatizing the OH with an acetyl group which acts as a suitable leaving group upon nucleophilic attack. The ribose Γ carbon attack is accomplished with a 1,2,4 triazole-3 -carboxymethyl ester, which directly attaches the nitrogen of the triazole to the carbon of the ribose, in the proper 1-β- D isomeric position. The bulky benzyl groups hinder attack at the other sugar carbons. Following purification of this intermediate, treatment with ammonia in methanolic conditions then simultaneously deblocks the ribose hydroxyls, and converts the triazole carboxymethyl ester to the carboxamide. Following this step, ribavirin may be recovered in good quantity by cooling and crystallization.
Ribavirin is possibly best viewed as a ribosyl purine analogue with an incomplete purine 6-membered ring. This structural resemblance historically prompted replacement of the 2' nitrogen of the triazole with a carbon (which becomes the 5' carbon in an imidazole), in an attempt to partly "fill out" the second ring, but to no great effect. Such 5' imidazole riboside derivatives show antiviral activity with 5' hydrogen or halide, but the larger the substituent, the smaller the activity, and all proved less active than ribavirin. Note that two natural products were already known with this imidazole riboside structure: substitution at the 5' carbon with OH results in pyrazomycin/pyrazofurin, an antibiotic with antiviral properties but unacceptable toxicity, and replacement with an amino group results in the natural purine
synthetic precursor 5-aminoimidazole-4-carboxamide-l- -D-ribofuranoside (AICAR), which has only modest antiviral properties.
Derivatization of the triazole 5' carbon, or replacement with a nitrogen (i.e., the 1,2,4,5 tetrazole 3-carboxamide), also results in substantial loss of activity, as does alkyl derivatization of the 3' carboxamide nitrogen. The 2' deoxyribose version of ribavirin (the DNA nucleoside analogue) is not active as an antiviral, suggesting strongly that ribavirin requires RNA-dependent enzymes for its antiviral activity. Antiviral activity is retained for acetate and phosphate derivation of the ribose hydroxyls, including the triphosphate and 3',5' cyclic phosphates, but these compounds are no more active than the parent molecule, reflecting the high efficiency of esterase and kinase activity in the body.
The most successful ribavirin derivative to date is the 3-carboxamidine derivative of the parent 3-carboxamide, first reported in 1973 and now called taribavirin (former names viramidine and ribamidine). This drug shows a similar spectrum of antiviral activity to ribavirin, which is not surprising as it is now known to be a pro-drug for ribavirin. Viramidine, however, has useful properties of less erythrocyte-trapping and better liver- targeting than ribavirin. The first property is due to viramidine's basic amidine group which inhibits drug entry into RBCs, and the second property is probably due to increased concentration of the enzymes which convert amidine to amide, in liver tissue. Viramidine is in phase III human trials and may one day be used in place of ribavirin, at least against certain kinds of viral hepatitis. Viramidine's slightly superior toxicological properties may eventually cause it to replace ribavirin in all uses of ribavirin.
Ribavirin is absorbed from the GI tract probably by nucleoside transporters. Absorption is about 45%, and this is modestly increased (to about 75%) by a fatty meal. Once in the plasma, ribavirin is transported through the cell membrane also by nucleoside transporters. Ribavirin is widely distributed in all tissues, including the CSF and brain. The pharmacokinetics of ribavirin is dominated by trapping of the phosphate form inside cells, particularly red blood cells (RBCs) which lack the enzyme to remove the phosphate once it has been added by kinases, and therefore attain high concentrations of the drug. Most of the kinase activity which converts the drug to active nucleotide form, is provided by adenine kinase. This enzyme is more active in virally infected cells.
The volume of distribution of ribavirin is large (2000 L/kg) and the length of time the drug is trapped varies greatly from tissue to tissue. The mean half-life for multiple doses in the body is about 12 days, but very long-term kinetics are dominated by the kinetics of RBCs (half-life 40 days). RBCs store ribavirin for the lifetime of the cells, releasing it into the
body's systems when old cells are degraded in the spleen. About a third of absorbed ribavirin is excreted into the urine unchanged, and the rest is excreted into urine as the de-ribosylated base 1,2,4-triazole 3-carboxamide, and the hydrolysis product of this, 1,2,4-triazole 3- carboxylic acid.
U.S. Patent Publication 20100144805 describes pharmaceutical small-molecule inhibitors of eIF4E that block the
wherein Ri is a hydrazone thiazole moiety of the structure:
or a barbituric acid moiety (or derivative thereof) of the structure:
R2 is hydrogen, hydroxyl or a nitro group present in one, two or three locations on the ring to which it is attached; R3 is a group individually present in one, two or three locations on the ring, wherein the group may be halo, hydrogen, conjugated or unconjugated aryl or heteroaryl, a alicyclic or polycyclic group, or R3, taken with the ring to which it is attached, forms a conjugated ring structure, e.g., a naphthalene ring; R4 is hydrogen, carboxyl, a lower alkyl ester, e.g. :
< ) <\
or carbonyl (in which case the dotted bond is present); R5 is N (in which case the dotted bond is present), NH, or carbonyl; and R-6 is NH or carbonyl.
Other possible inhibitors include MNK1/2 inhibitors (cercosporamide, S209, CGP57380, PF3644022) and mTOR ATP-active site inhibitors (rapamycin, RAD001, CCI- 779, PP242, Torinl, WYE-132, Ku-0063794, Palomid 529, and AZD8055, ΓΝΚ128, AZD2014 Palomid 529 BEZ235, PF-04691502, XL765).
B. Combination Therapies
The present invention also contemplates the treatment of SLE using standard therapeutic approaches where indicated in combination with the eIF4E therapies of the present disclosure. In general, the treatment of SLE involves treating elevated disease activity and trying to minimize the organ damage that can be associated with this increased inflammation and increased immune complex formation/deposition/complement activation. The compositions would be provided in a combined amount effective to exert a combined effect on the damaged tissue. This process may involve contacting the cells with the eIF4E inhibitor, in combination with a second therapeutic agent or factor(s) at the same time. This may be achieved by contacting the cell with a single composition or pharmacological formulation that includes both agents, or by contacting the cell with two distinct compositions or formulations, at the same time, wherein one composition includes the eIF4E inhibitor and the other includes the second agent.
Alternatively, treatment with the eIF4E inhibitor may precede or follow the additional agent treatment by intervals ranging from minutes to weeks. In embodiments where the second agent is applied separately to the target, one would generally ensure that a significant period of time did not expire between the time of each delivery, such that the agent would still be able to exert an advantageously combined effect on the target. In such instances, it is contemplated that one would contact the target with both modalities within about 12-24 hr of each other and, more preferably, within about 6-12 hr of each other. In some situations, it may be desirable to extend the time period for treatment significantly, however, where several days (Yokoyama et al, 2002, Kozyrev et al, 2012, Troutman et al, 2012, Christensen et al, 2005, Bernasconi et al, 2003 and Sun et al, 2007) to several weeks (Yokoyama et al, 2002, Kozyrev et al, 2012, Troutman et al, 2012, Christensen et al, 2005, Bernasconi et al, 2003, Sun et al, 2007 and Neininger et al, 2002) lapse between the respective administrations.
It also is conceivable that more than one administration of either the eIF4E inhibitor in combination with a second therapeutic agent will be desired. Various combinations may be employed, where the eIF4E inhibitor is "A" and the second therapeutic agent is "B", as exemplified below:
A/B/A B/A/B B/B/A A/A/B B/A/A A/B/B B/B/B/A B/B/A/B
A/A/B/B A/B/A/B A/B/B/A B/B/A/A B/A/B/A B/A/A/B B/B/B/A
A/A/A/B B/A/A/A A/B/A/A A/A/B/A A/B/B/B B/A/B/B B/B/A/B Other combinations are contemplated.
Foundational treatment for combination therapies as described above can include corticosteroids and anti-malarial drugs. Certain types of lupus nephritis such as diffuse proliferative glomerulonephritis require round of cytotoxic drugs. These drugs include, most commonly, cyclophosphamide and mycophenolate. Hydroxychloroquine (HCQ) was approved by the FDA for lupus in 1955. Some drugs approved for other diseases are used for SLE 'off-label' In November 2010, an FDA advisory panel recommended approving belimumab (Benlysta) as a treatment for elevated disease activity seen in autoantibody- positive lupus patients. The drug was approved by the FDA in March 201 1.
Due to the variety of symptoms and organ system involvement with SLE, its severity in an individual must be assessed in order to successfully treat SLE. Mild or remittent disease may, sometimes, be safely left minimally treated with hydroxychloroquine alone. If required, nonsteroidal anti-inflammatory drugs and low dose steroids may also be used. Hydroxychloroquine (HCQ) is an FDA-approved antimalarial used for constitutional, cutaneous, and articular manifestations. Hydroxychloroquine has relatively few side effects, and there is evidence that it improves survival among people who have SLE and stopping HCQ in stable SLE patients led to increased disease flares in Canadian lupus patients. Disease-modifying antirheumatic drugs (DMARDs) are oftentimes used off-label in SLE to decrease disease activity and lower the need for steroid use. DMARDs commonly in use are methotrexate and azathioprine. In more severe cases, medications that aggressively suppress the immune system (primarily high-dose corticosteroids and major immunosuppressants) are used to control the disease and prevent damage. Cyclophosphamide is used for severe glomerulonephritis, as well as other life-threatening or organ-damaging complications, such as vasculitis and lupus cerebritis. Mycophenolic acid is also used for treatment of lupus nephritis, but it is not FDA-approved for this indication.
Depending on the dosage, people who require steroids may develop Cushing's symptoms of truncal obesity, purple striae, buffalo hump and other associated symptoms. These may subside if and when the large initial dosage is reduced, but long-term use of even low doses can cause elevated blood pressure, glucose intolerance (including metabolic syndrome and/or diabetes), osteoporosis, insomnia, avascular necrosis and cataracts.
Numerous new immunosuppressive drugs are being actively tested for SLE. Rather than suppressing the immune system nonspecifically, as corticosteroids do, they target the responses of individual types of immune cells. Belimumab, or a humanized monoclonal antibody against B-lymphocyte stimulating factor (BlyS or BAFF), is FDA approved for lupus treatment and decreased SLE disease activity, especially in patients with baseline elevated disease activity and the presence of autoantibodies. Addition drugs, such as abatacept, epratuzimab, etanercept and others, are actively being studied in SLE patients and some of these drugs are already FDA-approved for treatment of rheumatoid arthritis or other disorders. Since a large percentage of people with SLE suffer from varying amounts of chronic pain, stronger prescription analgesics (pain killers) may be used if over-the-counter drugs (mainly nonsteroidal anti-inflammatory drugs) do not provide effective relief. Potent NSAIDs such as indomethacin and diclofenac are relatively contraindicated for patients with SLE because they increase the risk of kidney failure and heart failure.
Moderate pain is typically treated with mild prescription opiates such as dextropropoxyphene and co-codamol. Moderate to severe chronic pain is treated with stronger opioids, such as hydrocodone or longer-acting continuous-release opioids, such as oxycodone, MS Contin, or methadone. The fentanyl duragesic transdermal patch is also a widely used treatment option for the chronic pain caused by complications because of its long-acting timed release and ease of use. When opioids are used for prolonged periods, drug tolerance, chemical dependency, and addiction may occur. Opiate addiction is not typically a concern, since the condition is not likely to ever completely disappear. Thus, lifelong treatment with opioids is fairly common for chronic pain symptoms, accompanied by periodic titration that is typical of any long-term opioid regimen.
Intravenous immunoglobulins may be used to control SLE with organ involvement, or vasculitis. It is believed that they reduce antibody production or promote the clearance of immune complexes from the body, even though their mechanism of action is not well- understood. Unlike immunosuppressives and corticosteroids, IVIGs do not suppress the immune system, so there is less risk of serious infections with these drugs.
Avoiding sunlight is the primary change to the lifestyle of SLE sufferers, as sunlight is known to exacerbate the disease, as is the debilitating effect of intense fatigue. These two problems can lead to patients becoming housebound for long periods of time. Drugs unrelated to SLE should be prescribed only when known not to exacerbate the disease. Occupational exposure to silica, pesticides and mercury can also make the disease worsen.
Renal transplants are the treatment of choice for end-stage renal disease, which is one of the complications of lupus nephritis, but the recurrence of the full disease in the transplanted kidney is common in up to 30% of patients.
Antiphospholipid syndrome is also related to the onset of neural lupus symptoms in the brain. In this form of the disease the cause is very different from lupus: thromboses (blood clots or "sticky blood") form in blood vessels, which prove to be fatal if they move within the blood stream. If the thromboses migrate to the brain, they can potentially cause a stroke by blocking the blood supply to the brain. If this disorder is suspected in patients, brain scans are usually required for early detection. These scans can show localized areas of the brain where blood supply has not been adequate. The treatment plan for these patients requires anticoagulation. Often, low-dose aspirin is prescribed for this purpose, although for cases involving thrombosis anticoagulants such as warfarin are used.
C. Pharmaceutical Formulations and Delivery
Where therapeutic applications are contemplated, it will be necessary to prepare pharmaceutical compositions in a form appropriate for the intended application. Generally, this will entail preparing compositions that are essentially free of pyrogens, as well as other impurities that could be harmful to humans or animals.
One will generally desire to employ appropriate salts and buffers to render delivery vectors stable and allow for uptake by target cells. Buffers also will be employed when recombinant cells are introduced into a patient. Aqueous compositions of the present invention comprise an effective amount of the vector to cells, dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. Such compositions also are referred to as inocula. The phrases "pharmaceutically or pharmacologically acceptable" refer to molecular entities and compositions that do not produce adverse, allergic, or other untoward reactions when administered to an animal or a human. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except
insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions.
The active compositions of the present invention may include classic pharmaceutical preparations. Administration of these compositions according to the present invention will be via any common route so long as the target tissue is available via that route. Such routes include oral, nasal, buccal, rectal, vaginal or topical route. Alternatively, administration may be by orthotopic, intradermal, subcutaneous, intramuscular, intraperitoneal, intra-arterial or intravenous injection. Such compositions would normally be administered as pharmaceutically acceptable compositions.
The active compounds may also be administered parenterally or intraperitoneally. Solutions of the active compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must 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 carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various other ingredients enumerated above,
as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
For oral administration the polypeptides of the present invention may be incorporated with excipients and used in the form of non-ingestible mouthwashes and dentifrices. A mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution). Alternatively, the active ingredient may be incorporated into an antiseptic wash containing sodium borate, glycerin and potassium bicarbonate. The active ingredient may also be dispersed in dentifrices, including: gels, pastes, powders and slurries. The active ingredient may be added in a therapeutically effective amount to a paste dentifrice that may include water, binders, abrasives, flavoring agents, foaming agents, and humectants.
The compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically-acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like.
Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms such as injectable solutions, drug release capsules and the like. For parenteral administration in an aqueous solution, for example, the
solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion, (see for example, "Remington's Pharmaceutical Sciences," 15th Ed., 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biologies standards.
In a particular embodiment, microbes can be engineered to deliver drugs according to the present disclosure. Lactobacillus strains have been used for the purpose (drug delivery of paclitaxel). L. acidophilus and L. rhamnosus can form minicells in modified MRS broth. Nanoparticle size of obtained minicells is ~400 nm in diameter. These minicells have been packaged paclitaxel (10 μg/ml) and cephalosporin (10 μg/ml). Alternatively, for protein drugs and certain small molecules, synthetic machinery can be embedded in microorganisms for the production of drugs. Delivery of the microorganism therefore constitutes administration of the drug itself.
IV. Examples
The following examples are included to further illustrate various aspects of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques and/or compositions discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 - MATERIALS AND METHODS
Mice. Bankl ' mice were kindly provided by Dr T. Kurosaki (Riken Research Centre for Allergy and Immunology, Kyoto, Japan) and were backcrossed nine generations onto the
C57BL/6J background. C57BL/6J mice were purchased from Jackson Laboratory, Bar Harbor, Maine, USA. Mice were maintained under specific pathogen free (SPF) barrier conditions. This study was approved by the Oklahoma Medical Research Foundation Institutional Animal Care and Use Committee.
Antibodies and reagents. Phospho-specific antibodies to p38 (Thrl 80/Tyrl82, clonel2F8 #4631), JNK (Thrl83/Tyrl85, clone 81E11, #4668), ERK (Thr202/Tyr204, clone D13.14.4E, #4370), I /c B a (Ser32, clone 14D4, #2859), eIF4E (Ser209, #9741), M K1/2 (Thrl97/202, #211 1), 4E-BP1 (Thr37/46, clone 236B4, #2855), mTOR (Ser2448, clone D9C2, #5536), and AKT (Ser473, clone 193H12, #4058) and phosphorylation-state- independent antibodies to p38 (#9212), JNK, ERK, I /c B a , eIF4E (clone C46H6, #2067), MNK1 (clone C4C1, #2195), 4E-BP1 (clone 53H1 1, #9644), mTOR (clone 7C10, #2983), and AKT (#9272), were purchased from Cell Signaling Technology (Danvers, MA). Phosphorylation-state-independent antibody for MNK2 (S-20) was purchased from Sigma- Aldrich (St. Louis, MI).
Purification of splenic B cells. Splenic B cells were purified by negative selection using magnetic bead separation. Briefly, spleen cells from Bankl+/+ and Bankl"7" littermates were labeled with a cocktail of biotin-conjugated antibodies for 15 minutes. Cells were incubated an additional 15 minutes with anti-biotin micro beads (B-Cell Isolation Kit, mouse; Miltenyi Biotech, Auburn, CA) at 4° C. The labeled non-B-cells were depleted by magnetic retention in a MACS column while unlabeled B cells were recovered. The purity of the resulting cell population was typically more than 95% B220+CD3" as assessed by flow cytometry analysis.
Cell Culture. Cell culture was performed with RPMI 1640 medium supplemented with 10% FBS, L-glutamine (2 mM), 2-ME (50 μΜ) and antibiotics for all experiments, except for p38 analyses and the Akt axis where cells were serum-starved two hours prior to stimulation. Purified splenic B cells (106 cells/mL) were treated with 10 μg/ml F(ab')2 fragment of anti-mouse IgM (Jackson Immunoresearch, West Grove, PA), 2 μΜ CpG (ODN Type B; 1668, Invivogen, San Diego, CA) or combination of anti-mouse IgM and CpG, 20 μg/mL TLR4 agonist LPS or 1 μg/mL of the TLR7 agonist R848 (Invivogen, San Diego, CA), or were left unstimulated. Supernatants were collected at 0, 12, 24 and 48 hr, and 72 and 96 hours for anti-CD40 stimulation (low-endotoxin, azide-free anti-mouse CD40, Biolegend, San Diego, CA) was included (10 μg/ml final concentration), with or without CpG.
Western Blot. Stimulated splenic B cells were lysed in buffer containing 1% TritonX-100, 50 mM Tris pH 7.4, 50 mM NaCl, 1 mM EDTA, 2 mM Na3V04, and a protease inhibitor cocktail (Roche Applied Science, Indianapolis, ΓΝ). LDS sample buffer and reducing agent (Life Technologies, Carlsbad, CA) were added. Samples were then boiled, and protein was separated with NuPAGE 4-12% Bis-Tris Gels (Life Technologies, Carlsbad, CA). Proteins were blotted onto polyvinylidene difluoride membranes, blocked with 5% skimmed milk and the immunoblots were processed with specific antibodies diluted with 5% BSA or 5% skimmed milk solution, and detected with Novex ECL substrate (Invitrogen, Carlsbad CA).
ELISA Assays. Cytokine secretion by in vitro stimulated B cells was measured using the ELISA kit for IL-6 and IL-10 purchased from BD Bioscience (San Jose, CA). For this, 96-well plates were coated with antibody against mouse IL-6 or mouse IL-10 overnight at 4°C. Plates were then blocked for 1 h at room temperature with 10% FBS in PBS. Supernatants were then added to the plate and left for 2 h at room temperature. Following washing, biotinylated anti-IL-6 or anti-IL-10 was added and plates incubated for further an hour at room temperature. Streptavidine alkaline phosphatase was then added following incubation at room temperature and washing with tetramethylbenzidine liquid substrate. Absorbance was read at 450 nm, from which absorbance was subtracted at 570 nm within 30 min of stopping reaction. Cytokine concentration was determined by extrapolation from the standard curve. Standard curve was generated using recombinant mouse IL-6 or recombinant mouse IL-10 (BD Bioscience, San Jose, CA).
Splenic B cells (0.5 l06 cells/well) were stimulated for 6 days and supernatants were analyzed for immunoglobulin isotype antibodies as per the instructions from BD Pharmingen™ mouse immunoglobulin isotype ELISA kit (BD Biosciences, San Diego, CA, US). The inventors quantified IgG2a and IgG2c isotype antibodies using mouse IgG2a Ready-Set-Go ELISA kit (eBioscience Inc. San Diego CA, US) and mouse IgG2c ELISA Quantification Set (Bethyl Laboratories, Inc. TX, US) respectively and data are presented as ng/ml. For other antibodies, only OD's are shown.
Determination of Cell Viability and Proliferation Assay. Purified splenic B cells were resuspended in pre-warmed PBS/ 0.1% BSA and labeled with 2 μΜ CFSE (Life Technologies, Carlsbad, CA), and incubated at 37 °C in a water bath for 10 min. Five volumes of culture medium were added for quenching. After incubating 5 min on ice, the cell pellet was collected by centrifugation, washed 3 times with fresh complete RPMI medium and resuspended to the appropriate density. Cells were then stimulated with 10 μg/ml F(ab')2
fragment of anti-IgM, 2 μΜ CpG, 10 μg/ml anti-CD40, 20 μg/ml LPS, and 1 μg/ml R848, respectively, or the shown combinations and cultured for 48, 72 and 96 hr. After harvest, cells were stained with propidium iodide (PI, final concentration of 1 μg/ml; eBioscience Inc. San Diego CA, US) and acquired by LSR II or FACScalibur (BD bioscience, San Jose, CA) 36,000 events in total. For flow cytometry, debris and doublet cells were discriminated and viable cells (PI- negative) were gated and % of cell viability was determined. The histograms shown with FL1 channel represent overlaid data from un-stimulated CFSE-labeled B cells, stimulated Bankl+/+ and Bankl7" B cells, and the y-axis is presented as % viability of maximum. The analysis was conducted using FlowJo software (Tree Star Inc., Ashland, OR).
Inhibition of IL-6 production from splenic B cells. The MNK1 specific inhibitor,
CGP57380 [4-Amino-5-(4-fluoroanilino)-pyrazolo[3,4-d] pyrimidine] was obtained from Calbiochem®-Millipore (Billerica, MA), and MNKs inhibitor cercosporamide and MK2 inhibitor, PF-3644022 [(10R)-9,10,1 l,12-tetrahydro-10-methyl-3-(6-methyl-3-pyridinyl)-8H- [l,4]diazepino[5',6':4,5]thieno[3,2-f ]quinolin-8-one hydrate] were purchased from Sigma- Aldrich (St. Louis, MI). The purified splenic B cells (5 x 105 cells/ well) were seeded in complete RPMI1640 medium in a 48 well tissue culture plate. Then the cells were pretreated with or without inhibitor for 30 min or lh, respectively, before CpG stimulation for 24 hr time at which cells were harvested and subjected to determine % of viability by using propidium iodide staining (final concentration 1 μg/ml) and flow cytometry analysis. Supernatants were collected and stored at -80 °C until use and IL-6 was measured using a capture ELISA. The control well contained CpG and cell culture grade DMSO (Pierce, Rockford, IL) only (17).
Taqman RT-PCR and IL-6 mRNA Stability Assay. Total RNA was isolated with Trizol (Invitrogen, Carlsbad, CA) from triplicate cultures of 5 x 105 splenic B cells in the presence or absence of CpG treatment for the indicated times. After quantification and quality control of the RNA with a Nanodrop spectrophotometer (Thermo Scientific, West Palm Beach, FL), 400 ng RNA was subjected to first-strand cDNA synthesis for qRT-PCR (Origene, Rockvile MD). 5 ng total cDNA/RNA was used per reaction in TaqMan Fast Universal PCR Master Mix (Life Technologies, Carlsbad, CA). The qRT-PCR reactions were performed on a 7900 HT Fast Real-Time PCR system. The primers and probes for mouse TLR9 (Assay ID: Mm00446193_ml), Prdml (Mm00476128_ml) and IL-6 (Assay ID: Mm00446190_ml) and internal control gene 18S rRNA (4319413E) were purchased from Taqman Gene expression Assays (Life Technologies, Carlsbad, CA).
For the IL-6 mRNA stability assay, 5 x 105 splenic B cells were cultured in triplicate and treated with 2 μΜ CpG for 20h followed by addition of 1 μ^ηιΐ actinomycin D (Sigma- Aldrich, St. Louis, MI) for 0, 50, 100, 200 min. RNA was isolated and IL-6 transcription was determined with Taqman qRT-PCR using the primers as described above.
EXAMPLE 2 - RESULTS
BANK1 Deficiency Reduces CpG-Induced p38 Activation in Splenic B Cells. To discern the signaling cascades affected by BANK1, the inventors tested if BANK1 deficiency altered B cell proliferation after stimulation with CpG. Using purified splenic B-cells from Bankr7" and littermate control (+/+) mice, they did not observe any differences at any of the time points tested in proliferation with CpG or LPS, a TLR4 agonist (FIG. 1A). The inventors also did not observe differences in cell proliferation when using anti-IgM, anti-IgM+CpG, anti-CD40, CpG + anti-CD40, R848, R848+anti-CD40, LPS, LPS + anti-CD40, or anti- IgM+LPS (FIGS. 6A-C). They then tested the effects of BANK 1 deficiency on IgM-induced activation of NF κ B by analyzing phosphorylation and degradation of I κ B a , and activation of the MAPK pathways by studying phosphorylation of ERK and p38. BANK1 deficiency did not affect phosphorylation of MAPKs, or I κ B a following stimulation with anti-IgM alone (FIG. IB).
After CpG stimulation, p38 phosphorylation was importantly reduced (FIGS. 1C and ID), but ERK, ΓΝΚ and I /c B a phosphorylation were unchanged, in Bankl7" cells compared to cells from WT mice (FIG. 1C) or from Bankl+/+ littermates (data not shown). For all pathways, stimulation with anti-IgM+CpG (data not shown) yielded results identical to CpG alone (FIG. 1C). BANK1 deficiency had no effect on p38 signaling induced by LPS or R848 (FIG. IE). Therefore, BANK1 specifically modulates CpG-induced p38 signaling in vitro.
BANK1 Deficiency Reduces CpG-Stimulated IL-6 Secretion. It is known that the
MAPK p38-signaling pathway regulates the production of several cytokines including IL-6, TNFa, IFNy, and IL-10 (Enslen et al, 1998, Nagaleekar et al, 201 1 and Foey et al, 1998). Having shown that BANK1 deficiency reduced p38 phosphorylation after CpG stimulation, the inventors asked whether reduction in p38 phosphorylation leads to modulation of IL-6 or IL-10 secretion by Bankl7" B cells. As expected (Vanden and Bishop, 2008, Poudrier et al, 1998 and Barr et al, 2007), anti-IgM alone did not induce detectable IL-6 or IL-10 in splenic B-cells from WT, Bankl+/+ or Bankl7" littermates (data not shown), while CpG induced detectable levels of IL-10 and IL-6 production. Bankl7" B-cells showed consistently reduced
IL-6 but normal IL-10 secretion in response to CpG alone or the combination of CpG and BCR ligation (FIG. 2A).
In general, it has been observed that the combination of anti-CD40 and CpG leads to the most optimal secretion of IL-6 by B cells (Saito et al , 2002 and Haxhinasto and Bishop, 2004), and BANKl deficiency has been shown to lead to activation of Akt following stimulation through CD40. When the inventors investigated production of IL-6 by combining anti-CD40 and CpG, they clearly observed that the amount of IL-6 secreted was importantly increased when adding anti-CD40 to CpG as compared to CpG alone in Bankl+/+ B cells. Deficiency of BANKl reduced the levels of secreted IL-6 (FIG. 2A). The inventors tested if stimulation with CpG would lead to a reduction in cell viability by BANKl deficient cells and hence to reduced IL-6 secretion. This was not the case (FIG. 6). The inventors did observe slightly fluctuated cell viability when combining CpG with anti-CD40 in BANKl deficient cells, but proliferation was normal. Hence, the inventors conclude that reduced secretion of IL-6 is not due to changes in cell viability in BANKl deficient B cells.
CpG is a known agonist of Tlr9. To test if the reduced expression of IL-6 and reduced phosphorylation of p38 could be due to reduced expression of Tlr9, the inventors tested mRNA expression. Tlr9 gene expression was no different between Bankl+/+ and Bankl"7" mice (FIG. 2B). Next, the inventors examined whether BANKl deficiency had an effect on the production of IL-6 induced by TLR7 and TLR4. However, LPS and the TLR7 agonist R848 induced subtle amounts of IL-6 (Vanden and Bishop, 2008 and Poudrier et al, 1998) as compared to CpG stimulation and BANKl deficiency had no effect (FIG. 2C). Taken together, these results show that BANKl deficiency reduces secretion of IL-6 in response to CpG stimulation, overcomes the effect of anti-CD40 and is not due to changes in cell viability or to reduced Tlr9 expression.
Bankl affects translation initiation via the MNK1/2 and eIF4E pathway but not mRNA stability, which is controlled by MK2. The inventors suspected that BANKl deficiency could decrease IL-6 production by reducing IL-6 translation initiation and/or mRNA stability, since p38 directly controls these via the kinases MNK1/2 and MK2, respectively (Raught and Gingras, 1999; Ronkina et al, 2007). MK2 inhibitors are known to reduce IL-6 secretion by human peripheral blood mononuclear cells (Mourey et al, 2010). Consistent with this, the inventors observed reduced IL-6 production in CpG-stimulated WT mouse B cells treated with the MK2 inhibitor PF3644022 (FIG. 3 A). In addition, there was a significant and reproducible, though less marked reduction of IL-6 secretion after treatment with the MNKl/2/eIF4E inhibitors CGP57380 and cercosporamide using doses that
minimally affect cell viability (FIGS. 3B-D). These results show that MNK1/2 and MK2 inhibitors can reduce IL-6 secretion in mouse B cells and confirm that both MNK1/2 and MK2 are important for CpG-stimulated IL-6 secretion from mouse splenic B cells.
Since CpG-induced IL-6 production was reduced in Bankl7" cells, the inventors tested if IL-6 gene expression was altered by the deficiency of BANK1. As shown in FIG. 4A, IL-6 gene expression showed no differences between BANK 1 -sufficient and BANK 1 -deficient B cells following CpG stimulation. Since the p38-MK2 signal pathway is responsible in maintaining mRNA stability, the inventors tested the effect of BANK1 deficiency on MK2- mediated IL-6 secretion by testing the stability oiIL-6 mRNA. CpG-stimulated Bankl+/+ and Bankr7" splenic B-cells showed comparable IL-6 mRNA stability (FIG. 4B). Next, the inventors tested the effects of BANK1 on the activation of the p38-MNKl/2-mediated signaling pathway. MNK1/2 regulates the translation initiation factor eIF4E through phosphorylation (Raught and Gingras, 1999, Andersson and Sundler, 2006 and Shveygert et al, 2010). The inventors therefore analyzed phosphorylation of MNK1/2 and eIF4E. CpG- induced MNK1/2 and eIF4E phosphorylation were consistently reduced in Bankl7" cells (FIGS. 4C-D). These results show that BANK1 influences IL-6 secretion by its effects on the p38-regulated MNKl/2/eIF4E/eIF4G pathway of translation initiation while BANK1 does not affect IL-6 secretion via the MK2 pathway.
BANK1 has no effect on the AKT-mTORCl-4E-BPl Signaling Cascade. Activation of eIF4E is also controlled via the AKT-mTORCl-4E-BPl signaling cascade. Activation of this pathway leads to phosphorylation of 4E-BP1, which in turn releases eIF4E for it to be phosphorylated by MNK1/2 and initiate translation. In addition, stimulation through CD40 has been reported to lead to increased AKT phosphorylation in Bankl"7" purified B cells (Aiba et al, 2006). The cascade was induced with CpG alone (FIG. 5A), and the phosphorylation of AKT, mTOR and 4E-BP1 was strongly induced when anti-CD40 was included alone or in combination with CpG (FIGS. 5B and 5C). The inventors did observe an increase, albeit weak, in phospho-AKT following anti-CD40 and anti-CD40+CpG treatment on Bankl7" B cells (FIGS. 5B and 5C) (31). However, the inventors did not observe any differences in the downstream mTORCl to 4E-BP1, when the inventors used anti-CD40 alone or combined with CpG between Bankl7" and Bankl+/+ B cells or on phospho-AKT when using CpG alone (FIGS. 5A-C). Overall, these results show that BANK1 acts only in the MNK1/2 and eIF4E arm of p38 signaling to induce IL-6 secretion following CpG stimulation, and that absence of BANK1 does not affect activation of AKT, mTORCl or 4E- BP1 of induction of translation initiation.
BANKl deficiency leads to a tendency towards increased production of IgG2a/c subclass antibodies but does not affect expression of the Blimpl gene (Prdml). Finally, the inventors tested if BANKl had an effect on the in vitro secretion of antibodies following CpG stimulation. These results show a non-significant tendency towards increased, rather than decreased secretion of IgG2a and IgG2c antibodies by Bankl"7" B cells. Further, the inventors did not observe any difference in the expression of the Prdml gene that codifies for the transcription factor Blimpl (FIGS. 7A-B). These results suggest that secretion of IgG2a/2c antibodies is not significantly affected by reduced secretion of IL-6 by Bankl7" B cells.
Production of IL-6 is increased following CpG stimulation in a transgenic for the human BANKl gene. The inventors have shown that deficiency of BANKl specifically affects the phosphorylation of p38 and the downstream signaling of the translation initiation complex molecule eIF4E leading to decreased production of IL-6 protein (Wu et al, 2013). To show that the opposite occurs when one has over-expression of BANKl, the inventors produced a mouse transgenic for the human full-length isoform of BANKl . BANKl is expressed only in B cells thanks to the CD 19 promoter included in the construct. They observed increased levels of IL6 following in vitro stimulation with either CpG or CD40+CpG stimulation splenic B cells of BANKIFL-Tg mice as compared to normal C57B1/6 mice (WT) (FIGS. 8A-B).
Deficiency of Bankl suppresses the development of lupus-like disease. In order to analyze whether deficiency of BANKl can influence autoimmunity, and abrogate or delay the development of lupus, and to show whether BANKl could have a role in TLR7 signaling, the inventors crossed the BANKl -deficient animals with B6.Slelz/z.yaa, a congenic mouse containing the NZW locus Slel that leads to several lupus-related B cell phenotypes (Morel et al, 2001; Shi et al, 2007; Sobel et al, 2002; Vuyyuru et al, 2009). The yaa region in male animals amplifies the phenotypic effect, leading to the development of kidney disease in male mice (Hwang et al, 2012; Subramanian et al, 2006).
Recently, it has been shown that the yaa "mutation" is the result of a duplication of the genomic region in the X chromosome containing the TLR7 and TLR8 genes, which has naturally translocated onto the Y chromosome of the BXSB mouse (Subramanian et al, 2006), previously known as the BXSB.yaa strain Izui et al, 2000). The inventors performed experiments on the strain Be.Slel^Bankl ' .yaa and used as control the B6.Slelz/zBankl+/+.yaa littermate. The results show that the deficiency of BANKl does suppress the development of lupus phenotypes (FIGS. 9A-C).
Serum 11-6 is reduced in lupus prone mice deficient for Bankl. Among the lupus phenotypes is the increase in serum IL-6, which promotes IgG antibody production and inflammation. Recently, Darise it has been shown that the KO of IL-6 normalizes all the lupus phenotypes of the B6.Slel.yaa mouse. The inventors now show that lack of Bankl in this mouse led to a reduction of the in vivo serum levels of IL-6 excessively produced due to the presence of the yaa genomic segment, as detected in serum of the B6.Slelz/zBankl"A .yaa mice (FIG. 10). In conclusion, deficiency of Bankl reduces the effects induced by the presence of the yaa translocation (or TLR7 duplication) supporting that Bankl has an effect on TLR7 signaling.
Translation inhibitors can reduce the production of IL-6 induced by CpG in B cells from the BANKl Tg mouse. There are several inhibitors of translation initiation, such as Pateamine A (PatA), a small molecule isolated from the marine sponge Mycale s., and with immunosuppressive properties (Low et al, 2005; Di Marco et al, 2012). PatA alters the activity of the mRNA helicase EIF4A, needed for the assembly of the 40s ribosome subunit (Cencic et al, 2012). Other inhibitors, such as Cercosporamide or CGP57380, inhibit MNK1/2 (Altman et al, 2013), the kinase that following p38 activation, phosphorylates eIF4E. Inhibition of assembly of the complex by PatA at low doses has been shown to inhibit the production of pro-inflammatory cytokines (Di Marco et al, 2012; Gingras et al, 1999), but the effects of M K1 inhibition on IL-6 production, have never been shown in mice.
The inventors therefore tested the effect of Pateamine and the MNK1 inhibitor
CGP57380 in vitro, purifying splenic B cells from the transgenic mice following stimulation with CpG to detect IL-6 production and avoiding levels of the drugs that may be toxic to the cells (FIG. 1 1). FIG. 11 shows how 0.01 μΜ Pateamine is non-toxic (right panel) and is capable of inhibiting IL-6 secretion following CpG stimulation in purified B cells from the transgenic BANKIFL-Tg mice. In these experiments, the transgenic animals were crossed onto the Bankl-/- mice in order to have the human trans gene without the expression of the endogenous murine Bankl .
EXAMPLE 3 - DISCUSSION The inventors show here for the first time that the B cell adaptor with ankyrin repeats
BANKl influences signaling leading to the formation of the translation initiation eIF4E complex following stimulation with CpG, and that BANKl deficiency results in reduction of the translation of the proinflammatory cytokine IL-6. They have also shown that mRNA stability of IL-6 is not affected, nor the second axis of regulation of translation initiation via
AKT, which is also induced by CpG and strongly amplified with anti-CD40 treatment. Clearly, the combination of CpG and anti-CD40 treatment leads to stronger signaling of the AKT axis followed by strong activation of mTOR and 4E-BP1. However, absence of BANKl has no effect on the signaling pathway through this axis, except weak activation of AKT. While the secretion of IL-6 is optimal (Poudrier et al, 1998, Barr et al, 2007 and Barr et al, 2010) with the combination of CpG and anti-CD40, BANKl deficiency leads to its reduction, and anti-CD40 cannot overcome this effect. These results clearly show that overall BANKl controls the production of IL-6 via the p38-MNKl/2-eIF4E pathway. At the same time, activation of the CD40-induced pathway leads to strong phosphorylation of the AKT axis, which would eventually promote translation initiation of IL-6 through phosphorylation of 4E-BP 1 and the release of eIF4E for it to become phosphorylated by MNK1/2. However, the inventors clearly observe that this is not the case.
The involvement of BANKl in modulating the IL-6 response after CpG stimulation has important implications in the pathogenesis of autoimmunity and viral infection. Sera from autoimmunity -prone animals and cerebrospinal fluid from SLE patients have elevated levels of IL-6 (Alcocer-Varela et al, 1992 and Stuart et al, 1995) and peripheral blood cells from SLE patients spontaneously secrete increased levels of IL-6 (Linker-Israeli et al, 1991). Autoimmunity is ameliorated by IL-6 ablation (Barr et al, 2012). It is also important to note that IL-6, coordinated with IL-21, has been reported to control the differentiation towards plasma cells (Eto et al, 2011). More recently, B cell derived IL-6 was shown to function in an autocrine manner and trigger receptor revision through re-expression of RAG in the post germinal center response (Yan et al, 2012).
IL-6 has also important roles in infectious diseases. It has been reported that B cells release IL-6 to promote a follicular helper T cell (TFH) response to viral infection. B cell- derived IL-6 was necessary and sufficient to induce IL-21 from CD4+ T cells in vitro and to support TFH cell development in vivo upon acute influenza virus infection (Karnowski et al, 2012). IL-6 is also involved in the induction but not maintenance of plasma cells (Cassese et al, 2003). BANKl deficient mice have normal T cell-dependent humoral responses following immunization with NP-CGG (Aiba et al, 2006). One explanation for the unchanged primary humoral response reported by Aiba et al in Bankl-/- mice is likely to be the lack of TLR agonist challenge and IL-6 production by B cells in their in vivo system. Therefore, it will be worth to address if infection with DNA- or RNA-viruses, instead of NP- CGG immunization can initiate TLR9 or TLR7 activation that would probably result in reduced IL-6 production by Bankl-/- B cells, and suppressed germinal center responses.
Regarding humoral responses, at this point the inventors are unable to explain the tendency towards increased IgG2a/2c production induced by CpG and they do not know if this bears any relationship with the increased serum IgG2a observed by Aiba et al (Aiba et al, 2006).
BANKl has been genetically associated with SLE and other autoimmune diseases, and here the inventors observe that BANKl deficiency decreased IL-6 secretion by altering the translation initiation pathway upon CpG stimulation. These results therefore suggest that BANKl could be involved in controlling disease development through the control of IL-6 secretion. While there are several mechanisms through which IL-6 production is regulated, it is clear that the production of IL-6 is controlled through a multitude of pathways and through different genes.
BANKl was found to contain a conformational modular TIR domain at the N- terminus, similar to its relative, the molecule BCAP. BCAP is required for TLR-mediated activation of PI3K and AKT in macrophages (Troutman et al, 2012). In contrast, CpG- induced AKT activation is normal in Bankl-/- B cells (FIG. 5A). The data suggest that BCAP, rather than BANKl, may play a critical role in TLR-mediated activation of AKT, and further, BCAP and CD19 have complementary roles in BCR-mediated-PI3K activation (Aiba et al, 2008). While BANKl appears to mediate AKT activation upon CD40 ligation in B cells (Aiba et al, 2006; FIG. 5B), these results support a specific role for BANKl in transducing CpG-induced signals via p38-MNKl/2 and the translation initiation factor eIF4E in B cells. Thus, BANKl and BCAP rather than playing redundant roles, appear to have very different ones.
BANKl, with a putative TIR domain, would be prone to bind molecules containing TIR domains, and explain the very specific role of BANKl in p38 signaling and translation initiation. BANKl is an adaptor molecule with a modular structure. It has up to 23 tyrosines susceptible of phosphorylation, and their substrate could alternate among a variety of molecules forming specific complexes during CpG-induced activation, different from those occurring following BCR-induced ligation, for instance. One of those complexes induced by CpG could include p38. How BANKl controls the p38-MNKl/2 pathway downstream of TLR9 is at present not known, but is a subject of study in the inventors' laboratory.
The inventors have described that BANKl shows an interaction with the Src tyrosine kinase BLK (1), and that BLK serves to promote the interaction between BANKl and phospholipase C y2, a key molecule in signal transduction during BCR ligation (Bernal- Quiros et al, 2013). This phenomenon is apparently not linked to CpG-induced signaling, as the inventors do not observe phosphorylation of BANKl or changes in binding of BANKl to
BLK following CpG stimulation (data not shown). Nevertheless, the inventors provide here a role for BANK1 in CpG-induced signaling in the production of IL-6, which could be highly relevant in the study of autoimmunity and inflammation.
All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and/or methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
V. References
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
Aiba et al, Blood 11 1 : 1497-503, 2008.
Aiba et al, Immunity 24: 259-68, 2006.
Akashi et al, J. Rheumatol 17:375-379, 1990.
Alcocer-Varela <?i iz/., Lupus 1 : 1 11-7, 1992.
Altman et al, Blood 121 , 3675-81, 2013.
Andersson and Sundler, Cytokine 33: 52-7, 2006.
Banerjee et al, J. Virol 76: 5937-48, 2002.
Barr et al, Euro. J. Immunol 37: 3040-53, 2007.
Barr et al, J. Immunol. 185: 2783-9, 2010.
Barr el/., J. Exp. Med. 209: 1001-10, 2012.
Bernal-Quiros et al, PLoS ONE 8: e59842, 2013.
Bernasconi et al, Blood 101 : 4500-4, 2003.
Bodanszky et al, J. Antibiot., 29(5):549-53, 1976.
Bollig et al, Biochem. Biophys. Res. Comm. 301 : 665-70, 2003.
Bosher and Labouesse, Nat. Cell. Biol, 2:E31-E36, 2000.
Caplan et al . Gene, 252(l-2):95-l 05, 2000.
Cassese et al, J. Immunol. 171 : 1684-90, 2003.
Castillejo-Lopez et al, Annals Rheumatic Dis. 71 : 136-42, 2012.
Cencic et al, Meth. Enzymol. 51 1, 437-61, 2012.
Christensen et al, J. Exp. Med. 202: 321 -31, 2005.
Cook e? al, Cell, 27:487-496, 1981.
Di Marco et al, Nature Commun. 3, 896, 2012.
Elbashir et al, Nature, 411(6836):494-498, 2001.
Emlen et al, J. Biol. Chem. 273 : 1741-8, 1998.
Eto et al., PLoS ONE 6: el 7739, 2011.
Fire et al, Nature, 391 :806-81 1 , 1998.
Fischer, Med. Res. Rev., 27(6):755-796, 2007.
Foey et al, J. Immunol. 160: 920-8, 1998.
Forster and Symons, Cell, 49:211-220, 1987.
Gerlach et al, Nature (London), 328:802-805, 1987.
Gingras et al, Genes & Devel. 13 : 1422-37, 1999.
Gingras et al, Ann. Rev.Bbiochem. 68, 913-63, 1999.
Grishok et al, Science, 287:2494-2497, 2000.
Haxhinasto and Bishop, J. Biol. Chem. 279: 2575-82, 2004.
Hwang et al, J. Immunol 189, 5786-96, 2012.
Izui et al , Int. Rev. Immunol. 19, 447-72, 2000.
Jordan et al, Clin. Immunol. Immunopathol. 53 : S164-169, 1989.
Joyce, Nature, 338:217-244, 1989.
Karnowski et al, J. Exp. Med. 209: 2049-64, 2012.
Ketting e/ a/., Cell, 99: 133-141, 1999.
Kim and Cook, Proc. Natl Acad. Sci. USA, 84:8788-8792, 1987.
Kozyrev <¾ «/., Genes Immun. 13: 129-38, 2012.
Kozyrev et al, Nat. Genet. 40: 211-6, 2008.
Lin and Avery, Nature, 402: 128-129, 1999.
Linker- Israeli et al, J. Immunol. 147: 1 17-23, 1991.
Livingstone et al, Chemistry & Biology 16: 1240-9, 2009.
Low et al, Molecular Cell 20, 709-22, 2005.
Merrifield, J. Am. Chem. Soc, 85:2149-2154, 1963.
Michel and Westhof, J. Mol. Biol , 216:585-610, 1990.
Montgomery et al, Proc. Natl. Acad. Sci. USA, 95: 155-2-15507, 1998.
Morel et al, Proc Natl Acad Sci USA 98, 1787-92, 2001.
Morley, Biochem. Soc. Trans. 25: 503-9, 1997.
Mourey et al, J. Pharmacol. Exp. Ther. 333 : 797-807, 2010.
Nagaleekar et al, J. Immunol. 186: 4140-6, 201 1.
Neininger et al, J. Biol Chem. 277: 3065-8, 2002.
Peptide Synthesis, 1985
Poudrier et al, Euro. J. Immunol. 28: 3371-83, 1998.
Protective Groups in Organic Chemistry, 1973
Raught and Gingras, Int 'lJ. Biochem. & Cell Biol. 3 1 : 43-57, 1999.
Reinhold-Hurek and Shub, Nature, 357: 173-176, 1992.
Richter and Sonenberg, Nature 433 : 477-80, 2005.
Ronkina et al, i 27: 170-81, 2007.
Rowlett et al, American J. Physiology 294: G452-9, 2008.
Ruperto et al, Lupus 0: 1-10, 2010.
Saito h a/., J. Clinical Invest. 109: 1453-62, 2002.
Sarver et al, Science, 247: 1222-1225, 1990.
Scanlon et al, Proc. Natl. Acad. Sci. USA, 88: 10591-10595, 1991.
Sharp and Zamore, Science 287:2431-2433, 2000.
Sharp, Genes Dev., 13 : 139-141, 1999.
Shi et al. Arthritis Rheum 56, 3057-69, 2007.
Shveygert ei a/., Mol. Cell. Biol. 30: 5160-7, 2010.
Sobel et al, J. Immunol. 169, 2694-700, 2002.
Solid Phase Peptide Synthelia, 1984
Sonenberg, Biochem. Cell Biol. 86: 178-83, 2008.
Stuart et al, Clin. Exp. Rheumatol. 13 : 17-22, 1995.
Subramanian et al, Proc Natl Acad Sci USA 103, 9970-5, 2006.
Sun et al, lnflamm. Allergy Drug Targets 6: 223-35, 2007.
Tabara ei a/., Cell, 99: 123-132, 1999.
Troutman et al , Proc. Natl. Acad. Sci. USA, 109: 273-8, 2012.
U.S. Patent 4,415,732
U.S. Patent 4,458,066
U.S. Patent 5,354,855
U.S. Patent 5,795,715
U.S. Patent 5,889, 136
U.S. Patent 5,889, 155
U.S. Patent 6,261,569
U.S. Patent Publication 20100144805
Vanden Bush et al, Euro. J. Immunol. 38: 400-9, 2008.
Vuyyuru et al. , J. Immunol. 183, 5716-27, 2009.
Wincott et al, Nucleic Acids Res., 23(14):2677-2684, 1995.
WO 00/44914
WO 01/36646
WO 01/68836
WO 99/32619
Wu etal, J. Immunol 191, 6110-6, 2013. Yan etal, J. Autoimmun.38: 1-9, 2012. Yokoyama et al , EMBO J.21: 83-92, 2002.
Claims
1. A method of treating a subject having systemic lupus erythematosus (SLE) comprising administering to said subject an inhibitor of eIF4 function.
2. The method of claim 1, wherein administering comprises intravenous administration, intra-arterial administration, oral administration or intramuscular administration.
3. The method of claim 1, further comprising administering to said subject one or more standard SLE therapies.
4. The method of claim 3, wherein said one or more standard SLE therapies are selected from a corticosteroid, an NSAID, an immunosuppressant, and anti-malarial drug or an antibody.
5. The method of claim 3, wherein said one or more standard therapies are provided before or after said inhibitor.
6. The method of claim 3, wherein said one or more standard therapies are provided at the same time as said inhibitor.
7. The method of claim 1, wherein said inhibitor is an eIF4E inhibitor.
8. The method of claim 7, wherein said inhibitor is ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E.
9. The method of claim 1, wherein said inhibitor is an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor.
10. The method of claim 1, wherein said inhibitor is given more than once.
1 1. The method of claim 10, comprising chronic administration.
12. The method of claim 1, wherein administering comprises providing a microorganism to the subject that produces the inhibitor.
13. A method of reducing flare duration or severity in a subject having systemic lupus erythematosus (SLE) comprising administering to said subject an inhibitor of eIF4 function.
14. The method of claim 13, wherein administering comprises intravenous administration, intra-arterial administration, oral administration or intramuscular administration.
15. The method of claim 13, further comprising administering to said subject one or more standard SLE therapies.
16. The method of claim 15, wherein said one or more standard SLE therapies are selected from a corticosteroid, an NSAID, an immunosuppressant, an anti-malarial drug or an antibody.
17. The method of claim 15, wherein said one or more standard therapies are provided before or after said inhibitor.
18. The method of claim 15, wherein said one or more standard therapies are provided at the same time as said inhibitor.
19. The method of claim 13, wherein said inhibitor is an eIF4E inhibitor.
20. The method of claim 19, wherein said inhibitor is ribavirin, an antisense molecule or interfering RNA directed at eIF4E, a dominant negative eIF4E or a competing peptide fragment of eIF4E.
21. The method of claim 13, wherein said inhibitor is an inhibitor of eIF4E binding to eIF4G or eIF4A, and eIF4E function or expression inhibitor, a MNK1/2 inhibitor or an mTOR ATP active site inhibitor.
22. The method of claim 13, wherein said inhibitor is given more than once.
23. The method of claim 22, comprising chronic administration.
24. The method of claim 13, wherein administering comprises providing a microorganism to the subject that produces the inhibitor.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361885859P | 2013-10-02 | 2013-10-02 | |
US61/885,859 | 2013-10-02 |
Publications (3)
Publication Number | Publication Date |
---|---|
WO2015050957A2 true WO2015050957A2 (en) | 2015-04-09 |
WO2015050957A8 WO2015050957A8 (en) | 2015-09-11 |
WO2015050957A3 WO2015050957A3 (en) | 2015-11-12 |
Family
ID=52779279
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2014/058576 WO2015050957A2 (en) | 2013-10-02 | 2014-10-01 | Treatments for systemic lupus erythematosus |
Country Status (1)
Country | Link |
---|---|
WO (1) | WO2015050957A2 (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN105902533A (en) * | 2016-05-23 | 2016-08-31 | 浙江省医学科学院 | Application of compound cercosporamide in preparation of immunoenhancer |
US9458175B2 (en) | 2014-09-17 | 2016-10-04 | Celgene Avilomics Research, Inc. | MK2 inhibitors and uses thereof |
WO2021113364A1 (en) * | 2019-12-02 | 2021-06-10 | New York University | Selective inhibition of t follicular helper cells for treatment of autoimmune disorders |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9168286B2 (en) * | 2005-10-13 | 2015-10-27 | Human Genome Sciences, Inc. | Methods and compositions for use in treatment of patients with autoantibody positive disease |
US20110224223A1 (en) * | 2008-07-08 | 2011-09-15 | The Regents Of The University Of California, A California Corporation | MTOR Modulators and Uses Thereof |
CA2712747A1 (en) * | 2010-08-11 | 2012-02-11 | Universite De Montreal | Combinatory cancer treatment |
JP6130391B2 (en) * | 2011-11-23 | 2017-05-17 | インテリカイン, エルエルシー | Enhanced treatment regimen using MTOR inhibitors |
-
2014
- 2014-10-01 WO PCT/US2014/058576 patent/WO2015050957A2/en active Application Filing
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9458175B2 (en) | 2014-09-17 | 2016-10-04 | Celgene Avilomics Research, Inc. | MK2 inhibitors and uses thereof |
US9790235B2 (en) | 2014-09-17 | 2017-10-17 | Celgene Car Llc | MK2 inhibitors and uses thereof |
US10253040B1 (en) | 2014-09-17 | 2019-04-09 | Celgene Car Llc | MK2 inhibitors and uses thereof |
US10577380B2 (en) | 2014-09-17 | 2020-03-03 | Celgene Car Llc | MK2 inhibitors and uses thereof |
US11584757B2 (en) | 2014-09-17 | 2023-02-21 | Celgene Car Llc | MK2 inhibitors and uses thereof |
CN105902533A (en) * | 2016-05-23 | 2016-08-31 | 浙江省医学科学院 | Application of compound cercosporamide in preparation of immunoenhancer |
CN105902533B (en) * | 2016-05-23 | 2018-04-20 | 浙江省医学科学院 | Purposes of the compound cercosporamide in immunopotentiator is prepared |
WO2021113364A1 (en) * | 2019-12-02 | 2021-06-10 | New York University | Selective inhibition of t follicular helper cells for treatment of autoimmune disorders |
US11602524B2 (en) | 2019-12-02 | 2023-03-14 | New York University | Selective inhibition of T Follicular Helper cells for treatment of autoimmune disorders |
Also Published As
Publication number | Publication date |
---|---|
WO2015050957A3 (en) | 2015-11-12 |
WO2015050957A8 (en) | 2015-09-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10736910B2 (en) | Treatment of autophagy-based disorders and related pharmaceutical compositions, diagnostic and screening assays and kits | |
EP1768677B1 (en) | Nucleic acids for the treatment of hmgb1-related pathologies | |
Antonioli et al. | Adenosine deaminase in the modulation of immune system and its potential as a novel target for treatment of inflammatory disorders | |
ES2918501T3 (en) | Human mesothelin chimeric antigen receptors and uses thereof | |
JP6012591B2 (en) | Aptamers to β-NGF and their use in the treatment of β-NGF mediated diseases and disorders | |
US20160215042A1 (en) | Methods for modulating immune responses during chronic immune conditions by targeting metallothioneins | |
TW201934552A (en) | Diaryl substituted 5,5-fused ring compounds as C5aR inhibitors | |
JP2011514881A (en) | Use of TAM receptor inhibitors as immunopotentiators and use of TAM activators as immunosuppressants | |
Quinteiro et al. | 15-deoxy-Δ12, 14-prostaglandin J2 reduces albumin-induced arthritis in temporomandibular joint of rats | |
US20130017188A1 (en) | Modulation of sgk1 expression in th17 cells to modulate th17-mediated immune responses | |
KR20140019303A (en) | Compositions and methods for treating or preventing lupus | |
EP2895170A1 (en) | Inhibition of viral infection-triggered asthma with c-kit inhibitor | |
US20140127231A1 (en) | p38 MAPK Pathway Inhibitors as Female-Specific Therapeutics | |
CA3166135A1 (en) | The combination of cyclin dependent kinase 7 inhibitor and immunotherapy for treatment of cancer | |
US20180275123A1 (en) | Peptides for blocking il1rap protein-protein interaction and uses thereof for treatment of disease | |
WO2015050957A2 (en) | Treatments for systemic lupus erythematosus | |
JP2002501499A (en) | Uses of immunomodulators | |
CN115768479A (en) | Treating rheumatoid arthritis | |
EP3532494A2 (en) | Methods and compositions for modulaton of transforming growth factor beta-regulated functions | |
US8933011B2 (en) | Treatment of preterm labor with toll-like receptor 9 antagonists | |
US20210163609A1 (en) | Method for treating autoimmune disease | |
US11571427B2 (en) | Targeting the CBM signalosome complex induces regulatory T cells to inflame the tumor microenvironment | |
JP2020511418A (en) | Methods of treating diseases associated with ILC2 cells | |
JP6249453B2 (en) | Aptamers against IL-17 and use thereof | |
WO2024145365A2 (en) | Compositions for the diagnosis, treatment, prevention, and alleviation of neurodegenerative and autoimmune disorders |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14850884 Country of ref document: EP Kind code of ref document: A2 |
|
DPE1 | Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101) | ||
NENP | Non-entry into the national phase in: |
Ref country code: DE |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 14850884 Country of ref document: EP Kind code of ref document: A2 |