WO2020228360A1 - 一种治疗代谢疾病的融合蛋白 - Google Patents
一种治疗代谢疾病的融合蛋白 Download PDFInfo
- Publication number
- WO2020228360A1 WO2020228360A1 PCT/CN2020/070234 CN2020070234W WO2020228360A1 WO 2020228360 A1 WO2020228360 A1 WO 2020228360A1 CN 2020070234 W CN2020070234 W CN 2020070234W WO 2020228360 A1 WO2020228360 A1 WO 2020228360A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- seq
- fusion protein
- fragment
- amino acid
- acid sequence
- Prior art date
Links
- 102000037865 fusion proteins Human genes 0.000 title claims abstract description 115
- 108020001507 fusion proteins Proteins 0.000 title claims abstract description 115
- 238000011282 treatment Methods 0.000 title abstract description 19
- 208000030159 metabolic disease Diseases 0.000 title abstract description 6
- 208000016097 disease of metabolism Diseases 0.000 title abstract 2
- 239000012634 fragment Substances 0.000 claims abstract description 105
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon Chemical group C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 MASNOZXLGMXCHN-ZLPAWPGGSA-N 0.000 claims abstract description 81
- 108090000765 processed proteins & peptides Proteins 0.000 claims abstract description 75
- 102000004196 processed proteins & peptides Human genes 0.000 claims abstract description 69
- 229920001184 polypeptide Polymers 0.000 claims abstract description 65
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 57
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 49
- 238000002360 preparation method Methods 0.000 claims abstract description 21
- 150000001413 amino acids Chemical group 0.000 claims description 81
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 claims description 46
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 claims description 45
- 108010075254 C-Peptide Proteins 0.000 claims description 41
- 102000007079 Peptide Fragments Human genes 0.000 claims description 38
- 239000003814 drug Substances 0.000 claims description 25
- 238000000034 method Methods 0.000 claims description 24
- 229940079593 drug Drugs 0.000 claims description 23
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 16
- 201000010099 disease Diseases 0.000 claims description 15
- 230000014509 gene expression Effects 0.000 claims description 15
- 108060003951 Immunoglobulin Proteins 0.000 claims description 12
- 210000004899 c-terminal region Anatomy 0.000 claims description 12
- 102000018358 immunoglobulin Human genes 0.000 claims description 12
- 108091033319 polynucleotide Proteins 0.000 claims description 10
- 102000040430 polynucleotide Human genes 0.000 claims description 10
- 239000002157 polynucleotide Substances 0.000 claims description 10
- 239000008194 pharmaceutical composition Substances 0.000 claims description 8
- 230000002503 metabolic effect Effects 0.000 claims description 6
- 238000012258 culturing Methods 0.000 claims description 5
- 229940072221 immunoglobulins Drugs 0.000 claims description 4
- 238000012217 deletion Methods 0.000 claims description 3
- 230000037430 deletion Effects 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 229910052698 phosphorus Inorganic materials 0.000 claims description 3
- 230000000694 effects Effects 0.000 abstract description 55
- 210000004369 blood Anatomy 0.000 abstract description 24
- 239000008280 blood Substances 0.000 abstract description 24
- 241000699670 Mus sp. Species 0.000 abstract description 22
- 230000004580 weight loss Effects 0.000 abstract description 14
- 230000009467 reduction Effects 0.000 abstract description 2
- 125000003275 alpha amino acid group Chemical group 0.000 abstract 2
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 46
- 102000007446 Glucagon-Like Peptide-1 Receptor Human genes 0.000 description 46
- 108060003199 Glucagon Proteins 0.000 description 45
- 102000051325 Glucagon Human genes 0.000 description 44
- 229960004666 glucagon Drugs 0.000 description 44
- 108010063919 Glucagon Receptors Proteins 0.000 description 41
- 102100040890 Glucagon receptor Human genes 0.000 description 41
- 235000018102 proteins Nutrition 0.000 description 40
- 210000004027 cell Anatomy 0.000 description 38
- 235000001014 amino acid Nutrition 0.000 description 29
- 230000001270 agonistic effect Effects 0.000 description 28
- 206010012601 diabetes mellitus Diseases 0.000 description 27
- 239000000556 agonist Substances 0.000 description 22
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 22
- DTHNMHAUYICORS-KTKZVXAJSA-N Glucagon-like peptide 1 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 DTHNMHAUYICORS-KTKZVXAJSA-N 0.000 description 19
- 102100040918 Pro-glucagon Human genes 0.000 description 19
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 18
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 18
- 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 16
- 208000008589 Obesity Diseases 0.000 description 16
- 238000001514 detection method Methods 0.000 description 16
- 239000008103 glucose Substances 0.000 description 16
- 235000020824 obesity Nutrition 0.000 description 16
- 230000037396 body weight Effects 0.000 description 15
- 230000002218 hypoglycaemic effect Effects 0.000 description 15
- 230000004927 fusion Effects 0.000 description 14
- 210000002966 serum Anatomy 0.000 description 14
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 description 13
- 239000013612 plasmid Substances 0.000 description 13
- 208000016261 weight loss Diseases 0.000 description 13
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 12
- 230000006870 function Effects 0.000 description 11
- 230000001965 increasing effect Effects 0.000 description 11
- 239000002609 medium Substances 0.000 description 11
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 description 9
- 108010011459 Exenatide Proteins 0.000 description 9
- 102100039997 Gastric inhibitory polypeptide receptor Human genes 0.000 description 9
- 101000886866 Homo sapiens Gastric inhibitory polypeptide receptor Proteins 0.000 description 9
- 102000004877 Insulin Human genes 0.000 description 9
- 108090001061 Insulin Proteins 0.000 description 9
- 229960001519 exenatide Drugs 0.000 description 9
- 229940125396 insulin Drugs 0.000 description 9
- 150000003626 triacylglycerols Chemical class 0.000 description 9
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 206010022489 Insulin Resistance Diseases 0.000 description 8
- 108010019598 Liraglutide Proteins 0.000 description 8
- YSDQQAXHVYUZIW-QCIJIYAXSA-N Liraglutide Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCNC(=O)CC[C@H](NC(=O)CCCCCCCCCCCCCCC)C(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=C(O)C=C1 YSDQQAXHVYUZIW-QCIJIYAXSA-N 0.000 description 8
- 238000005516 engineering process Methods 0.000 description 8
- 229960002701 liraglutide Drugs 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 7
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 7
- 239000000539 dimer Substances 0.000 description 7
- 238000000338 in vitro Methods 0.000 description 7
- 230000035772 mutation Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 6
- 108010007622 LDL Lipoproteins Proteins 0.000 description 6
- 102000007330 LDL Lipoproteins Human genes 0.000 description 6
- 208000001145 Metabolic Syndrome Diseases 0.000 description 6
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 6
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 6
- JUFFVKRROAPVBI-PVOYSMBESA-N chembl1210015 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)N[C@H]1[C@@H]([C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO[C@]3(O[C@@H](C[C@H](O)[C@H](O)CO)[C@H](NC(C)=O)[C@@H](O)C3)C(O)=O)O2)O)[C@@H](CO)O1)NC(C)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 JUFFVKRROAPVBI-PVOYSMBESA-N 0.000 description 6
- 230000009977 dual effect Effects 0.000 description 6
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 5
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 5
- 206010016654 Fibrosis Diseases 0.000 description 5
- 238000009825 accumulation Methods 0.000 description 5
- 230000004913 activation Effects 0.000 description 5
- 210000001367 artery Anatomy 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 235000012000 cholesterol Nutrition 0.000 description 5
- 238000006731 degradation reaction Methods 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- 239000013604 expression vector Substances 0.000 description 5
- 229940126864 fibroblast growth factor Drugs 0.000 description 5
- 230000037406 food intake Effects 0.000 description 5
- 235000012631 food intake Nutrition 0.000 description 5
- 208000006454 hepatitis Diseases 0.000 description 5
- 239000000859 incretin Substances 0.000 description 5
- 230000003914 insulin secretion Effects 0.000 description 5
- 210000004185 liver Anatomy 0.000 description 5
- 210000005229 liver cell Anatomy 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 238000012986 modification Methods 0.000 description 5
- 208000008338 non-alcoholic fatty liver disease Diseases 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 201000001320 Atherosclerosis Diseases 0.000 description 4
- 102100020683 Beta-klotho Human genes 0.000 description 4
- 108010077544 Chromatin Proteins 0.000 description 4
- 208000032928 Dyslipidaemia Diseases 0.000 description 4
- 108010004460 Gastric Inhibitory Polypeptide Proteins 0.000 description 4
- 102100039994 Gastric inhibitory polypeptide Human genes 0.000 description 4
- 208000013016 Hypoglycemia Diseases 0.000 description 4
- 208000017170 Lipid metabolism disease Diseases 0.000 description 4
- 108060001084 Luciferase Proteins 0.000 description 4
- 102400000319 Oxyntomodulin Human genes 0.000 description 4
- 101800001388 Oxyntomodulin Proteins 0.000 description 4
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 4
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 4
- 210000000577 adipose tissue Anatomy 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 210000003483 chromatin Anatomy 0.000 description 4
- 230000003247 decreasing effect Effects 0.000 description 4
- 235000014113 dietary fatty acids Nutrition 0.000 description 4
- 239000013613 expression plasmid Substances 0.000 description 4
- 229930195729 fatty acid Natural products 0.000 description 4
- 239000000194 fatty acid Substances 0.000 description 4
- 150000004665 fatty acids Chemical class 0.000 description 4
- 231100000283 hepatitis Toxicity 0.000 description 4
- 229940088597 hormone Drugs 0.000 description 4
- 239000005556 hormone Substances 0.000 description 4
- 230000005847 immunogenicity Effects 0.000 description 4
- MGXWVYUBJRZYPE-YUGYIWNOSA-N incretin Chemical class C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(N)=O)C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)[C@@H](C)O)[C@@H](C)CC)C1=CC=C(O)C=C1 MGXWVYUBJRZYPE-YUGYIWNOSA-N 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 210000004962 mammalian cell Anatomy 0.000 description 4
- PXZWGQLGAKCNKD-DPNMSELWSA-N molport-023-276-326 Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O)[C@@H](C)O)C(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C1=CC=CC=C1 PXZWGQLGAKCNKD-DPNMSELWSA-N 0.000 description 4
- RXWNCPJZOCPEPQ-NVWDDTSBSA-N puromycin Chemical compound C1=CC(OC)=CC=C1C[C@H](N)C(=O)N[C@H]1[C@@H](O)[C@H](N2C3=NC=NC(=C3N=C2)N(C)C)O[C@@H]1CO RXWNCPJZOCPEPQ-NVWDDTSBSA-N 0.000 description 4
- 230000004044 response Effects 0.000 description 4
- 230000037390 scarring Effects 0.000 description 4
- 231100000240 steatosis hepatitis Toxicity 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 206010003210 Arteriosclerosis Diseases 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- HTQBXNHDCUEHJF-XWLPCZSASA-N Exenatide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(=O)NCC(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CO)C(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)CNC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 HTQBXNHDCUEHJF-XWLPCZSASA-N 0.000 description 3
- 239000012739 FreeStyle 293 Expression medium Substances 0.000 description 3
- 239000004471 Glycine Substances 0.000 description 3
- 108010010234 HDL Lipoproteins Proteins 0.000 description 3
- 102000015779 HDL Lipoproteins Human genes 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 3
- 108010028554 LDL Cholesterol Proteins 0.000 description 3
- 239000005089 Luciferase Substances 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 108700008625 Reporter Genes Proteins 0.000 description 3
- DLSWIYLPEUIQAV-UHFFFAOYSA-N Semaglutide Chemical compound CCC(C)C(NC(=O)C(Cc1ccccc1)NC(=O)C(CCC(O)=O)NC(=O)C(CCCCNC(=O)COCCOCCNC(=O)COCCOCCNC(=O)CCC(NC(=O)CCCCCCCCCCCCCCCCC(O)=O)C(O)=O)NC(=O)C(C)NC(=O)C(C)NC(=O)C(CCC(N)=O)NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(CC(C)C)NC(=O)C(Cc1ccc(O)cc1)NC(=O)C(CO)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(Cc1ccccc1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)C(C)(C)NC(=O)C(N)Cc1cnc[nH]1)C(C)O)C(C)O)C(C)C)C(=O)NC(C)C(=O)NC(Cc1c[nH]c2ccccc12)C(=O)NC(CC(C)C)C(=O)NC(C(C)C)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CCCNC(N)=N)C(=O)NCC(O)=O DLSWIYLPEUIQAV-UHFFFAOYSA-N 0.000 description 3
- 230000001154 acute effect Effects 0.000 description 3
- 235000004279 alanine Nutrition 0.000 description 3
- 239000003472 antidiabetic agent Substances 0.000 description 3
- 230000036528 appetite Effects 0.000 description 3
- 235000019789 appetite Nutrition 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 238000004113 cell culture Methods 0.000 description 3
- 238000010382 chemical cross-linking Methods 0.000 description 3
- 230000007882 cirrhosis Effects 0.000 description 3
- 208000019425 cirrhosis of liver Diseases 0.000 description 3
- 150000001875 compounds Chemical group 0.000 description 3
- 238000010276 construction Methods 0.000 description 3
- 230000001419 dependent effect Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 230000030136 gastric emptying Effects 0.000 description 3
- 230000012010 growth Effects 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 238000006460 hydrolysis reaction Methods 0.000 description 3
- 230000010354 integration Effects 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 230000004060 metabolic process Effects 0.000 description 3
- 230000017074 necrotic cell death Effects 0.000 description 3
- 230000001766 physiological effect Effects 0.000 description 3
- 239000013641 positive control Substances 0.000 description 3
- 229940044601 receptor agonist Drugs 0.000 description 3
- 239000000018 receptor agonist Substances 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 108010060325 semaglutide Proteins 0.000 description 3
- 229950011186 semaglutide Drugs 0.000 description 3
- 150000003384 small molecules Chemical class 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- 208000024891 symptom Diseases 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- -1 trade name ) Chemical compound 0.000 description 3
- 238000001890 transfection Methods 0.000 description 3
- YEKUUBPJRPXMBM-PTCFZACGSA-N (2S)-5-[[(5S)-5-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S,3R)-2-[[2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]acetyl]amino]-3-hydroxybutanoyl]amino]-3-phenylpropanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-6-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-5-carbamimidamido-1-[[(2S)-1-[[(2S)-5-carbamimidamido-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-carboxy-1-[[(2S)-1-[[2-(carboxymethylamino)-2-oxoethyl]amino]-1-oxopropan-2-yl]amino]-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopentan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-6-oxohexyl]amino]-2-(hexadecanoylamino)-5-oxopentanoic acid Chemical compound CCCCCCCCCCCCCCCC(=O)N[C@@H](CCC(=O)NCCCC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)Cc1c[nH]cn1)[C@@H](C)O)[C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)NCC(=O)NCC(O)=O)C(O)=O YEKUUBPJRPXMBM-PTCFZACGSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 101710104526 Beta-klotho Proteins 0.000 description 2
- 101800001415 Bri23 peptide Proteins 0.000 description 2
- 101800000655 C-terminal peptide Proteins 0.000 description 2
- 102400000107 C-terminal peptide Human genes 0.000 description 2
- 241000282693 Cercopithecidae Species 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000004930 Fatty Liver Diseases 0.000 description 2
- 108010023302 HDL Cholesterol Proteins 0.000 description 2
- 206010019708 Hepatic steatosis Diseases 0.000 description 2
- 102100031000 Hepatoma-derived growth factor Human genes 0.000 description 2
- 101000886868 Homo sapiens Gastric inhibitory polypeptide Proteins 0.000 description 2
- 101001083798 Homo sapiens Hepatoma-derived growth factor Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 238000008214 LDL Cholesterol Methods 0.000 description 2
- XVVOERDUTLJJHN-UHFFFAOYSA-N Lixisenatide Chemical compound C=1NC2=CC=CC=C2C=1CC(C(=O)NC(CC(C)C)C(=O)NC(CCCCN)C(=O)NC(CC(N)=O)C(=O)NCC(=O)NCC(=O)N1C(CCC1)C(=O)NC(CO)C(=O)NC(CO)C(=O)NCC(=O)NC(C)C(=O)N1C(CCC1)C(=O)N1C(CCC1)C(=O)NC(CO)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(=O)NC(CCCCN)C(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)CC)NC(=O)C(NC(=O)C(CC(C)C)NC(=O)C(CCCNC(N)=N)NC(=O)C(NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(CCC(O)=O)NC(=O)C(CCC(O)=O)NC(=O)C(CCSC)NC(=O)C(CCC(N)=O)NC(=O)C(CCCCN)NC(=O)C(CO)NC(=O)C(CC(C)C)NC(=O)C(CC(O)=O)NC(=O)C(CO)NC(=O)C(NC(=O)C(CC=1C=CC=CC=1)NC(=O)C(NC(=O)CNC(=O)C(CCC(O)=O)NC(=O)CNC(=O)C(N)CC=1NC=NC=1)C(C)O)C(C)O)C(C)C)CC1=CC=CC=C1 XVVOERDUTLJJHN-UHFFFAOYSA-N 0.000 description 2
- 102000003729 Neprilysin Human genes 0.000 description 2
- 108090000028 Neprilysin Proteins 0.000 description 2
- 239000012124 Opti-MEM Substances 0.000 description 2
- 208000001132 Osteoporosis Diseases 0.000 description 2
- 241000283984 Rodentia Species 0.000 description 2
- 239000007983 Tris buffer Substances 0.000 description 2
- 230000002159 abnormal effect Effects 0.000 description 2
- 229960004733 albiglutide Drugs 0.000 description 2
- OGWAVGNOAMXIIM-UHFFFAOYSA-N albiglutide Chemical compound O=C(O)C(NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)CNC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)CNC(=O)C(NC(=O)CNC(=O)C(N)CC=1(N=CNC=1))CCC(=O)O)C(O)C)CC2(=CC=CC=C2))C(O)C)CO)CC(=O)O)C(C)C)CO)CO)CC3(=CC=C(O)C=C3))CC(C)C)CCC(=O)O)CCC(=O)N)C)C)CCCCN)CCC(=O)O)CC4(=CC=CC=C4))C(CC)C)C)CC=6(C5(=C(C=CC=C5)NC=6)))CC(C)C)C(C)C)CCCCN)CCCNC(=N)N OGWAVGNOAMXIIM-UHFFFAOYSA-N 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 2
- 230000008827 biological function Effects 0.000 description 2
- 210000004204 blood vessel Anatomy 0.000 description 2
- 239000004202 carbamide Substances 0.000 description 2
- 239000000969 carrier Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 210000004978 chinese hamster ovary cell Anatomy 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 108091005205 cotadutide Proteins 0.000 description 2
- 229940121426 cotadutide Drugs 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 230000002354 daily effect Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000002950 deficient Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 208000010706 fatty liver disease Diseases 0.000 description 2
- 230000004761 fibrosis Effects 0.000 description 2
- 230000036541 health Effects 0.000 description 2
- 208000019622 heart disease Diseases 0.000 description 2
- 238000001727 in vivo Methods 0.000 description 2
- 210000003000 inclusion body Anatomy 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 230000002427 irreversible effect Effects 0.000 description 2
- 102000005861 leptin receptors Human genes 0.000 description 2
- 108010019813 leptin receptors Proteins 0.000 description 2
- 210000000265 leukocyte Anatomy 0.000 description 2
- 108010004367 lixisenatide Proteins 0.000 description 2
- 229960001093 lixisenatide Drugs 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 238000006386 neutralization reaction Methods 0.000 description 2
- 230000003647 oxidation Effects 0.000 description 2
- 238000007254 oxidation reaction Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 230000003285 pharmacodynamic effect Effects 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 229950010131 puromycin Drugs 0.000 description 2
- 108700027806 rGLP-1 Proteins 0.000 description 2
- 238000003259 recombinant expression Methods 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000010532 solid phase synthesis reaction Methods 0.000 description 2
- 230000007863 steatosis Effects 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 238000010254 subcutaneous injection Methods 0.000 description 2
- 239000007929 subcutaneous injection Substances 0.000 description 2
- 238000001356 surgical procedure Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 2
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 1
- NGJOFQZEYQGZMB-KTKZVXAJSA-N (4S)-5-[[2-[[(2S,3R)-1-[[(2S)-1-[[(2S,3R)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-5-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[2-[[(1S)-4-carbamimidamido-1-carboxybutyl]amino]-2-oxoethyl]amino]-1-oxohexan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-1-oxohexan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-1,5-dioxopentan-2-yl]amino]-2-oxoethyl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-3-carboxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-2-oxoethyl]amino]-4-[[(2S)-2-[[(2S)-2-amino-3-(1H-imidazol-4-yl)propanoyl]amino]propanoyl]amino]-5-oxopentanoic acid Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 NGJOFQZEYQGZMB-KTKZVXAJSA-N 0.000 description 1
- NFGXHKASABOEEW-UHFFFAOYSA-N 1-methylethyl 11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate Chemical compound COC(C)(C)CCCC(C)CC=CC(C)=CC(=O)OC(C)C NFGXHKASABOEEW-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 208000004611 Abdominal Obesity Diseases 0.000 description 1
- 102000011690 Adiponectin Human genes 0.000 description 1
- 108010076365 Adiponectin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 108010074051 C-Reactive Protein Proteins 0.000 description 1
- 102100032752 C-reactive protein Human genes 0.000 description 1
- 101800001453 C-terminal extension peptide Proteins 0.000 description 1
- 101100337060 Caenorhabditis elegans glp-1 gene Proteins 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 206010065941 Central obesity Diseases 0.000 description 1
- 108020004705 Codon Proteins 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
- 208000030453 Drug-Related Side Effects and Adverse reaction Diseases 0.000 description 1
- 102000005593 Endopeptidases Human genes 0.000 description 1
- 108010059378 Endopeptidases Proteins 0.000 description 1
- 241000283086 Equidae Species 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 1
- 108010049003 Fibrinogen Proteins 0.000 description 1
- 102000008946 Fibrinogen Human genes 0.000 description 1
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 description 1
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 description 1
- 101150089905 Gcgr gene Proteins 0.000 description 1
- 229940123232 Glucagon receptor agonist Drugs 0.000 description 1
- 108010088406 Glucagon-Like Peptides Proteins 0.000 description 1
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 1
- 229940089838 Glucagon-like peptide 1 receptor agonist Drugs 0.000 description 1
- 101800004295 Glucagon-like peptide 1(7-36) Proteins 0.000 description 1
- 101800004266 Glucagon-like peptide 1(7-37) Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 101000846529 Homo sapiens Fibroblast growth factor 21 Proteins 0.000 description 1
- 101001015516 Homo sapiens Glucagon-like peptide 1 receptor Proteins 0.000 description 1
- 206010020710 Hyperphagia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 206010062767 Hypophysitis Diseases 0.000 description 1
- 108010009817 Immunoglobulin Constant Regions Proteins 0.000 description 1
- 102000009786 Immunoglobulin Constant Regions Human genes 0.000 description 1
- 102000001706 Immunoglobulin Fab Fragments Human genes 0.000 description 1
- 108010054477 Immunoglobulin Fab Fragments Proteins 0.000 description 1
- 108010021625 Immunoglobulin Fragments Proteins 0.000 description 1
- 102000008394 Immunoglobulin Fragments Human genes 0.000 description 1
- 208000026350 Inborn Genetic disease Diseases 0.000 description 1
- 239000012097 Lipofectamine 2000 Substances 0.000 description 1
- 102000004895 Lipoproteins Human genes 0.000 description 1
- 108090001030 Lipoproteins Proteins 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 206010033307 Overweight Diseases 0.000 description 1
- 241001494479 Pecora Species 0.000 description 1
- 208000018262 Peripheral vascular disease Diseases 0.000 description 1
- 102000003827 Plasma Kallikrein Human genes 0.000 description 1
- 108090000113 Plasma Kallikrein Proteins 0.000 description 1
- 108010022233 Plasminogen Activator Inhibitor 1 Proteins 0.000 description 1
- 102100039418 Plasminogen activator inhibitor 1 Human genes 0.000 description 1
- 108010058003 Proglucagon Proteins 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 241000282887 Suidae Species 0.000 description 1
- 229940100389 Sulfonylurea Drugs 0.000 description 1
- 229940123464 Thiazolidinedione Drugs 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 244000000188 Vaccinium ovalifolium Species 0.000 description 1
- 206010047700 Vomiting Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 210000001789 adipocyte Anatomy 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 229940127003 anti-diabetic drug Drugs 0.000 description 1
- 230000001315 anti-hyperlipaemic effect Effects 0.000 description 1
- 239000000883 anti-obesity agent Substances 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000002220 antihypertensive agent Substances 0.000 description 1
- 229940127088 antihypertensive drug Drugs 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000003143 atherosclerotic effect Effects 0.000 description 1
- 238000007681 bariatric surgery Methods 0.000 description 1
- 239000003613 bile acid Substances 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000008512 biological response Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 235000019577 caloric intake Nutrition 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000019522 cellular metabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011097 chromatography purification Methods 0.000 description 1
- 239000012539 chromatography resin Substances 0.000 description 1
- 208000037998 chronic venous disease Diseases 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 239000012228 culture supernatant Substances 0.000 description 1
- 238000009109 curative therapy Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000006240 deamidation Effects 0.000 description 1
- 238000000354 decomposition reaction Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000018823 dietary intake Nutrition 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 108010005794 dulaglutide Proteins 0.000 description 1
- 229960005175 dulaglutide Drugs 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 108010048367 enhanced green fluorescent protein Proteins 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 210000003158 enteroendocrine cell Anatomy 0.000 description 1
- 230000007071 enzymatic hydrolysis Effects 0.000 description 1
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 229950003499 fibrin Drugs 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 238000001917 fluorescence detection Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000003629 gastrointestinal hormone Substances 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 208000016361 genetic disease Diseases 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000004104 gestational diabetes Diseases 0.000 description 1
- 230000004110 gluconeogenesis Effects 0.000 description 1
- 230000014101 glucose homeostasis Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 230000004116 glycogenolysis Effects 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 235000009200 high fat diet Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000003054 hormonal effect Effects 0.000 description 1
- 102000053391 human F Human genes 0.000 description 1
- 108700031895 human F Proteins 0.000 description 1
- 102000056713 human FGF21 Human genes 0.000 description 1
- 201000001421 hyperglycemia Diseases 0.000 description 1
- 210000003016 hypothalamus Anatomy 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000016784 immunoglobulin production Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 210000004969 inflammatory cell Anatomy 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002576 ketones Chemical class 0.000 description 1
- 239000003446 ligand Substances 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 208000018191 liver inflammation Diseases 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000003468 luciferase reporter gene assay Methods 0.000 description 1
- 235000012054 meals Nutrition 0.000 description 1
- 210000001704 mesoblast Anatomy 0.000 description 1
- 230000006371 metabolic abnormality Effects 0.000 description 1
- 230000011278 mitosis Effects 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- GNZCSGYHILBXLL-UHFFFAOYSA-N n-tert-butyl-6,7-dichloro-3-methylsulfonylquinoxalin-2-amine Chemical compound ClC1=C(Cl)C=C2N=C(S(C)(=O)=O)C(NC(C)(C)C)=NC2=C1 GNZCSGYHILBXLL-UHFFFAOYSA-N 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 238000007857 nested PCR Methods 0.000 description 1
- 235000020925 non fasting Nutrition 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 238000007410 oral glucose tolerance test Methods 0.000 description 1
- 230000036542 oxidative stress Effects 0.000 description 1
- 238000002638 palliative care Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000000737 periodic effect Effects 0.000 description 1
- 230000004526 pharmaceutical effect Effects 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 210000003635 pituitary gland Anatomy 0.000 description 1
- 229940012957 plasmin Drugs 0.000 description 1
- 230000004983 pleiotropic effect Effects 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- GCYXWQUSHADNBF-AAEALURTSA-N preproglucagon 78-108 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC=1N=CNC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 GCYXWQUSHADNBF-AAEALURTSA-N 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000003449 preventive effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000770 proinflammatory effect Effects 0.000 description 1
- 210000001236 prokaryotic cell Anatomy 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000012562 protein A resin Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 230000005180 public health Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000011808 rodent model Methods 0.000 description 1
- 230000036186 satiety Effects 0.000 description 1
- 235000019627 satiety Nutrition 0.000 description 1
- 230000036573 scar formation Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000000391 smoking effect Effects 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 230000010473 stable expression Effects 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 208000023516 stroke disease Diseases 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 108010048573 taspoglutide Proteins 0.000 description 1
- WRGVLTAWMNZWGT-VQSPYGJZSA-N taspoglutide Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)NC(C)(C)C(=O)N[C@@H](CCCNC(N)=N)C(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@H](CC=1C=CC=CC=1)NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)C(C)(C)NC(=O)[C@@H](N)CC=1NC=NC=1)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 WRGVLTAWMNZWGT-VQSPYGJZSA-N 0.000 description 1
- 229950007151 taspoglutide Drugs 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 150000001467 thiazolidinediones Chemical class 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 239000003053 toxin Substances 0.000 description 1
- 231100000765 toxin Toxicity 0.000 description 1
- 108700012359 toxins Proteins 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- 230000010080 triple agonism Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 210000001186 vagus nerve Anatomy 0.000 description 1
- 230000002792 vascular Effects 0.000 description 1
- 208000019553 vascular disease Diseases 0.000 description 1
- 230000024883 vasodilation Effects 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/04—Anorexiants; Antiobesity agents
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/605—Glucagons
-
- 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
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- 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
- A61K38/22—Hormones
- A61K38/26—Glucagons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
- A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
- A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
- A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/475—Growth factors; Growth regulators
- C07K14/50—Fibroblast growth factor [FGF]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/62—DNA sequences coding for fusion proteins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0684—Cells of the urinary tract or kidneys
- C12N5/0686—Kidney cells
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/106—Plasmid DNA for vertebrates
- C12N2800/107—Plasmid DNA for vertebrates for mammalian
Definitions
- the invention relates to the field of biotechnology, in particular to a fusion protein for treating metabolic diseases, and a preparation method and application thereof.
- Type 1 diabetes is mainly manifested by insufficient insulin secretion, which requires daily injections of insulin; while type 2 diabetes is caused by the body's inefficient use of insulin.
- type 2 diabetes patients account for the vast majority. It is estimated that about 80-90% of patients with type 2 diabetes are obviously obese (Center for disease control and prevention (CDC) National Diabetes Fact Sheet, 2014).
- GLP-1R GLP-1 receptor
- Dulaglutide trade name:
- Albiglutide Albiglutide, trade name
- Liraglutide Liraglutide, trade name and Respectively used to treat obesity and diabetes
- Exenatide Exenatide, trade name
- Lixisenatide Lixisenatide
- Semaglutide Semaglutide
- GLP-1R agonists have a significant hypoglycemic effect, and unlike insulin, the hypoglycemic effect of GLP-1R agonists is strictly blood sugar-dependent, not easy to cause hypoglycemia, and has a weight-lowering effect.
- these drugs have side effects in treatment, mainly gastrointestinal effects, such as nausea, and the major part of the reduction is not more than 10% of the average body weight.
- Bariatric surgery can significantly improve obesity and treat diabetes, it is not widely used, because most patients are unwilling to accept this type of surgery due to the risks of surgery and long-term sequelae (Obesity and Diabetes, New Surgical and Nonsurgical Approaches, Springer Press, 2015).
- the current new generation of diabetes drugs is mainly focused on the research of dual- or multi-acting incretin receptor agonists, such as GLP-1R/GCGR and GLP-1R/GIPR dual-acting agonists, and even GLP-1R/ GCGR/GIPR triple-acting agonist (Nature Medicine, 22(7):694-695, 2016).
- GCG glucagon
- GLP-1 Glucagon-like peptide-1
- GLP-1 and GLP-1 Glucagon-like peptide-1
- Clinically, GLP-1 and its analogues are mainly used for blood sugar control in diabetic patients, while Glucagon (GCG) is used for acute hypoglycemia.
- Glucagon can effectively reduce weight; more importantly, GLP-1 and Glucagon seem to have a positive addition or synergy Physiological effects, such as Glucagon receptor (GCGR) and GLP-1 receptor (GLP-1R) dual agonists can reduce weight more effectively than GLP-1R single agonists.
- GCGR Glucagon receptor
- GLP-1R GLP-1 receptor
- GCGR activation may lead to increased blood glucose levels, this risk can be appropriately offset by GLP-1R activation and/or GIPR activation.
- Matthias H. A review article by et al. described in detail the various hybrid peptide forms currently in clinical or pre-clinical conditions (Cell Metab, 24(1):51-62, 2016). Alessandro Pocai et al.
- MEDI0382 although the second position of MEDI0382 also retains the natural Ser amino acid, it can only support the once-a-day dosing frequency.
- PEG cross-linking or fatty acid cross-linking are currently more commonly used technical means, such as Semaglutide (Semaglutide), but it involves chemical synthesis and cross-linking steps, so the preparation process is more complicated.
- FGF21 As an important metabolic regulator, FGF21 has been proven to improve a variety of metabolic abnormalities in preclinical model of type II diabetes (T2DM) experiments. As a therapeutic drug for diabetic patients, FGF21 improves insulin sensitivity, improves blood sugar control, reduces weight, reduces low-density lipoprotein cholesterol (LDL-C) and triglycerides, and increases high-density lipoprotein cholesterol (HDL-C) levels. There is a potential role. In diabetic mice and monkeys, human FGF21 can reduce the fasting serum glucose concentration and reduce the fasting serum triglyceride, insulin and glucagon concentrations. In addition, in rodent models of diet-induced obesity, FGF21 administration resulted in a dose-dependent overall weight loss. Therefore, FGF21 has the potential to treat diseases such as diabetes, obesity, dyslipidemia and metabolic syndrome.
- T2DM type II diabetes
- FGF21 has a very short serum half-life: 30 minutes in mice and 2 hours in monkeys. Therefore, in order to maintain the biological activity in the body, daily injection or continuous infusion of the corresponding FGF21 protein is required.
- circulating FGF21 levels tend to increase in patients with obesity, dyslipidemia, TDM2 and other diseases related to insulin resistance. Studies have shown that increased FGF21 concentration is associated with increased CVD risk and can also lead to osteoporosis. Affect reproduction (promote metabolism and lead to insufficient energy), etc. (Proc Natl Acad Sci USA, 109(8): 3143-8, 2012; Mol Metab, 5(8): 690-8, 2016).
- the homology of the FGF family sequence and the wide distribution of FGFR1 receptors have also brought attention to the potential safety problems caused by the clinical high-dose use of FGF21 (J Intern Med, 281(3):233-246, 2017).
- GCGR/GLP-1R double-acting agonist, GLP-1R/GIPR double-acting agonist, GLP-1R/GCGR/GIPR triple-acting agonist and FGF21 analogues are used for the treatment of diabetes and weight loss, in addition to GLP -1 analog and FGF21 are fused with Fc to prepare a dual-active protein (The American Diabetes Association, 2016).
- the current double-acting or even triple-acting peptides modified based on Glucagon or oxyntomodulin generally require partial amino acids to be substituted for unnatural amino acids to improve stability and activity, and even require fatty acid or PEG modification.
- FGF21 analogues it is extremely difficult to fusion expression with FGF21 analogues to prepare a single molecule. At present, there is no report on the combination of double-acting or multi-acting agonist polypeptides and FGF21 analogs.
- the purpose of the present invention is to provide a fusion protein for treating metabolic diseases and a preparation method and application thereof to solve the problems in the prior art.
- a fusion protein for the treatment of metabolic diseases including a Glucagon analog fragment and a long-acting protein unit fragment, the Glucagon analog fragment includes:
- the polypeptide fragment in a) is selected from amino acid sequences such as SEQ ID NO.29, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.38 , SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44 shown in one of the polypeptide fragments.
- the long-acting protein fragments derived from unit F C portion of mammalian immunoglobulins are derived from unit F C portion of mammalian immunoglobulins.
- the long-acting protein unit fragment includes:
- a polypeptide fragment whose amino acid sequence has more than 90% sequence identity with one of SEQ ID Nos. 4 to 13 and has the function of the polypeptide fragment defined in c).
- the fusion protein further includes a first connecting peptide fragment located between the Glucagon analog fragment and the long-acting protein unit fragment.
- the first connecting peptide Fragments are rich in G, S and/or A.
- the first connecting peptide fragment includes a polypeptide fragment with an amino acid sequence as shown in one of SEQ ID NOs. 14-23.
- the fusion protein includes Glucagon analog fragments, first connecting peptide fragments and long-acting protein unit fragments in sequence from N-terminus to C-terminus.
- the amino acid sequence of the fusion protein is SEQ ID NO. 56, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 65, SEQ ID NO. 69 , SEQ ID NO.70, SEQ ID NO.71 shown in one of them.
- the fusion protein further includes FGF21 analog fragments.
- the FGF21 analog fragment includes:
- N-terminal HPIPDSS can be deleted or partly deleted;
- X 19 is selected from R, Y, V, E or C;
- X 31 is selected from A or C;
- X 36 is selected from R or K;
- X 43 is selected from G or C;
- X 45 is selected X is selected from A, K, E or V;
- X 56 is selected from K, R, V or I;
- X 98 is selected from L, R or D;
- X 118 is selected from L or C;
- X 122 is selected from K or R;
- X 134 is selected From A or C;
- X 167 is selected from S, A or R;
- X 170 is selected from G or E;
- X 171 is selected from P or G;
- X 174 is selected from G, A or L;
- X 179 is selected from Y, A or F ;
- X 180 is selected from A or E;
- X 181 is selected from
- the polypeptide fragment in e) is selected from the polypeptide fragments whose amino acid sequence is as shown in one of SEQ ID NOs. 87 to 90.
- the fusion protein further includes a second connecting peptide fragment, the second connecting peptide fragment is located between the long-acting protein unit fragment and the FGF21 analog fragment, preferably, the second connecting peptide Fragments are rich in G, S and/or A.
- the second connecting peptide fragment includes a polypeptide fragment whose amino acid sequence is as shown in one of SEQ ID NOs. 14-23.
- the fusion protein includes Glucagon analog fragments, first connecting peptide fragments, long-acting protein unit fragments, and FGF21 analog fragments in sequence from N-terminal to C-terminal;
- the fusion protein includes Glucagon analog fragment, first connecting peptide fragment, long-acting protein unit fragment, second connecting peptide fragment and FGF21 analog fragment in sequence from N-terminal to C-terminal.
- the amino acid sequence of the fusion protein is shown in one of SEQ ID NOs. 91-115.
- Another aspect of the present invention provides an isolated polynucleotide encoding the fusion protein.
- Another aspect of the present invention provides a construct containing the isolated polynucleotide.
- Another aspect of the present invention provides an expression system containing the construct or the exogenous polynucleotide integrated into the genome.
- Another aspect of the present invention provides a method for preparing the fusion protein, which comprises: culturing the expression system according to claim 17 under suitable conditions to express the fusion protein, and isolating and purifying to provide the fusion protein .
- Another aspect of the present invention provides a pharmaceutical composition comprising the fusion protein or the culture of the expression system.
- Another aspect of the present invention provides the use of the fusion protein and the pharmaceutical composition in the preparation of medicines.
- Another aspect of the present invention provides that the drug is selected from drugs for the treatment of metabolic-related diseases.
- Figure 1A shows a schematic diagram of the results of serum stability over time.
- Figure 1B shows a schematic diagram of the results of serum stability over time.
- Figure 2A is a schematic diagram showing the hypoglycemic effect of the fusion protein in Example 6 in db/db mice.
- Figure 2B is a schematic diagram showing the hypoglycemic effect of the fusion protein in Example 6 in db/db mice.
- Figure 3 is a schematic diagram showing the effect of the fusion protein in Example 7 on the body weight of DIO mice.
- the inventors of the present invention unexpectedly discovered that after fusion of GCG analogues with Fc, the effects on the activities of GLP-1R and GCGR are completely different, thereby providing a new fusion protein with The invention has good stability, and has good hypoglycemic and weight loss effects for mice, and thus has a good industrialization prospect. On this basis, the present invention has been completed.
- diabetes generally includes type 1 diabetes, type 2 diabetes, gestational diabetes, and other symptoms that cause hyperglycemia.
- the term is used for metabolic disorders, in which the pancreas does not produce enough insulin or the body's cells fail to respond properly to insulin, so the efficiency of tissue cells in absorbing glucose decreases and glucose accumulates in the blood.
- Type 1 diabetes is also called insulin-dependent diabetes and juvenile-onset diabetes. It is caused by the destruction of ⁇ cells and usually leads to absolute insulin deficiency.
- Type 2 diabetes also known as non-insulin-dependent diabetes and adult-onset diabetes, is generally associated with insulin resistance.
- Incretin is usually a gastrointestinal hormone that regulates blood sugar by enhancing glucose-stimulated insulin secretion (also known as glucose-dependent insulin secretion, GSIS) (Lancet, 368:1696-705, 2006). Incretin can also slow down the rate of nutrient absorption by delaying gastric emptying and directly reduce food absorption. At the same time, incretin also inhibits the secretion of glucagon from intestinal alpha cells. There are two known incretins so far: glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP).
- GLP-1 glucagon-like peptide-1
- GIP glucose-dependent insulinotropic polypeptide
- GIP is usually a 42-amino acid peptide obtained by proteolytic processing from a 133-amino acid precursor (pre-pro-GIP). These molecules are involved in a variety of biological functions, including glucose homeostasis and insulin secretion. , Gastric emptying and intestinal growth and regulation of food intake.
- GLP-1 is a 30 or 31 amino acid polypeptide incretin hormone secreted from intestinal L-cells, including GLP-1 (7-36) and GLP- 1(7-37) Two active forms. GLP-1 is released into the circulation after a meal and exerts biological activity by activating the GLP-1 receptor. GLP-1 has many biological effects, including glucose-dependent insulin secretion, inhibition of glucagon production, delaying gastric emptying and suppressing appetite (Trends Pharmacol Sci, 32(1): 8-15, 2011), etc.
- Natural GLP-1 can be rapidly degraded by dipeptidyl peptidase-4 (DPP-IV), neutral endopeptidase (NEP), plasma kallikrein or plasmin, which limits its therapeutic potential. Since natural GLP-1 has only an ultra-short half-life of about 2 minutes in the body, there has been a method of treating diabetes and obesity by using chemical modification and/or preparation to improve its efficacy (Bioorg Med Chem Lett, 23(14) ): 4011-8, 2013; Expert Opin Investig Drugs, 25(2):145-58, 2016).
- Glucagon is usually a 29 amino acid peptide, which corresponds to amino acids 53-81 of preproglucagon, and the sequence is shown in SEQ ID NO: 24 (Nutrition, Metabolism & Cardiovascular Diseases, 16: S28-S34, 2006). Glucagon receptor activation has been shown to increase energy expenditure and reduce food intake in both rodents and humans (Nat. Rev. Endocrinol, 6: 689-697, 2010) and these effects are in rodents Stable and continuous.
- Glucagon has many physiological effects, such as stimulating glycogen decomposition and gluconeogenesis, increasing blood glucose levels under hypoglycemic conditions, regulating liver ketone production, regulating bile acid metabolism and satiety effects through the vagus nerve.
- glucagon has been used for acute hypoglycemia, glucagon receptor activation reduces food intake and promotes lipolysis and weight loss in animals and humans.
- receptor agonist can generally be a polypeptide, protein or other small molecule that binds to a receptor and triggers the usual response of a natural ligand.
- the "GLP-1 receptor (GLP-1R) agonist” can generally be a polypeptide, protein or other small molecule that binds to GLP-1R and can trigger a characteristic response identical or similar to that of natural GLP-1.
- GLP-1R agonists activate GLP-1R completely or partially, and then cause a series of downstream signaling pathways in cells to produce corresponding cellular activity: such as ⁇ cells secrete insulin; typical GLP-1R agonists include natural GLP-1 And its mutants and analogs, such as Exenatide, Liraglutide, etc.
- GLP-1 analogue or “GLP-1 mutant” both mean GLP-1R agonists and can be used interchangeably.
- a "glucagon receptor (GCGR) agonist” is a Glucagon receptor agonist, usually a polypeptide that binds to GCGR and can trigger the same or similar characteristic response as natural glucagon (Glucagon) , Protein or other small molecules.
- GCGR agonists activate GCGR completely or partially, and then cause a series of downstream signaling pathways in cells to produce corresponding cell activities: such as glycogenolysis, glycogenogenesis, fatty acid oxidation and ketogenesis in liver cells.
- Glucagon analogue means Glucagon receptor agonists and can be used interchangeably.
- FGF21 usually refers to fibroblast growth factor (FGF), also called heparin binding growth factor (heparin binding growth factor), which is a type of polypeptide substance mainly secreted by the pituitary gland and hypothalamus.
- FGF has many functions, such as promoting the mitosis of fibroblasts, the growth of mesoderm cells, and stimulating the formation of blood vessels.
- FGF21 is an important member of the FGF family. At present, the hormone is used as a drug to be developed into a weight-loss drug and a drug for the treatment of diabetes, and the drug has entered the clinical trial stage.
- FGF21 plays a physiological role through FGF21-related receptors (such as FGFR1c) and its co-receptor ⁇ -klotho (KLB).
- FGF21-related receptors such as FGFR1c
- KLB co-receptor ⁇ -klotho
- dimer is typically formed by an immunoglobulin constant region (F C) and natural non-covalent covalent interaction. If no other otherwise indicated, are all formed dimer F C homodimers, as the dimer of the present invention provides.
- F C immunoglobulin constant region
- EC 50 concentration for 50% of maximal effect usually refers to the concentration required for a certain drug or substance to stimulate 50% of its corresponding biological response.
- concentration for 50% of maximal effect usually refers to the concentration required for a certain drug or substance to stimulate 50% of its corresponding biological response.
- low-density lipoprotein generally belongs to a type of plasma lipoprotein, and is the main carrier of cholesterol in the blood, which tends to deposit cholesterol on the arterial wall.
- White blood cells try to digest low-density lipoproteins, but in the process they turn them into toxins. More and more white blood cells are attracted to the changed area, causing the arterial wall to become inflamed. Over time, as the process continues, these plaque deposits can accumulate on the arterial wall, making the channel very narrow and lacking flexibility. If too much plaque accumulates, the artery can be completely blocked.
- LDL-C complex of LDL and cholesterol
- HDL high-density protein
- triglycerides is usually another type of fat used to store energy from excessive eating.
- High levels of triglycerides in the blood are related to atherosclerosis.
- High triglycerides can be caused by overweight and obesity, physical inactivity, smoking, excessive alcohol consumption and high carbohydrate (over 60% of total calories) intake.
- Sometimes underlying diseases or genetic diseases are the cause of high triglycerides.
- People with high triglycerides generally have high total cholesterol levels, including high LDL cholesterol and low HDL cholesterol. Many people with heart disease or diabetes also have high triglyceride levels.
- the first aspect of the present invention provides a fusion protein, including a Glucagon analog fragment and a long-acting protein unit fragment, the Glucagon analog fragment includes:
- b) a polypeptide fragment whose amino acid sequence has more than 90% sequence identity with SEQ ID NO. 81 and has the function of the polypeptide fragment defined in a).
- the amino acid sequence in b) specifically refers to: the amino acid sequence shown in SEQ ID No. 81 has been substituted, deleted or added one or more (specifically, 1-50, 1-30, 1- 20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or add one or more at the N-terminal and/or C-terminal ( Specifically, it can be 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, and have A polypeptide fragment whose amino acid is the function of the polypeptide fragment shown in SEQ ID No.
- 81 may simultaneously have GCGR/GLP-1R dual agonistic activity, GLP-1R/GIPR dual agonistic activity, or GLP-1R/GCGR/GIPR triple agonism Active, and can resist protease hydrolysis in the body.
- the amino acid sequence in b) may have 90%, 93%, 95%, 97%, or 99% or more sequence identity with SEQ ID No. 81.
- the fragment in a) may be an amino acid sequence such as SEQ ID NO.29, SEQ ID NO.32, SEQ ID NO.33, SEQ ID NO.35, SEQ ID NO.38 , SEQ ID NO.42, SEQ ID NO.43, SEQ ID NO.44 a polypeptide fragment shown in one of them.
- the present inventors have found that the present invention is obtained by screening GCG analogs retained even if the second native Ser, after integration with F C, sufficient to support the stability of the frequency of administration once a week, reduced immunogenicity risk potential .
- the modification at positions 16, 17 and 18 can also better maintain the GCGR agonistic activity.
- the present invention provides a fusion protein, said fragment may be derived from long-acting protein unit F C portion of a mammalian immunoglobulin, the immunoglobulin molecule is a polypeptide chain containing disulfide bonds, typically having two light chains And two heavy chains.
- F C portion of an immunoglobulin used herein have the usual meaning in the field of immunology terms. Specifically, the term refers to an antibody fragment obtained by removing two antigen binding regions (Fab fragments) from an antibody.
- F. C portion may include a hinge region and the C-terminal extension reaches through antibody CH 2 and CH 3 domains.
- F portion C may further comprise one or more glycosylation sites.
- the human body has 5 kinds of human immunoglobulins with different effect characteristics and pharmacokinetic characteristics: IgG, IgA, IgM, IgD and IgE.
- IgG is the highest content of immunoglobulin in serum.
- IgG is also the longest serum half-life of all immunoglobulins (about 23 days).
- the long-acting protein fragment optionally mutant unit F C F C portion of the complete portion of an immunoglobulin, fragment F C portion of an immunoglobulin or immunoglobulin self.
- F C portion of an immunoglobulin used in the present invention may be derived from mammalian IgG1, IgG2 or IgG4, or a mutant F C region; preferred, and may be sourced from human IgG1, IgG2 or IgG4, or the F C region mutant; more preferably, it may be originated from human F C region of human IgG1 or IgG4, or a mutant thereof.
- position 297 is glycine or alanine replacement F C domain.
- Kabat's EU index number Sequences of proteins of immunological interest, fifth edition, public health service, National Institutes of Health, Bethesda, MD (1991)).
- the long-acting protein unit fragment may include: c) a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID NOs. 4 to 13; or, d) an amino acid sequence and SEQ ID NO .A polypeptide fragment with one of 4 to 13 having more than 90% sequence identity and the function of the polypeptide fragment defined in c).
- the amino acid sequence in d) specifically refers to: the amino acid sequence shown in one of SEQ ID Nos.
- 4 to 13 has been substituted, deleted or added one or more (specifically, 1-50, 1- 30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids, or added at the N-terminal and/or C-terminal One or more (specifically 1-50, 1-30, 1-20, 1-10, 1-5, 1-3, 1, 2, or 3) amino acids and
- the obtained polypeptide fragment having the function of the polypeptide fragment shown in one of SEQ ID Nos. 4 to 13, for example, can improve the overall DPP-IV resistance of the fusion protein.
- the amino acid sequence in d) may be 90%, 93%, 95%, 97%, or 99% similar to one of SEQ ID Nos. 4-13.
- F C domain may be derived from human IgG1, and as SEQ ID NO.12, SEQ ID NO.13 FIG.
- the end of the chain K F C may be removed to facilitate improved uniformity of the expression product, such as SEQ ID NO.5, SEQ ID NO.9.
- the present invention provides fusion GCG analogue F C, to obtain a high resistance to DPP-IV, there is no prior art discloses the like can be increased by fusion GCG F C DPP-IV enzyme resistance, And pharmacodynamic experiments show that stability can support the once-a-week administration frequency.
- the fusion protein provided by the present invention may also include a first connecting peptide fragment, which is usually located between the Glucagon analog fragment and the long-acting protein unit fragment.
- the first connecting peptide fragment may generally be a fragment rich in G, S and/or A.
- the first connecting peptide fragment may be composed of glycine G, serine S and alanine A, and those skilled in the art can Choose an appropriate content ratio of G, S and A, preferably 12:3:1, or 12:1:3, or 12:1:2; or 12:2:1, etc., and S or A can be missing,
- the ratio of G to A can be 4:1 or the ratio of G to S can be 4:1.
- the first connecting peptide fragment includes a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID NOs. 14-23.
- the fusion protein may sequentially include Glucagon analog fragments, first connecting peptide fragments and long-acting protein unit fragments from N-terminus to C-terminus.
- the amino acid sequence of the fusion protein is as SEQ ID NO. 56, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 62, SEQ ID NO. 65, SEQ ID NO. .69, SEQ ID NO.70, SEQ ID NO.71 shown in one of them.
- the fusion protein further includes FGF21 analog fragments, and the FGF21 analog fragments may include:
- N-terminal HPIPDSS can be deleted or partly deleted; X 19 is selected from R, Y, V, E or C; X 31 is selected from A or C; X 36 is selected from R or K; X 43 is selected from G or C; X 45 is selected X is selected from A, K, E or V; X 56 is selected from K, R, V or I; X 98 is selected from L, R or D; X 118 is selected from L or C; X 122 is selected from K or R; X 134 is selected From A or C; X 167 is selected from S, A or R; X 170 is selected from G or E; X 171 is selected from P or G; X 174 is selected from G, A or L; X 179 is selected from Y, A or F X 180 is selected from A or E; X 181 is selected from S, K or deletion; or, f) a polypeptide whose amino acid sequence has more than 90% sequence identity with SEQ ID NO.
- polypeptide fragment having the function of the polypeptide fragment shown in SEQ ID No. 119 may have the same or similar biological function as natural FGF21 (SEQ ID NO. 87).
- the amino acid sequence in f) can have more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% sequence identity with SEQ ID No. 119.
- the amino acid sequence of the FGF21 analog fragment can be selected from those described in patents or patent applications such as US20140213512, US8188040, US9493530, WO 2016114633, US 20150291677, US 9422353, US 8541369, US7622445, US7576190, US20070142278, US 9006400 or US 20130252884 Amino acid sequence of FGF21 analog or mutant.
- the polypeptide fragment in e) is selected from polypeptide fragments whose amino acid sequence is shown in one of SEQ ID NOs. 87 to 90.
- the fusion protein provided by the present invention may also include a second connecting peptide fragment, which is usually located between the long-acting protein unit fragment and the FGF21 analog fragment.
- the second connecting peptide fragment may generally be a fragment rich in G, S and/or A, for example, the second connecting peptide fragment may be composed of glycine G, serine S and alanine A, and those skilled in the art can Choose an appropriate content ratio of G, S and A, preferably 12:3:1, or 12:1:3, or 12:1:2; or 12:2:1, etc., and S or A can be missing, For example, the ratio of G to A can be 4:1 or the ratio of G to S can be 4:1.
- the second connecting peptide fragment includes a polypeptide fragment having an amino acid sequence as shown in one of SEQ ID NOs. 14-23.
- the fusion protein may include Glucagon analog fragments, first connecting peptide fragments, long-acting protein unit fragments and FGF21 analog fragments in sequence from N-terminal to C-terminal, and may also include Glucagon analogs in sequence.
- the amino acid sequence of the fusion protein is shown in one of SEQ ID NOs. 91-115.
- the fusion protein group of formula II (A352L1F6L5M2) and the same dose of the fusion protein of formula I (A352L1F6) + FGF21 analog (F6L5M2) combined administration group are similar than has a more significant weight loss effect. It proved that the fusion protein A352L1F6L5M2 may have lower side effects and improved safety (Example 7).
- the second aspect of the present invention provides an isolated polynucleotide encoding the fusion protein provided by the first aspect of the present invention.
- the third aspect of the present invention provides a construct containing the isolated polynucleotide provided by the second aspect of the present invention.
- the construct can usually be constructed by inserting the isolated polynucleotide into a suitable expression vector.
- suitable expression vector may include, but is not limited to, pcDNA3.1 , PBudCE4.1, pEE14.1, pPIC9, etc.
- the fourth aspect of the present invention provides an expression system that contains the construct provided by the third aspect of the present invention or the polynucleotide provided by the second aspect of the present invention integrated with exogenous in the genome.
- the expression system can be a host cell, the host cell can express the fusion protein as described above, the host cell can be a eukaryotic cell and/or a prokaryotic cell, more specifically a mammalian cell, E. coli, yeast, etc. , More specifically, HEK293, CHO, etc.
- the fifth aspect of the present invention provides a method for preparing the fusion protein provided by the first aspect of the present invention.
- a suitable method to prepare the fusion protein For example, solid phase synthesis may be used.
- the preparation method of the fusion protein may include: culturing the expression system provided in the fourth aspect of the present invention under suitable conditions to express the fusion protein, and then isolating and purifying to provide the fusion protein.
- the sixth aspect of the present invention provides a pharmaceutical composition comprising the fusion protein provided in the first aspect of the present invention or the culture of the expression system provided in the fourth aspect of the present invention.
- the pharmaceutical composition may also include a pharmaceutically acceptable carrier.
- the carrier may include various excipients and diluents, and these carriers themselves are not essential active ingredients and do not have excessive toxicity after administration. Suitable carriers should be well-known to those skilled in the art, for example, a full discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991).
- the seventh aspect of the present invention provides the use of the fusion protein provided in the first aspect of the present invention and the pharmaceutical composition provided in the sixth aspect of the present invention in the preparation of drugs, and the drugs can be selected from drugs used for the treatment of metabolic related diseases.
- Metabolic-related diseases can be metabolic syndrome.
- the characteristics of metabolic syndrome can usually include the following risk factors (for example, three or more): (1) abdominal obesity (excessive adipose tissue in or around the abdomen), (2) causing Atherosclerotic dyslipidemia, dyslipidemia, including high triglycerides, low HDL cholesterol and high LDL cholesterol, which enhance the accumulation of plaque in the arterial wall, (3) increased blood pressure, (4) insulin resistance or glucose tolerance Suffering, (5) thrombus-like state, such as high fibrin or plasminogen activator inhibitor-1 in the blood, and (6) pro-inflammatory state, such as elevated C-reactive protein in the blood.
- risk factors can include aging, hormonal imbalances, and genetic factors.
- the fusion protein provided by the present invention has good hypoglycemic and weight loss effects on mice, and thus proved to be useful for treating obesity or diabetes.
- the fusion protein of the present invention can usually be Reduce appetite, reduce food intake, reduce patient's body fat level, increase energy consumption and other mechanisms for treatment.
- the eighth aspect of the present invention provides a treatment method comprising: administering to an individual a therapeutically effective amount of the fusion protein provided in the first aspect of the present invention, the culture of the expression system provided in the fourth aspect of the present invention, or the sixth aspect of the present invention The pharmaceutical composition provided.
- treatment includes preventive, curative or palliative treatments that can lead to the desired pharmaceutical and/or physiological effects.
- the effect preferably refers to medically reducing one or more symptoms of the disease or completely eliminating the disease, or blocking or delaying the occurrence of the disease and/or reducing the risk of disease development or deterioration.
- mammals generally include humans, non-human primates, or other mammals (such as dogs, cats, horses, sheep, pigs, cows, etc.), which can be based on the use of the preparation, kit or combination The preparations benefit from treatment.
- therapeutically effective amount generally refers to an amount that can achieve the effect of treating the diseases listed above after a proper administration period.
- sequence identity refers to the percentage of identical residues in the sequences participating in the comparison.
- sequence identity of the sequence of two or more entries can be calculated using calculation software well known in the art, such software can be obtained from NCBI.
- Incretin hormone proteins such as GLP-1 analogs and Exendin-4 can cause side effects such as nausea and vomiting in patients, and they are dose-related. Therefore, in the case of maintaining the ideal blood sugar level, reducing the dose as much as possible will theoretically alleviate the uncomfortable side effects of the patient.
- reports on the effects of FGF21 on osteoporosis and reproduction are also reported from time to time (Scand J Clin Lab Invest, 75(2): 121-5, 2015; Mol Metab, 5(8): 690-8, 2016).
- the risk of side effects of drugs is proportional to the administered dose.
- the safety of drugs is extremely high.
- the inventors found that the fusion protein of the present invention has a good effect of controlling blood sugar and body weight at a very low dose, and has a small impact on the gastrointestinal tract, greatly reducing the administered dose, thereby significantly reducing the risk of potential side effects.
- the in vitro cell activity determination of GLP-1R and GCGR agonistic activity adopts the luciferase reporter gene detection method. This method is based on the principle that GLP-1R and GCGR can activate the downstream cAMP pathway after being excited.
- the activity determination of FGF21 and its analogs is obtained by co-transfecting FGF21 signaling pathway-related genes into HEK293T cells and detecting the changes in fluorescence caused by the signal. Joseph R. Chabenne et al. and Richard D. DiMarchi et al.
- the inventors unexpectedly discovered that after fusion of GCG analogs of different sequences with Fc, the effects on the activities of GLP-1R and GCGR are completely different.
- the C-terminus of the added additional GCG like or similar cex sequences (SEQ ID NO.2-3) fused to a further chain C F.
- SEQ ID NO.2-3 fused to a further chain C F.
- the retention rate of -1R agonistic activity is significantly improved, and the highest increase is more than 200 times (Example Table 2), but the retention rate of GCGR agonistic activity is basically unchanged, or even slightly decreased.
- Glucagon A001L1F6 native active protein fused to the F C (SEQ ID NO.51) and Joseph R.Chabenne reported Glucagon-cex with the fusion activity of F C protein A012L1F6 (SEQ ID NO.52) of the present invention is prepared as a control verify F C integration to improve the stability.
- A001L1F6 (SEQ ID NO. 51) nor A012L1F6 (SEQ ID NO. 52) showed obvious signs of resistance to DPP-IV (Example 4). Meanwhile, in Example 4 it can also be seen, it is also a 16-18 GCG mutation analogs of the fusion protein F C, but the stability was a great difference.
- GCG analogs provided by the present invention have been shown to be sufficient to support the once-a-week dosing frequency, rather than the once-a-day commonly reported (such as albumin-bound liraglutide).
- the preservation of natural amino acids further reduces the risk of immunogenicity, avoids chemical cross-linking and makes the preparation process easier and more convenient.
- the present invention also carried out a random blood glucose experiment in mice with leptin receptor-deficient type 2 diabetes (db/db).
- db/db leptin receptor-deficient type 2 diabetes
- a db/db random blood glucose experiment was carried out, and the fusion protein of the present invention was administered.
- Rats showed a significant hypoglycemic effect than liraglutide, as well as a better weight loss effect.
- the present invention also carried out weight loss experiments in DIO mice, and there have been many reports on the potential weight loss effects of GCGR agonists.
- natural Glucagon is easy to be degraded and has a small molecular weight, so its potential to become a medicine is not high.
- Glucagon analogs are mainly used for acute symptoms of hypoglycemia.
- Clinical reports of long-acting GCG analogs for weight loss in diabetic patients are also emerging. As we all know, obesity is one of the causes of insulin resistance in diabetic patients, and weight loss is an important indicator for evaluating a hypoglycemic drug.
- the fusion protein of the present invention caused a significant decrease in body weight after administration of DIO mice.
- the fusion protein of the present invention has potential pharmacokinetic properties suitable for administration once a week or more.
- the dosage depends on the frequency and mode of administration, the age, sex, weight, and general condition of the subject being treated, the condition and severity of the treatment, any concomitant diseases to be treated, and other factors obvious to those skilled in the art. Meanwhile, according to the condition of the subject and other pathological conditions, the fusion protein of the present invention can be administered or applied in combination with one or more other therapeutically active compounds or substances.
- other therapeutically active compounds that can be selected include but are not limited to Anti-diabetic drugs, anti-hyperlipidemic drugs, anti-obesity drugs, anti-hypertensive drugs and reagents for the treatment of diabetes or complications related to diabetes.
- Metabolic-related diseases are associated with an increased risk of coronary heart disease and other conditions related to the accumulation of vascular plaque, such as stroke and peripheral vascular disease, known as atherosclerotic cardiovascular disease (ASCVD).
- ASCVD atherosclerotic cardiovascular disease
- Patients with metabolic syndrome can develop fully mature type 2 diabetes from an early insulin resistance state, and the risk of ASCVD is further increased.
- the relationship between insulin resistance, metabolic syndrome, and vascular disease may involve one or more common pathogenesis, including insulin-stimulated vasodilation disorders, caused by increased oxidative stress Decreased availability of insulin resistance-related and abnormal adipocyte-derived hormones (such as adiponectin) (Lteif, Mather, Can. J. Cardiol. 20 (Supplement B): 66B-76B, 2004)
- the fusion protein of the present invention can also be used to treat obesity.
- the fusion protein of the present invention treats obesity through mechanisms such as reducing appetite, reducing food intake, reducing the patient's body fat level, and increasing energy expenditure.
- the fusion protein of the present invention can be used to treat non-alcoholic fatty liver disease (NAFLD).
- NAFLD refers to a broad spectrum of liver diseases, ranging from simple fatty liver (steatosis) to non-alcoholic steatotic hepatitis (NASH) to cirrhosis (irreversible advanced scarring of the liver). All stages of NAFLD have fat accumulation in liver cells. Simple fatty liver is the abnormal accumulation of certain types of fat and triglycerides in liver cells, but no inflammation or scar formation. In NASH, fat accumulation is associated with varying degrees of liver inflammation (hepatitis) and scarring (fibrosis). Inflammatory cells can destroy liver cells (hepatocyte necrosis).
- steatosis refers to fatty infiltration
- hepatitis refers to inflammation in the liver
- necrosis refers to destroyed liver cells.
- NASH can eventually lead to scarring of the liver (fibrosis) and then irreversible advanced scarring (cirrhosis). Cirrhosis caused by NASH is the last and most severe stage of the NAFLD spectrum.
- the fusion protein provided by the present invention has the advantage of a long half-life, and can generally be considered to support a once-a-week or longer dosing frequency.
- the fusion protein has a significant effect of reducing blood sugar and weight.
- it has good stability in vivo and in vitro, and low immunogenicity.
- the fusion protein since the fusion protein does not need to introduce unnatural amino acids, and does not involve chemical synthesis and cross-linking steps, it can be prepared by a recombinant method, which greatly simplifies the preparation process.
- MOLECULAR CLONING A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel, etc., Current PROTOCOLS IN MOLECULAR BI, John Wi , New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998;METHODS Chromatin, Vol. ENZYMOLOGY, Vol. and AP Wolfe, eds.), Academic Press, San Diego, 1999; and Methods IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, etc.
- the fusion protein prepared in this example is expressed by formula I, and the specific structure is: ALF (formula I), preferably consisting of a GCG analog fragment (corresponding to A in formula I) and a connecting peptide fragment (corresponding to L in formula I)
- ALF formula I
- GCG analog fragment corresponding to A in formula I
- connecting peptide fragment corresponding to L in formula I
- the amino acid sequence of the dimeric fusion protein obtained by fusion of) and Fc (corresponding to F in formula I) is shown in Table 1 below.
- the preparation process is as follows:
- coli Top10F' after identification of the positive clone, inoculate it in 500ml LB medium, cultivate it overnight, and collect the bacteria by centrifugation. Omega Endo-Free Plasmid Maxi Kit or similar method to extract plasmid.
- the fusion protein obtained in Example 1 was subjected to in vitro activity determination, including GLP-1R agonistic activity detection and GCGR agonistic activity detection.
- the GLP-1R agonistic activity is detected by the luciferase reporter gene detection method.
- the human GLP-1R gene was cloned into mammalian cell expression plasmid pCDNA3.1 to construct a recombinant expression plasmid pCDNA3.1-GLP-1R, and the full-length luciferase gene was cloned into pCRE-EGFP plasmid. Replace the EGFP gene to obtain the pCRE-Luc recombinant plasmid.
- the pCDNA3.1-GLP-1R and pCRE-Luc plasmids were transfected into CHO cells at a molar ratio of 1:10, and stable expression strains were screened to obtain recombinant CHO/GLP-1R stable transfected cell lines.
- the purified recombinant protein (Tables 1 and 2) or natural Glucagon (Hangzhou Zhongpi Biochemical Co., Ltd., GLUC-004) and natural GLP-1 (Hangzhou Zhongpi Biochemical Co., Ltd.) Company, GLUC-016B) as a control, diluted with 0.2% FBS-containing DMEM/F12 medium to a series of specified concentrations, added to the cell culture wells, 100 ⁇ l/well, tested after 6h stimulation.
- the detection was performed according to the instructions of the Lucifersae reporter kit (Lucifersae reporter kit, Ray Biotech, Cat: 68-LuciR-S200).
- GCGR agonistic activity detection also uses luciferase reporter gene detection method.
- the GCGR gene was cloned into mammalian cell expression plasmid pCDNA3.1, and the recombinant expression plasmid pCDNA3.1-GCGR was constructed.
- the construction of transfected CHO cells and stable transfected cell line CHO/GCGR was the same as above.
- Table 1 protein are numbered according to the following rules: GCG peptide analogs of polypeptide connection code code + + F C code, such as A382L1F6, and A382 indicates GCG code for polypeptide analogs IgG Fc F6 L1 connecting peptide fusion is obtained by the code.
- dimer fusion proteins without Exendin-4 C-terminal sequence G232L1F6 (SEQ ID NO.82), G352L1F6 (SEQ ID NO.83), G382L1F6 (SEQ ID NO.84), G395L1F6 (SEQ ID NO. ID NO.85) and G402L1F6 (SEQ ID NO.86) were tested for in vitro activity (preparation and purification methods are the same as in Example 1), including GLP-1R agonistic activity detection and GCGR agonistic activity detection.
- Explanation: a. is the ratio of GLP-1R agonistic activity before and after the addition of the C-terminal extension peptide Cex of Exendin-4 (in the present invention, any of SEQ ID NO. 2-3).
- GDSerGS H-D-Ser-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO.79);
- GAibGS H-Aib-QGTFTSDYSKYLDSQAAQDFVQWLMNGGPSSGAPPPS (SEQ ID NO.80);
- the fusion protein concentrate it by ultrafiltration, and then dilute it to 1.6mg/ml with 20mM PB pH7.4. After sterilization and filtration, the serum (FBS, GEMINI 900-108, A97E00G) is diluted 10 times, mixed and divided. Put it into a sterile centrifuge tube;
- Residual activity Take the 0 hour activity value as 100%, and compare it with the value measured at the subsequent time point.
- the fusion protein prepared in this example is expressed by Formula II, and the specific structure is: AL 1 -FL 2 -B (Formula II), where A is a GCG analog fragment, F is a long-acting protein unit fragment, and B is FGF21
- the analog fragment can be natural FGF21 (SEQ ID NO. 87) or its derivatives, L 1 is a connecting peptide fragment, and the sequence is selected from any of SEQ ID NO. 14-23; L 2 is a connecting peptide fragment, which can be Not present or selected from any one of SEQ ID NO. 14-23.
- the prepared amino acids are shown in SEQ ID NO. 91-115, and the corresponding codes are shown in Table 4.
- the inclusion bodies were washed four times with washing solution (50mM Tris, 150mM NaCl, 2M urea, pH 8.0) and weighed; according to (1:10 mass-volume ratio), 1ml denaturation solution (50mM Tris, 150mM NaCl) was added for every 0.1g inclusion body , 8M urea, 10mM DTT, pH 8.5), gently mix and dissolve on a shaker at room temperature for more than 5 hours; dilute and refold according to the ratio of 1:100-200.
- washing solution 50mM Tris, 150mM NaCl, 2M urea, pH 8.0
- 1ml denaturation solution 50mM Tris, 150mM NaCl
- the puromycin resistance gene pac was amplified by PCR and cloned into pcDNA3.1(+) to replace the original G418 resistance gene.
- the GAL4DBD-ELK1, IRES, KLB ( ⁇ -klotho) genes were amplified by PCR, and cloned into the pcDNA-Puro plasmid to construct the plasmid pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro for cell transfection screening.
- the constructed recombinant plasmid and pFR-Luc plasmid use Omega Extract with Endo-Free Plasmid Midi Kit for use.
- the cell transfection process is as follows: HEK293T cells are spread on a 6-well plate with 3 ⁇ 10 5 cells per well and cultured overnight.
- the cell transfection reagent was prepared according to the following ratio: Lipofectamine 2000 (6 ⁇ l): pFR-Luc (4.6 ⁇ g): pcDNA-GAL4DBD-ELK1-IRES-KLB-Puro (1 ⁇ g). After standing for 20 minutes, slowly add to the 6-well plate and mix while adding. After culturing for 6 hours, change to DMEM+10% FBS medium, and continue culturing at 37°C and 5% CO 2 . Screening and obtaining stable transgenic cell lines with FGF21 activity response.
- db/db mice were screened according to three indicators: body weight, non-fasting blood glucose, and pre-medicine OGTT reaction, and were divided into groups with 10 mice in each group. Try to exclude individuals who were too large or too small.
- the dose shown in Table 5 subcutaneous injection is administered every 4 days, the first time is the 0th day, and the last time is the 16th day; the negative control group is normal saline (PBS) (5 ⁇ l/gram body weight), the positive control group is Laglutide (10nmol/kg body weight) was administered by subcutaneous injection once a day for 18 consecutive days. Random blood glucose values were measured at 9 a.m. before the first injection and on the 2nd, 6th, 10d, 14d, and 18d. The results of random blood glucose changes are shown in Figure 2.
- Figures 2A and 2B show that the fusion protein in Table 5 has a significantly better hypoglycemic effect in db/db mice than the positive control liraglutide.
- the purpose of this example is to study the effect of different dual-effect fusion proteins on the body weight of DIO mice.
- 7-week-old male C57BL/6J male mice were given high-fat diet (60% kcal from fat) for 16 weeks (23 weeks in total), and the test was conducted when the body weight was about 55 g.
- Feeding conditions 12h light/12h dark, ad libitum feeding, single cage rearing, the mice were grouped according to body weight and body weight growth curve the day before administration (8 mice/group), and subcutaneous administration treatment the next day. It was administered at a dose of 20nmol/kg body weight, once every 4 days.
- the present invention effectively overcomes various shortcomings in the prior art and has high industrial value.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Health & Medical Sciences (AREA)
- Zoology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Biophysics (AREA)
- Gastroenterology & Hepatology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- General Engineering & Computer Science (AREA)
- Diabetes (AREA)
- Endocrinology (AREA)
- Toxicology (AREA)
- Microbiology (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Epidemiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Hematology (AREA)
- Obesity (AREA)
- Immunology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Urology & Nephrology (AREA)
- Emergency Medicine (AREA)
- Cell Biology (AREA)
- Child & Adolescent Psychology (AREA)
Abstract
Description
样品 | SEQ ID NO. | 剂量(nmol/kg) |
A137L1F6 | 59 | 30 |
A175L1F6 | 60 | 30 |
A352L1F6 | 69 | 30 |
A382L1F6 | 70 | 30 |
A232L1F9L5M2 | 98 | 6 |
A089L1F9L5M2 | 100 | 6 |
A352L1F10L5M2 | 106 | 6 |
A175L1F2L5M2 | 109 | 6 |
Claims (21)
- 一种融合蛋白,包括Glucagon类似物片段和长效蛋白单元片段,所述Glucagon类似物片段包括:a)氨基酸序列如SEQ ID No.81所示的多肽片段:X 1SX 3GTFTSDYSKYLDX 16X 17X 18AQDFVQWLX 27X 28X 29X z SEQ ID No.81;X 1选自H或Y;X 3选自Q或E;X 16选自除Y、N、W、和H以外的任一氨基酸;X 17选自除P、L、T、F和H以外的任一氨基酸;X 18选自除P、F、H和W以外的任一氨基酸;X 17与X 18不同时为R;X 27选自M或L;X 28选自D或A;X 29为T或缺失;X z选自GGPSSGAPPPS或GPSSGAPPPS;或,b)氨基酸序列与SEQ ID NO.81具有90%以上序列同一性且具有a)限定的多肽片段的功能的多肽片段。
- 如权利要求1所述的融合蛋白,其特征在于,所述a)中的多肽片段选自氨基酸序列如SEQ ID NO.29、SEQ ID NO.32、SEQ ID NO.33、SEQ ID NO.35、SEQ ID NO.38、SEQ ID NO.42、SEQ ID NO.43、SEQ ID NO.44其中之一所示的多肽片段。
- 如权利要求1所述的融合蛋白,其特征在于,所述长效蛋白单元片段源自哺乳动物免疫球蛋白的F C部分。
- 如权利要求3所述的融合蛋白,其特征在于,所述长效蛋白单元片段包括:c)氨基酸序列如SEQ ID NO.4~13其中之一所示的多肽片段;或,d)氨基酸序列与SEQ ID NO.4~13其中之一具有90%以上序列同一性且具有c)限定的多肽片段的功能的多肽片段。
- 如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白还包括第一连接肽片段,所述第一连接肽片段位于Glucagon类似物片段和长效蛋白单元片段之间,优选的,所述第一连接肽片段富含G、S和/或A。
- 如权利要求5所述的融合蛋白,其特征在于,所述第一连接肽片段包括氨基酸序列如SEQ ID NO.14~23其中之一所示的多肽片段。
- 如权利要求5所述的融合蛋白,其特征在于,所述融合蛋白自N端至C端依次包括Glucagon类似物片段、第一连接肽片段和长效蛋白单元片段。
- 如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO.56、SEQ ID NO.59、SEQ ID NO.60、SEQ ID NO.62、SEQ ID NO.65、SEQ ID NO.69、SEQ ID NO.70、SEQ ID NO.71其中之一所示。
- 如权利要求1所述的融合蛋白,其特征在于,所述融合蛋白还包括FGF21类似物片段。
- 如权利要求9所述的融合蛋白,其特征在于,所述FGF21类似物片段包括:e)氨基酸序列如SEQ ID NO.119所示的多肽片段:HPIPDSSPLLQFGGQVRQ X 19YLYTDDAQQTE X 31HLEI X 36EDGTVG X 43A X 45DQSPESLLQL X 56ALKPGVIQILGVKTSRFLCQRPDGALYGSLHFDPEACSFRE X 98LLEDGYNVYQSEAHGLPLH X 118PGN X 122SPHRDPAPRGP X 134RFLPLPGLPPALPEPPGILAPQPPDVGSSDPL X 167MV X 170 X 171SQ X 174RSPS X 179 X 18 0 X 181 SEQ ID NO.119;N端HPIPDSS可缺失或部分缺失;X 19选自R、Y、V、E或C;X 31选自A或C;X 36选自R或K;X 43选自G或C;X 45选自A、K、E或V;X 56选自K、R、V或I;X 98选自L、R或D;X 118选自L或C;X 122选自K或R;X 134选自A或C;X 167选自S、A或R;X 170选自G或E;X 171选自P或G;X 174选自G、A或L;X 179选自Y、A或F;X 180选自A或E;X 181选自S、K或缺失;或,f)氨基酸序列与SEQ ID NO.119具有80%以上序列同一性且具有e)限定的多肽片段的功能的多肽片段;优选的,所述e)中的多肽片段选自氨基酸序列如SEQ ID NO.87~90其中之一所示的多肽片段。
- 如权利要求9所述的融合蛋白,其特征在于,所述融合蛋白还包括第二连接肽片段,所述第二接肽片段位于长效蛋白单元片段和FGF21类似物片段之间,优选的,所述第二连接肽片段富含G、S和/或A。
- 如权利要求11所述的融合蛋白,其特征在于,所述第二连接肽片段包括氨基酸序列如SEQ ID NO.14~23其中之一所示的多肽片段。
- 如权利要求11所述的融合蛋白,其特征在于,所述融合蛋白自N端至C端依次包括Glucagon类似物片段、第一连接肽片段、长效蛋白单元片段和FGF21类似物片段;或,所述融合蛋白自N端至C端依次包括Glucagon类似物片段、第一连接肽片段、长效蛋白单元片段、第二连接肽片段和FGF21类似物片段。
- 如权利要求13所述的融合蛋白,其特征在于,所述融合蛋白的氨基酸序列如SEQ ID NO.91~115其中之一所示。
- 一种分离的多核苷酸,编码如权利要求1~14任一权利要求所述的融合蛋白。
- 一种构建体,所述构建体含有如权利要求15所述的分离的多核苷酸。
- 一种表达***,所述表达***含有如权利要求16所述的构建体或基因组中整合有外源的如权利要求15所述的多核苷酸。
- 如权利要求1-14任一权利要求所述的融合蛋白的制备方法,包括:在合适的条件下 培养如权利要求17所述的表达***,使之表达所述融合蛋白,分离、纯化以提供所述融合蛋白。
- 一种药物组合物,包括如权利要求1-14之任一权利要求所述的融合蛋白或如权利要求17所述的表达***的培养物。
- 如权利要求1-14任一权利要求所述的融合蛋白、如权利要求19所述的药物组合物在制备药物中的用途。
- 如权利要求20所述的用途,其特征在于,所述药物选自用于治疗代谢相关疾病的药物。
Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
AU2020275797A AU2020275797A1 (en) | 2019-05-16 | 2020-01-03 | Fusion protein for treatment of metabolic disease |
JP2021566567A JP7268202B2 (ja) | 2019-05-16 | 2020-01-03 | 代謝性疾患を治療するための融合タンパク質 |
EP20805380.1A EP3971214A4 (en) | 2019-05-16 | 2020-01-03 | Fusion protein for treatment of metabolic disease |
US17/610,451 US20220340635A1 (en) | 2019-05-16 | 2020-01-03 | Fusion protein for treatment of metabolic disease |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910407446.9A CN111944055B (zh) | 2019-05-16 | 2019-05-16 | 一种治疗代谢疾病的融合蛋白 |
CN201910407446.9 | 2019-05-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2020228360A1 true WO2020228360A1 (zh) | 2020-11-19 |
Family
ID=73290037
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/CN2020/070234 WO2020228360A1 (zh) | 2019-05-16 | 2020-01-03 | 一种治疗代谢疾病的融合蛋白 |
Country Status (6)
Country | Link |
---|---|
US (1) | US20220340635A1 (zh) |
EP (1) | EP3971214A4 (zh) |
JP (1) | JP7268202B2 (zh) |
CN (2) | CN114853908B (zh) |
AU (1) | AU2020275797A1 (zh) |
WO (1) | WO2020228360A1 (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114621327A (zh) * | 2020-12-10 | 2022-06-14 | 江苏中新医药有限公司 | GLP-1、GIP和Gcg多重受体激动蛋白质 |
Families Citing this family (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114981294B (zh) * | 2020-12-23 | 2024-04-26 | 浙江道尔生物科技有限公司 | 一种长效胰高血糖素衍生物 |
CN112625093B (zh) * | 2020-12-29 | 2022-12-23 | 清远市图微安创科技开发有限公司 | 用于预防和/或治疗非酒精性脂肪肝炎的多肽化合物 |
CN114014940B (zh) * | 2021-11-25 | 2022-11-15 | 华兰基因工程有限公司 | 一种2019-nCoV表面蛋白受体结合区融合蛋白制备方法 |
WO2023207107A1 (zh) * | 2022-04-29 | 2023-11-02 | 苏州星洲生物科技有限公司 | Glp-1/gcg受体共激动剂、包含其的药物组合物及其用途 |
CN115536739B (zh) * | 2022-07-04 | 2023-04-14 | 北京惠之衡生物科技有限公司 | 一种glp-1受体和gcg受体共激动多肽衍生物的制备方法 |
Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999057151A2 (en) * | 1998-04-30 | 1999-11-11 | Boehringer Ingelheim International Gmbh | FAP α-SPECIFIC ANTIBODY WITH IMPROVED PRODUCIBILITY |
US20070142278A1 (en) | 2003-12-10 | 2007-06-21 | Beals John M | Muteins of fibroblast growth factor 21 |
US7576190B2 (en) | 2004-05-13 | 2009-08-18 | Eli Lilly And Company | FGF-21 fusion proteins |
US7622445B2 (en) | 2004-09-02 | 2009-11-24 | Eli Lilly And Company | Muteins of fibroblast growth factor 21 |
US8188040B2 (en) | 2009-05-05 | 2012-05-29 | Amgen Inc. | FGF21 mutants and uses thereof |
US8541369B2 (en) | 2011-10-04 | 2013-09-24 | Eli Lilly And Company | Fibroblast growth factor 21 variants having improved pharmacological potency and/or improved pharmaceutical stability |
US20130252884A1 (en) | 2009-06-11 | 2013-09-26 | Novo Nordisk A/S | Fgf21 analogues and derivatives |
US20140213512A1 (en) | 2011-08-31 | 2014-07-31 | Amgen Inc. | Method of Treating or Ameliorating Type 1 Diabetes Using FGF21 |
US9006400B2 (en) | 2011-09-26 | 2015-04-14 | Novartis Ag | Fibroblast growth factor-21-Fc fusion proteins |
US9018164B2 (en) | 2005-11-07 | 2015-04-28 | Indiana University Research And Technology Corporation | Glucagon analogs exhibiting physiological solubility and stability |
US9150632B2 (en) | 2009-06-16 | 2015-10-06 | Indiana University Research And Technology Corporation | GIP receptor-active glucagon compounds |
US20150291677A1 (en) | 2012-11-28 | 2015-10-15 | Ngm Biopharmaceuticals, Inc. | Compositions and methods for treatment of metabolic disorders and diseases |
WO2016114633A1 (en) | 2015-01-16 | 2016-07-21 | Yuhan Corporation | Long-acting fgf21 fusion proteins and pharmaceutical composition comprising the same |
US9422353B2 (en) | 2012-06-11 | 2016-08-23 | Eli Lilly And Company | Fibroblast growth factor 21 variant, composition , and uses thereof |
US9493530B2 (en) | 2009-05-05 | 2016-11-15 | Amgen Inc. | FGF21 mutants comprising a mutation at position 98, 171 and/or 180 |
CN107995914A (zh) * | 2016-08-19 | 2018-05-04 | 安源医药科技(上海)有限公司 | 人成纤维细胞生长因子21融合蛋白及其制备方法与用途 |
CN109306015A (zh) * | 2011-06-17 | 2019-02-05 | 韩美科学株式会社 | 包括泌酸调节肽和免疫球蛋白片段的结合物以及其应用 |
Family Cites Families (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20150116465A (ko) | 2007-02-15 | 2015-10-15 | 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 | 글루카곤/glp-1 수용체 공동-항진물질 |
CN101993496B (zh) * | 2009-08-20 | 2013-06-05 | 重庆富进生物医药有限公司 | 双重调节血糖血脂融合蛋白及其制法和用途 |
US8551946B2 (en) | 2010-01-27 | 2013-10-08 | Indiana University Research And Technology Corporation | Glucagon antagonist-GIP agonist conjugates and compositions for the treatment of metabolic disorders and obesity |
CN103458920B (zh) * | 2010-12-22 | 2016-07-06 | 印第安那大学科技研究公司 | 表现出gip受体活性的胰高血糖素类似物 |
US9458214B2 (en) * | 2011-09-26 | 2016-10-04 | Novartis Ag | Dual function fibroblast growth factor 21 proteins |
WO2014037373A1 (en) * | 2012-09-07 | 2014-03-13 | Sanofi | Fusion proteins for treating a metabolic syndrome |
TWI802396B (zh) | 2014-09-16 | 2023-05-11 | 南韓商韓美藥品股份有限公司 | 長效glp-1/高血糖素受體雙促效劑治療非酒精性脂肝疾病之用途 |
WO2016049190A1 (en) * | 2014-09-24 | 2016-03-31 | Indiana University Research And Technology Corporation | Incretin-insulin conjugates |
TW201718629A (zh) * | 2015-09-25 | 2017-06-01 | 韓美藥品股份有限公司 | 包含多個生理多肽及免疫球蛋白Fc區之蛋白質接合物 |
KR102670157B1 (ko) * | 2015-10-28 | 2024-05-29 | 주식회사유한양행 | 이중 작용 단백질 및 이를 포함하는 약학적 조성물 |
PE20181494A1 (es) | 2015-12-31 | 2018-09-18 | Hanmi Pharm Ind Co Ltd | Conjugado persistente de triple activador que activa el receptor de glucagon, glp-1 u gip |
CN108440668A (zh) * | 2017-02-16 | 2018-08-24 | 瑞阳(苏州)生物科技有限公司 | Fgf21与igf-1的融合蛋白及其应用 |
EP3596130A4 (en) * | 2017-03-14 | 2020-12-30 | Sunshine Lake Pharma Co., Ltd. | DOUBLE TARGET FUSION PROTEINS WITH FC PORTION OF AN IMMUNE LOBULIN |
-
2019
- 2019-05-16 CN CN202210475324.5A patent/CN114853908B/zh active Active
- 2019-05-16 CN CN201910407446.9A patent/CN111944055B/zh active Active
-
2020
- 2020-01-03 WO PCT/CN2020/070234 patent/WO2020228360A1/zh unknown
- 2020-01-03 AU AU2020275797A patent/AU2020275797A1/en active Pending
- 2020-01-03 US US17/610,451 patent/US20220340635A1/en active Pending
- 2020-01-03 JP JP2021566567A patent/JP7268202B2/ja active Active
- 2020-01-03 EP EP20805380.1A patent/EP3971214A4/en active Pending
Patent Citations (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1999057151A2 (en) * | 1998-04-30 | 1999-11-11 | Boehringer Ingelheim International Gmbh | FAP α-SPECIFIC ANTIBODY WITH IMPROVED PRODUCIBILITY |
US20070142278A1 (en) | 2003-12-10 | 2007-06-21 | Beals John M | Muteins of fibroblast growth factor 21 |
US7576190B2 (en) | 2004-05-13 | 2009-08-18 | Eli Lilly And Company | FGF-21 fusion proteins |
US7622445B2 (en) | 2004-09-02 | 2009-11-24 | Eli Lilly And Company | Muteins of fibroblast growth factor 21 |
US9018164B2 (en) | 2005-11-07 | 2015-04-28 | Indiana University Research And Technology Corporation | Glucagon analogs exhibiting physiological solubility and stability |
US9493530B2 (en) | 2009-05-05 | 2016-11-15 | Amgen Inc. | FGF21 mutants comprising a mutation at position 98, 171 and/or 180 |
US8188040B2 (en) | 2009-05-05 | 2012-05-29 | Amgen Inc. | FGF21 mutants and uses thereof |
US20130252884A1 (en) | 2009-06-11 | 2013-09-26 | Novo Nordisk A/S | Fgf21 analogues and derivatives |
US9150632B2 (en) | 2009-06-16 | 2015-10-06 | Indiana University Research And Technology Corporation | GIP receptor-active glucagon compounds |
CN109306015A (zh) * | 2011-06-17 | 2019-02-05 | 韩美科学株式会社 | 包括泌酸调节肽和免疫球蛋白片段的结合物以及其应用 |
US20140213512A1 (en) | 2011-08-31 | 2014-07-31 | Amgen Inc. | Method of Treating or Ameliorating Type 1 Diabetes Using FGF21 |
US9006400B2 (en) | 2011-09-26 | 2015-04-14 | Novartis Ag | Fibroblast growth factor-21-Fc fusion proteins |
US8541369B2 (en) | 2011-10-04 | 2013-09-24 | Eli Lilly And Company | Fibroblast growth factor 21 variants having improved pharmacological potency and/or improved pharmaceutical stability |
US9422353B2 (en) | 2012-06-11 | 2016-08-23 | Eli Lilly And Company | Fibroblast growth factor 21 variant, composition , and uses thereof |
US20150291677A1 (en) | 2012-11-28 | 2015-10-15 | Ngm Biopharmaceuticals, Inc. | Compositions and methods for treatment of metabolic disorders and diseases |
WO2016114633A1 (en) | 2015-01-16 | 2016-07-21 | Yuhan Corporation | Long-acting fgf21 fusion proteins and pharmaceutical composition comprising the same |
CN107995914A (zh) * | 2016-08-19 | 2018-05-04 | 安源医药科技(上海)有限公司 | 人成纤维细胞生长因子21融合蛋白及其制备方法与用途 |
Non-Patent Citations (28)
Title |
---|
"METHODS IN MOLECULAR BIOLOGY", vol. 119, 1999, ACADEMIC PRESS, article "Chromatin Protocols" |
"Sequences of proteins of immunological interest", 1991, NATIONAL INSTITUTES OF HEALTH |
ALESSANDRO POCAI ET AL.: "reported a GLP-1R/GCGR dual agonist based on Oxyntomodulin (OXM", DIABETES, vol. 58, no. 10, 2009, pages 2258 - 2266 |
AUSUBEL ET AL.: "CURRENT PROTOCOLS IN MOLECULAR BIOLOGY", 1987, JOHN WILEY & SONS |
BIOORG MED CHEM LETT, vol. 23, no. 14, 2013, pages 4011 - 8 |
CELL METABOLISM, vol. 24, 2016, pages 51 - 62 |
DIABETES CARE, vol. 36, 2013, pages 498 - 504 |
DIABETES OBESMETAB, vol. 18, no. 12, 2016, pages 1176 - 1190 |
EVERS A ET AL., J MED CHEM., vol. 60, no. 10, 2017, pages 4293 - 4303 |
EXPERT OPINLNVESTIG DRUGS, vol. 25, no. 2, 2016, pages 145 - 58 |
J INTERN MED, vol. 281, no. 3, 2017, pages 233 - 246 |
J. PEPT. SCI., vol. 14, 2008, pages 588 - 595 |
JOSEPH R. CHABENNE ET AL., J DIABETES SCI TECHNOL., vol. 4, no. 6, 2010, pages 1322 - 1331 |
LANCET, vol. 368, 2006, pages 1696 - 705 |
LTEIF, MATHER, CAN. J. CARDIOL., vol. 20, 2004, pages 66B - 76B |
MATTHIAS H. TSCHOP ET AL., CELL METAB, vol. 24, no. 1, 2016, pages 51 - 62 |
MOLMETAB, vol. 5, no. 8, 2016, pages 690 - 8 |
N ENGL J MED, vol. 355, no. 23, 2006, pages 2427 - 43 |
NAT. REV. ENDOCRINOL, vol. 6, 2010, pages 689 - 697 |
NATURE MEDICINE, vol. 22, no. 7, 2016, pages 694 - 695 |
PROC NATL ACADSCI USA, vol. 109, no. 8, 2012, pages 3143 - 8 |
RICHARD D. DIMARCHI ET AL., REPORTED A GCG-BASED GLP-1R/GCGR DUAL AGONIST |
SAMBROOK ET AL.: "MOLECULAR CLONING: A LABORATORY MANUAL", 1989, COLD SPRING HARBOR LABORATORY PRESS |
SCAND J CLIN LAB INVEST, vol. 75, no. 2, 2015, pages 121 - 5 |
See also references of EP3971214A4 |
TRENDS PHARMACOLSCI, vol. 32, no. 1, 2011, pages 8 - 15 |
WOLFFE: "CHROMATIN STRUCTURE AND FUNCTION", 1998, ACADEMIC PRESS |
XU J ET AL., BIOCONJUG CHEM., vol. 24, no. 6, 2013, pages 915 - 25 |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114621327A (zh) * | 2020-12-10 | 2022-06-14 | 江苏中新医药有限公司 | GLP-1、GIP和Gcg多重受体激动蛋白质 |
CN114621327B (zh) * | 2020-12-10 | 2023-08-18 | 江苏中新医药有限公司 | GLP-1、GIP和Gcg多重受体激动蛋白质 |
Also Published As
Publication number | Publication date |
---|---|
CN111944055A (zh) | 2020-11-17 |
CN111944055B (zh) | 2022-08-02 |
AU2020275797A1 (en) | 2022-01-20 |
EP3971214A4 (en) | 2023-06-28 |
EP3971214A9 (en) | 2022-05-18 |
JP7268202B2 (ja) | 2023-05-02 |
CN114853908A (zh) | 2022-08-05 |
EP3971214A1 (en) | 2022-03-23 |
US20220340635A1 (en) | 2022-10-27 |
JP2022532332A (ja) | 2022-07-14 |
CN114853908B (zh) | 2024-06-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
WO2020228360A1 (zh) | 一种治疗代谢疾病的融合蛋白 | |
US11858975B2 (en) | Multi-domain active protein for treating metabolic diseases | |
JP2022031787A (ja) | グルカゴン及びglp-1共アゴニスト化合物 | |
CN109836488B (zh) | 一种治疗代谢疾病的胰高血糖素类似物 | |
KR102460198B1 (ko) | 비만 치료를 위한 글루카곤 및 glp-1 공동-작용제 | |
US20080032932A1 (en) | Method of reducing mortality and morbidity associated with critical illnesses | |
CN109836503B (zh) | 一种治疗代谢疾病的多重活性蛋白 | |
JP2013506628A (ja) | 延長された半減期を有する薬物融合物及びコンジュゲート | |
TW201420606A (zh) | 同源二聚體蛋白 | |
AU2017358289A1 (en) | Pharmaceutical composition for preventing or treating hepatitis, hepatic fibrosis, and hepatic cirrhosis comprising fusion proteins | |
WO2023246928A1 (zh) | 一种改进的glp-1受体激动剂的融合蛋白和应用 | |
KR20210103265A (ko) | 신규 비알코올성 지방간염 치료용 약학 조성물 | |
NZ794313A (en) | Pharmaceutical composition for preventing or treating hepatitis, hepatic fibrosis, and hepatic cirrhosis comprising fusion proteins | |
AU2002326815A1 (en) | Glucagon-like Peptides (GLP-1) and Treatment of Respiratory Distress |
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: 20805380 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2021566567 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
ENP | Entry into the national phase |
Ref document number: 2020805380 Country of ref document: EP Effective date: 20211216 |
|
ENP | Entry into the national phase |
Ref document number: 2020275797 Country of ref document: AU Date of ref document: 20200103 Kind code of ref document: A |