CA2557151C - Peptide analogues of gip for treatment of diabetes, insulin resistance and obesity - Google Patents
Peptide analogues of gip for treatment of diabetes, insulin resistance and obesity Download PDFInfo
- Publication number
- CA2557151C CA2557151C CA2557151A CA2557151A CA2557151C CA 2557151 C CA2557151 C CA 2557151C CA 2557151 A CA2557151 A CA 2557151A CA 2557151 A CA2557151 A CA 2557151A CA 2557151 C CA2557151 C CA 2557151C
- Authority
- CA
- Canada
- Prior art keywords
- gip
- insulin
- glucose
- mice
- pro3
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims abstract description 129
- 208000001072 type 2 diabetes mellitus Diseases 0.000 title claims description 56
- 206010022489 Insulin Resistance Diseases 0.000 title claims description 53
- 208000008589 Obesity Diseases 0.000 title claims description 29
- 235000020824 obesity Nutrition 0.000 title claims description 22
- 206010012601 diabetes mellitus Diseases 0.000 title abstract description 95
- 238000011282 treatment Methods 0.000 title abstract description 76
- 230000004048 modification Effects 0.000 claims abstract description 27
- 238000012986 modification Methods 0.000 claims abstract description 27
- 238000006467 substitution reaction Methods 0.000 claims abstract description 24
- 108010036598 gastric inhibitory polypeptide receptor Proteins 0.000 claims description 54
- 108010004460 Gastric Inhibitory Polypeptide Proteins 0.000 claims description 51
- 108010049869 gastric inhibitory polypeptide (1-42) Proteins 0.000 claims description 42
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 claims description 33
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 32
- 229930195729 fatty acid Natural products 0.000 claims description 32
- 239000000194 fatty acid Substances 0.000 claims description 32
- 150000004665 fatty acids Chemical class 0.000 claims description 30
- 150000001413 amino acids Chemical group 0.000 claims description 28
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 claims description 25
- 108010020107 gastric inhibitory polypeptide (3-42) Proteins 0.000 claims description 24
- 125000003277 amino group Chemical group 0.000 claims description 17
- 125000000539 amino acid group Chemical group 0.000 claims description 14
- -1 palmitate fatty acid Chemical class 0.000 claims description 11
- 230000021736 acetylation Effects 0.000 claims description 9
- 238000006640 acetylation reaction Methods 0.000 claims description 9
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 claims description 8
- 239000002464 receptor antagonist Substances 0.000 claims description 8
- 229940044551 receptor antagonist Drugs 0.000 claims description 8
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 claims description 8
- 125000001429 N-terminal alpha-amino-acid group Chemical group 0.000 claims description 6
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 claims description 4
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 claims description 4
- 239000004472 Lysine Substances 0.000 claims description 4
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 claims description 4
- PMMYEEVYMWASQN-DMTCNVIQSA-N Hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 claims description 3
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 claims description 3
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 claims description 3
- PMMYEEVYMWASQN-UHFFFAOYSA-N dl-hydroxyproline Natural products OC1C[NH2+]C(C([O-])=O)C1 PMMYEEVYMWASQN-UHFFFAOYSA-N 0.000 claims description 3
- 108010071400 gastric inhibitory polypeptide (1-30) Proteins 0.000 claims description 3
- 108010073571 gastric inhibitory polypeptide (1-39) Proteins 0.000 claims description 3
- 229960002591 hydroxyproline Drugs 0.000 claims description 3
- FGMPLJWBKKVCDB-UHFFFAOYSA-N trans-L-hydroxy-proline Natural products ON1CCCC1C(O)=O FGMPLJWBKKVCDB-UHFFFAOYSA-N 0.000 claims description 3
- 102100025012 Dipeptidyl peptidase 4 Human genes 0.000 abstract description 76
- 230000015556 catabolic process Effects 0.000 abstract description 73
- 108010067722 Dipeptidyl Peptidase 4 Proteins 0.000 abstract description 69
- 238000006731 degradation reaction Methods 0.000 abstract description 69
- 238000000034 method Methods 0.000 abstract description 26
- 102000004196 processed proteins & peptides Human genes 0.000 abstract description 17
- 239000005557 antagonist Substances 0.000 abstract description 16
- 101000930822 Giardia intestinalis Dipeptidyl-peptidase 4 Proteins 0.000 abstract description 7
- 230000002496 gastric effect Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 102000016622 Dipeptidyl Peptidase 4 Human genes 0.000 abstract 1
- 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 348
- 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 257
- 239000008103 glucose Substances 0.000 description 254
- 102000004877 Insulin Human genes 0.000 description 174
- 108090001061 Insulin Proteins 0.000 description 174
- 241000699670 Mus sp. Species 0.000 description 174
- 229940125396 insulin Drugs 0.000 description 174
- 108700037808 Pro(3)- glucose-dependent insulinotropic polypeptide Proteins 0.000 description 150
- 230000000694 effects Effects 0.000 description 101
- 108010093255 N-AcGIP(LysPAL37) Proteins 0.000 description 79
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 73
- 239000011780 sodium chloride Substances 0.000 description 67
- 239000007924 injection Substances 0.000 description 63
- 238000002347 injection Methods 0.000 description 63
- 230000003914 insulin secretion Effects 0.000 description 63
- 230000037396 body weight Effects 0.000 description 55
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 51
- 210000004027 cell Anatomy 0.000 description 49
- 229940090044 injection Drugs 0.000 description 49
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 40
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 34
- 230000003578 releasing effect Effects 0.000 description 32
- 238000011534 incubation Methods 0.000 description 31
- 238000001727 in vivo Methods 0.000 description 27
- 230000001154 acute effect Effects 0.000 description 26
- 238000007912 intraperitoneal administration Methods 0.000 description 26
- 230000004044 response Effects 0.000 description 26
- 230000009471 action Effects 0.000 description 25
- 230000001684 chronic effect Effects 0.000 description 25
- 230000006362 insulin response pathway Effects 0.000 description 25
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 25
- 235000001014 amino acid Nutrition 0.000 description 24
- 210000004369 blood Anatomy 0.000 description 23
- 239000008280 blood Substances 0.000 description 23
- 102400000322 Glucagon-like peptide 1 Human genes 0.000 description 22
- 230000037406 food intake Effects 0.000 description 22
- 238000012360 testing method Methods 0.000 description 22
- 241000282414 Homo sapiens Species 0.000 description 21
- 241001465754 Metazoa Species 0.000 description 20
- 238000004128 high performance liquid chromatography Methods 0.000 description 20
- 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 20
- 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
- 230000003247 decreasing effect Effects 0.000 description 19
- 230000002641 glycemic effect Effects 0.000 description 19
- 239000000203 mixture Substances 0.000 description 19
- IVOMOUWHDPKRLL-KQYNXXCUSA-N Cyclic adenosine monophosphate Chemical compound C([C@H]1O2)OP(O)(=O)O[C@H]1[C@@H](O)[C@@H]2N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-KQYNXXCUSA-N 0.000 description 18
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 18
- 101800000224 Glucagon-like peptide 1 Proteins 0.000 description 18
- IVOMOUWHDPKRLL-UHFFFAOYSA-N UNPD107823 Natural products O1C2COP(O)(=O)OC2C(O)C1N1C(N=CN=C2N)=C2N=C1 IVOMOUWHDPKRLL-UHFFFAOYSA-N 0.000 description 18
- 229940095074 cyclic amp Drugs 0.000 description 18
- 238000000338 in vitro Methods 0.000 description 18
- 230000003389 potentiating effect Effects 0.000 description 18
- 235000012631 food intake Nutrition 0.000 description 17
- 230000002503 metabolic effect Effects 0.000 description 17
- JNTMAZFVYNDPLB-PEDHHIEDSA-N (2S,3S)-2-[[[(2S)-1-[(2S,3S)-2-amino-3-methyl-1-oxopentyl]-2-pyrrolidinyl]-oxomethyl]amino]-3-methylpentanoic acid Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JNTMAZFVYNDPLB-PEDHHIEDSA-N 0.000 description 16
- 101100533230 Caenorhabditis elegans ser-2 gene Proteins 0.000 description 16
- 101800004266 Glucagon-like peptide 1(7-37) Proteins 0.000 description 16
- 229940024606 amino acid Drugs 0.000 description 16
- 230000001965 increasing effect Effects 0.000 description 16
- 108010054812 diprotin A Proteins 0.000 description 15
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 15
- 230000006872 improvement Effects 0.000 description 15
- 230000002473 insulinotropic effect Effects 0.000 description 15
- 230000010030 glucose lowering effect Effects 0.000 description 14
- 230000001976 improved effect Effects 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 14
- 150000003839 salts Chemical class 0.000 description 14
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 14
- 101000886868 Homo sapiens Gastric inhibitory polypeptide Proteins 0.000 description 13
- 239000008194 pharmaceutical composition Substances 0.000 description 13
- 230000002058 anti-hyperglycaemic effect Effects 0.000 description 12
- 210000000227 basophil cell of anterior lobe of hypophysis Anatomy 0.000 description 12
- 239000000872 buffer Substances 0.000 description 12
- 231100000673 dose–response relationship Toxicity 0.000 description 12
- 102000005962 receptors Human genes 0.000 description 12
- 108020003175 receptors Proteins 0.000 description 12
- 210000002237 B-cell of pancreatic islet Anatomy 0.000 description 11
- 208000002705 Glucose Intolerance Diseases 0.000 description 11
- 102000017011 Glycated Hemoglobin A Human genes 0.000 description 11
- 230000015572 biosynthetic process Effects 0.000 description 11
- 238000006243 chemical reaction Methods 0.000 description 11
- 230000001419 dependent effect Effects 0.000 description 11
- 238000002474 experimental method Methods 0.000 description 11
- 108091005995 glycated hemoglobin Proteins 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 11
- 239000000126 substance Substances 0.000 description 11
- 230000001225 therapeutic effect Effects 0.000 description 11
- 239000003981 vehicle Substances 0.000 description 11
- 238000002679 ablation Methods 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- 230000009286 beneficial effect Effects 0.000 description 10
- 230000004071 biological effect Effects 0.000 description 10
- 230000007423 decrease Effects 0.000 description 10
- 230000014101 glucose homeostasis Effects 0.000 description 10
- 239000007928 intraperitoneal injection Substances 0.000 description 10
- 238000004519 manufacturing process Methods 0.000 description 10
- 238000002360 preparation method Methods 0.000 description 10
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 9
- 230000004075 alteration Effects 0.000 description 9
- 239000012634 fragment Substances 0.000 description 9
- 230000006870 function Effects 0.000 description 9
- 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 9
- 239000000859 incretin Substances 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 239000011159 matrix material Substances 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 238000003127 radioimmunoassay Methods 0.000 description 9
- 208000011580 syndromic disease Diseases 0.000 description 9
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 8
- 206010060378 Hyperinsulinaemia Diseases 0.000 description 8
- 206010020880 Hypertrophy Diseases 0.000 description 8
- 108091006629 SLC13A2 Proteins 0.000 description 8
- 239000003875 Wang resin Substances 0.000 description 8
- 238000000586 desensitisation Methods 0.000 description 8
- 229940088597 hormone Drugs 0.000 description 8
- 239000005556 hormone Substances 0.000 description 8
- 102000050325 human granulocyte inhibitory Human genes 0.000 description 8
- 230000003451 hyperinsulinaemic effect Effects 0.000 description 8
- 201000008980 hyperinsulinism Diseases 0.000 description 8
- 239000003112 inhibitor Substances 0.000 description 8
- 230000014759 maintenance of location Effects 0.000 description 8
- 238000005259 measurement Methods 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 238000003786 synthesis reaction Methods 0.000 description 8
- 210000001519 tissue Anatomy 0.000 description 8
- 101100315624 Caenorhabditis elegans tyr-1 gene Proteins 0.000 description 7
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 7
- KRHYYFGTRYWZRS-UHFFFAOYSA-M Fluoride anion Chemical compound [F-] KRHYYFGTRYWZRS-UHFFFAOYSA-M 0.000 description 7
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 7
- 229930182555 Penicillin Natural products 0.000 description 7
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 7
- 239000003814 drug Substances 0.000 description 7
- 230000036252 glycation Effects 0.000 description 7
- 229920000669 heparin Polymers 0.000 description 7
- 229960002897 heparin Drugs 0.000 description 7
- 239000007788 liquid Substances 0.000 description 7
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 7
- 229940049954 penicillin Drugs 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 229960005322 streptomycin Drugs 0.000 description 7
- WFDIJRYMOXRFFG-UHFFFAOYSA-N Acetic anhydride Chemical compound CC(=O)OC(C)=O WFDIJRYMOXRFFG-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 6
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 6
- 241000700159 Rattus Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000008485 antagonism Effects 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000003776 cleavage reaction Methods 0.000 description 6
- 239000007857 degradation product Substances 0.000 description 6
- 210000002950 fibroblast Anatomy 0.000 description 6
- 230000002068 genetic effect Effects 0.000 description 6
- 206010020718 hyperplasia Diseases 0.000 description 6
- 230000007017 scission Effects 0.000 description 6
- 230000003248 secreting effect Effects 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 239000003104 tissue culture media Substances 0.000 description 6
- 239000003643 water by type Substances 0.000 description 6
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 5
- 241000699802 Cricetulus griseus Species 0.000 description 5
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 5
- 239000004480 active ingredient Substances 0.000 description 5
- 210000000577 adipose tissue Anatomy 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 239000003472 antidiabetic agent Substances 0.000 description 5
- 230000008901 benefit Effects 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 238000004587 chromatography analysis Methods 0.000 description 5
- 238000001212 derivatisation Methods 0.000 description 5
- 230000006866 deterioration Effects 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 201000001421 hyperglycemia Diseases 0.000 description 5
- 230000003834 intracellular effect Effects 0.000 description 5
- 238000011866 long-term treatment Methods 0.000 description 5
- 239000002609 medium Substances 0.000 description 5
- 229920000747 poly(lactic acid) Polymers 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 239000011347 resin Substances 0.000 description 5
- 229920005989 resin Polymers 0.000 description 5
- 230000028327 secretion Effects 0.000 description 5
- 238000000926 separation method Methods 0.000 description 5
- 230000000638 stimulation Effects 0.000 description 5
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 5
- 125000001493 tyrosinyl group Chemical class [H]OC1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 5
- FUOOLUPWFVMBKG-UHFFFAOYSA-N 2-Aminoisobutyric acid Chemical compound CC(C)(N)C(O)=O FUOOLUPWFVMBKG-UHFFFAOYSA-N 0.000 description 4
- 108010016626 Dipeptides Proteins 0.000 description 4
- 239000004366 Glucose oxidase Substances 0.000 description 4
- 108010015776 Glucose oxidase Proteins 0.000 description 4
- 241000699666 Mus <mouse, genus> Species 0.000 description 4
- KSPIYJQBLVDRRI-UHFFFAOYSA-N N-methylisoleucine Chemical compound CCC(C)C(NC)C(O)=O KSPIYJQBLVDRRI-UHFFFAOYSA-N 0.000 description 4
- MUBZPKHOEPUJKR-UHFFFAOYSA-N Oxalic acid Chemical compound OC(=O)C(O)=O MUBZPKHOEPUJKR-UHFFFAOYSA-N 0.000 description 4
- 239000012980 RPMI-1640 medium Substances 0.000 description 4
- 241000283984 Rodentia Species 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 230000003178 anti-diabetic effect Effects 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 230000006735 deficit Effects 0.000 description 4
- 239000012909 foetal bovine serum Substances 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- 229940116332 glucose oxidase Drugs 0.000 description 4
- 235000019420 glucose oxidase Nutrition 0.000 description 4
- IPCSVZSSVZVIGE-UHFFFAOYSA-N hexadecanoic acid Chemical group CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 description 4
- 230000002779 inactivation Effects 0.000 description 4
- 230000005764 inhibitory process Effects 0.000 description 4
- 150000002500 ions Chemical class 0.000 description 4
- 210000004153 islets of langerhan Anatomy 0.000 description 4
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 4
- 210000004072 lung Anatomy 0.000 description 4
- 235000012054 meals Nutrition 0.000 description 4
- 230000001404 mediated effect Effects 0.000 description 4
- 230000004060 metabolic process Effects 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 150000007524 organic acids Chemical class 0.000 description 4
- 210000004923 pancreatic tissue Anatomy 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 230000010656 regulation of insulin secretion Effects 0.000 description 4
- 238000004007 reversed phase HPLC Methods 0.000 description 4
- 239000002904 solvent Substances 0.000 description 4
- 238000010254 subcutaneous injection Methods 0.000 description 4
- 239000007929 subcutaneous injection Substances 0.000 description 4
- 239000003826 tablet Substances 0.000 description 4
- APIXJSLKIYYUKG-UHFFFAOYSA-N 3 Isobutyl 1 methylxanthine Chemical compound O=C1N(C)C(=O)N(CC(C)C)C2=C1N=CN2 APIXJSLKIYYUKG-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- ZKHQWZAMYRWXGA-KQYNXXCUSA-N Adenosine triphosphate Chemical compound C1=NC=2C(N)=NC=NC=2N1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)[C@H]1O ZKHQWZAMYRWXGA-KQYNXXCUSA-N 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 3
- 102100039994 Gastric inhibitory polypeptide Human genes 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101000684208 Homo sapiens Prolyl endopeptidase FAP Proteins 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 3
- 206010020710 Hyperphagia Diseases 0.000 description 3
- 102000016267 Leptin Human genes 0.000 description 3
- 108010092277 Leptin Proteins 0.000 description 3
- 208000001145 Metabolic Syndrome Diseases 0.000 description 3
- 125000000729 N-terminal amino-acid group Chemical group 0.000 description 3
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 3
- NERFNHBZJXXFGY-UHFFFAOYSA-N [4-[(4-methylphenyl)methoxy]phenyl]methanol Chemical compound C1=CC(C)=CC=C1COC1=CC=C(CO)C=C1 NERFNHBZJXXFGY-UHFFFAOYSA-N 0.000 description 3
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 3
- 230000005856 abnormality Effects 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 239000012298 atmosphere Substances 0.000 description 3
- 239000002585 base Substances 0.000 description 3
- UCMIRNVEIXFBKS-UHFFFAOYSA-N beta-alanine Chemical compound NCCC(O)=O UCMIRNVEIXFBKS-UHFFFAOYSA-N 0.000 description 3
- 229940098773 bovine serum albumin Drugs 0.000 description 3
- 239000007853 buffer solution Substances 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000001413 cellular effect Effects 0.000 description 3
- 239000003610 charcoal Substances 0.000 description 3
- 238000007385 chemical modification Methods 0.000 description 3
- 239000007795 chemical reaction product Substances 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- XVOYSCVBGLVSOL-UHFFFAOYSA-N cysteic acid Chemical compound OC(=O)C(N)CS(O)(=O)=O XVOYSCVBGLVSOL-UHFFFAOYSA-N 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 235000005911 diet Nutrition 0.000 description 3
- 230000037213 diet Effects 0.000 description 3
- 239000003085 diluting agent Substances 0.000 description 3
- 230000003292 diminished effect Effects 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 230000002124 endocrine Effects 0.000 description 3
- 230000002708 enhancing effect Effects 0.000 description 3
- 238000011156 evaluation Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000030136 gastric emptying Effects 0.000 description 3
- IPCSVZSSVZVIGE-UHFFFAOYSA-M hexadecanoate Chemical compound CCCCCCCCCCCCCCCC([O-])=O IPCSVZSSVZVIGE-UHFFFAOYSA-M 0.000 description 3
- 229940039781 leptin Drugs 0.000 description 3
- NRYBAZVQPHGZNS-ZSOCWYAHSA-N leptin Chemical compound O=C([C@H](CO)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)CNC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(C)C)CCSC)N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CS)C(O)=O NRYBAZVQPHGZNS-ZSOCWYAHSA-N 0.000 description 3
- 230000007774 longterm Effects 0.000 description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 3
- 235000019341 magnesium sulphate Nutrition 0.000 description 3
- 238000012423 maintenance Methods 0.000 description 3
- 230000007246 mechanism Effects 0.000 description 3
- 150000007522 mineralic acids Chemical class 0.000 description 3
- 235000020925 non fasting Nutrition 0.000 description 3
- 230000035764 nutrition Effects 0.000 description 3
- 235000016709 nutrition Nutrition 0.000 description 3
- 235000005985 organic acids Nutrition 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 229920001467 poly(styrenesulfonates) Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 238000011533 pre-incubation Methods 0.000 description 3
- 230000000750 progressive effect Effects 0.000 description 3
- 230000002035 prolonged effect Effects 0.000 description 3
- 230000004043 responsiveness Effects 0.000 description 3
- 230000002441 reversible effect Effects 0.000 description 3
- 239000007790 solid phase Substances 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 230000004936 stimulating effect Effects 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000013268 sustained release Methods 0.000 description 3
- 239000012730 sustained-release form Substances 0.000 description 3
- 238000011191 terminal modification Methods 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- VZCYOOQTPOCHFL-UHFFFAOYSA-N trans-butenedioic acid Natural products OC(=O)C=CC(O)=O VZCYOOQTPOCHFL-UHFFFAOYSA-N 0.000 description 3
- 238000011870 unpaired t-test Methods 0.000 description 3
- MRTPISKDZDHEQI-YFKPBYRVSA-N (2s)-2-(tert-butylamino)propanoic acid Chemical compound OC(=O)[C@H](C)NC(C)(C)C MRTPISKDZDHEQI-YFKPBYRVSA-N 0.000 description 2
- NPDBDJFLKKQMCM-SCSAIBSYSA-N (2s)-2-amino-3,3-dimethylbutanoic acid Chemical compound CC(C)(C)[C@H](N)C(O)=O NPDBDJFLKKQMCM-SCSAIBSYSA-N 0.000 description 2
- ODHCTXKNWHHXJC-VKHMYHEASA-N 5-oxo-L-proline Chemical compound OC(=O)[C@@H]1CCC(=O)N1 ODHCTXKNWHHXJC-VKHMYHEASA-N 0.000 description 2
- 229930024421 Adenine Natural products 0.000 description 2
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 description 2
- 102000004400 Aminopeptidases Human genes 0.000 description 2
- 108090000915 Aminopeptidases Proteins 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 238000012935 Averaging Methods 0.000 description 2
- 101100455752 Caenorhabditis elegans lys-3 gene Proteins 0.000 description 2
- 101100315627 Caenorhabditis elegans tyr-3 gene Proteins 0.000 description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 2
- 241000557626 Corvus corax Species 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 2
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- OHCQJHSOBUTRHG-KGGHGJDLSA-N FORSKOLIN Chemical compound O=C([C@@]12O)C[C@](C)(C=C)O[C@]1(C)[C@@H](OC(=O)C)[C@@H](O)[C@@H]1[C@]2(C)[C@@H](O)CCC1(C)C OHCQJHSOBUTRHG-KGGHGJDLSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- QEFRNWWLZKMPFJ-ZXPFJRLXSA-N L-methionine (R)-S-oxide Chemical compound C[S@@](=O)CC[C@H]([NH3+])C([O-])=O QEFRNWWLZKMPFJ-ZXPFJRLXSA-N 0.000 description 2
- QEFRNWWLZKMPFJ-UHFFFAOYSA-N L-methionine sulphoxide Natural products CS(=O)CCC(N)C(O)=O QEFRNWWLZKMPFJ-UHFFFAOYSA-N 0.000 description 2
- LRQKBLKVPFOOQJ-YFKPBYRVSA-N L-norleucine Chemical compound CCCC[C@H]([NH3+])C([O-])=O LRQKBLKVPFOOQJ-YFKPBYRVSA-N 0.000 description 2
- 108010019598 Liraglutide Proteins 0.000 description 2
- 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 2
- 241000124008 Mammalia Species 0.000 description 2
- 206010028813 Nausea Diseases 0.000 description 2
- 101100426589 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) trp-3 gene Proteins 0.000 description 2
- 206010067482 No adverse event Diseases 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 2
- 229920000954 Polyglycolide Polymers 0.000 description 2
- ODHCTXKNWHHXJC-GSVOUGTGSA-N Pyroglutamic acid Natural products OC(=O)[C@H]1CCC(=O)N1 ODHCTXKNWHHXJC-GSVOUGTGSA-N 0.000 description 2
- 239000008156 Ringer's lactate solution Substances 0.000 description 2
- 102000005157 Somatostatin Human genes 0.000 description 2
- 108010056088 Somatostatin Proteins 0.000 description 2
- 238000003639 Student–Newman–Keuls (SNK) method Methods 0.000 description 2
- 102000003629 TRPC3 Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- ODHCTXKNWHHXJC-UHFFFAOYSA-N acide pyroglutamique Natural products OC(=O)C1CCC(=O)N1 ODHCTXKNWHHXJC-UHFFFAOYSA-N 0.000 description 2
- 230000010933 acylation Effects 0.000 description 2
- 238000005917 acylation reaction Methods 0.000 description 2
- 229960000643 adenine Drugs 0.000 description 2
- 210000001789 adipocyte Anatomy 0.000 description 2
- 230000029936 alkylation Effects 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- 229940127003 anti-diabetic drug Drugs 0.000 description 2
- 239000003963 antioxidant agent Substances 0.000 description 2
- 235000006708 antioxidants Nutrition 0.000 description 2
- 230000003190 augmentative effect Effects 0.000 description 2
- 210000000467 autonomic pathway Anatomy 0.000 description 2
- 210000003719 b-lymphocyte Anatomy 0.000 description 2
- XMIIGOLPHOKFCH-UHFFFAOYSA-N beta-phenylpropanoic acid Natural products OC(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 150000001649 bromium compounds Chemical class 0.000 description 2
- 244000309466 calf Species 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000005341 cation exchange Methods 0.000 description 2
- OHCQJHSOBUTRHG-UHFFFAOYSA-N colforsin Natural products OC12C(=O)CC(C)(C=C)OC1(C)C(OC(=O)C)C(O)C1C2(C)C(O)CCC1(C)C OHCQJHSOBUTRHG-UHFFFAOYSA-N 0.000 description 2
- 230000001447 compensatory effect Effects 0.000 description 2
- 230000001010 compromised effect Effects 0.000 description 2
- 229920001577 copolymer Polymers 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000003937 drug carrier Substances 0.000 description 2
- 230000000081 effect on glucose Effects 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 230000007515 enzymatic degradation Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 230000000763 evoking effect Effects 0.000 description 2
- 238000000605 extraction Methods 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000003629 gastrointestinal hormone Substances 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 235000011167 hydrochloric acid Nutrition 0.000 description 2
- 230000003345 hyperglycaemic effect Effects 0.000 description 2
- 230000002218 hypoglycaemic effect Effects 0.000 description 2
- 230000001771 impaired effect Effects 0.000 description 2
- 206010022498 insulinoma Diseases 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 150000004694 iodide salts Chemical class 0.000 description 2
- 238000005040 ion trap Methods 0.000 description 2
- 125000001449 isopropyl group Chemical group [H]C([H])([H])C([H])(*)C([H])([H])[H] 0.000 description 2
- 238000011813 knockout mouse model Methods 0.000 description 2
- 239000004310 lactic acid Substances 0.000 description 2
- 235000014655 lactic acid Nutrition 0.000 description 2
- 229960002701 liraglutide Drugs 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- VZCYOOQTPOCHFL-UPHRSURJSA-N maleic acid Chemical compound OC(=O)\C=C/C(O)=O VZCYOOQTPOCHFL-UPHRSURJSA-N 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 230000008693 nausea Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 239000003921 oil Substances 0.000 description 2
- 235000019198 oils Nutrition 0.000 description 2
- 208000021255 pancreatic insulinoma Diseases 0.000 description 2
- 230000036961 partial effect Effects 0.000 description 2
- 238000010647 peptide synthesis reaction Methods 0.000 description 2
- 235000011007 phosphoric acid Nutrition 0.000 description 2
- 230000001766 physiological effect Effects 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000010149 post-hoc-test Methods 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 201000009104 prediabetes syndrome Diseases 0.000 description 2
- 125000001500 prolyl group Chemical group [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000009467 reduction Effects 0.000 description 2
- 230000029054 response to nutrient Effects 0.000 description 2
- FSYKKLYZXJSNPZ-UHFFFAOYSA-N sarcosine Chemical compound C[NH2+]CC([O-])=O FSYKKLYZXJSNPZ-UHFFFAOYSA-N 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 238000012163 sequencing technique Methods 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- BEOOHQFXGBMRKU-UHFFFAOYSA-N sodium cyanoborohydride Chemical compound [Na+].[B-]C#N BEOOHQFXGBMRKU-UHFFFAOYSA-N 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
- 229960000553 somatostatin Drugs 0.000 description 2
- 230000003595 spectral effect Effects 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- KDYFGRWQOYBRFD-UHFFFAOYSA-N succinic acid Chemical compound OC(=O)CCC(O)=O KDYFGRWQOYBRFD-UHFFFAOYSA-N 0.000 description 2
- 230000008093 supporting effect Effects 0.000 description 2
- WROMPOXWARCANT-UHFFFAOYSA-N tfa trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F.OC(=O)C(F)(F)F WROMPOXWARCANT-UHFFFAOYSA-N 0.000 description 2
- 229940124597 therapeutic agent Drugs 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- GETQZCLCWQTVFV-UHFFFAOYSA-N trimethylamine Chemical compound CN(C)C GETQZCLCWQTVFV-UHFFFAOYSA-N 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 210000003462 vein Anatomy 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- AFVLVVWMAFSXCK-UHFFFAOYSA-N α-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(C#N)=CC1=CC=C(O)C=C1 AFVLVVWMAFSXCK-UHFFFAOYSA-N 0.000 description 2
- LSPHULWDVZXLIL-UHFFFAOYSA-N (+/-)-Camphoric acid Chemical compound CC1(C)C(C(O)=O)CCC1(C)C(O)=O LSPHULWDVZXLIL-UHFFFAOYSA-N 0.000 description 1
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 description 1
- BVAUMRCGVHUWOZ-ZETCQYMHSA-N (2s)-2-(cyclohexylazaniumyl)propanoate Chemical compound OC(=O)[C@H](C)NC1CCCCC1 BVAUMRCGVHUWOZ-ZETCQYMHSA-N 0.000 description 1
- ZSOPWZQRZHWYFY-NUXPJIRBSA-N (2s,3s)-2-amino-3-methyl-1-(1,3-thiazolidin-3-yl)pentan-1-one;(e)-but-2-enedioic acid Chemical compound OC(=O)\C=C\C(O)=O.CC[C@H](C)[C@H](N)C(=O)N1CCSC1 ZSOPWZQRZHWYFY-NUXPJIRBSA-N 0.000 description 1
- WSEVKKHALHSUMB-RYVRVIGHSA-N (4S)-4-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2R)-5-amino-2-[[(2S)-6-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-carboxypropanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxypropanoyl]amino]hexanoyl]amino]-5-oxopentanoyl]amino]-4-methylsulfanylbutanoyl]amino]-4-carboxybutanoyl]amino]-4-carboxybutanoyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S,3S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-6-amino-1-[[(2S)-4-amino-1-[[2-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[[2-[[(2S)-1-[(2S)-2-[(2S)-2-[(2S)-2-[[(2S)-1-amino-3-hydroxy-1-oxopropan-2-yl]carbamoyl]pyrrolidine-1-carbonyl]pyrrolidine-1-carbonyl]pyrrolidin-1-yl]-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-2-oxoethyl]amino]-1,4-dioxobutan-2-yl]amino]-1-oxohexan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-3-(1H-indol-3-yl)-1-oxopropan-2-yl]amino]-4-carboxy-1-oxobutan-2-yl]amino]-3-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC[C@H](C)[C@H](NC(=O)[C@H](Cc1ccccc1)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](N)CC(O)=O)C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)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)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(N)=O WSEVKKHALHSUMB-RYVRVIGHSA-N 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- VFWCMGCRMGJXDK-UHFFFAOYSA-N 1-chlorobutane Chemical class CCCCCl VFWCMGCRMGJXDK-UHFFFAOYSA-N 0.000 description 1
- VUQPJRPDRDVQMN-UHFFFAOYSA-N 1-chlorooctadecane Chemical class CCCCCCCCCCCCCCCCCCCl VUQPJRPDRDVQMN-UHFFFAOYSA-N 0.000 description 1
- MIJDSYMOBYNHOT-UHFFFAOYSA-N 2-(ethylamino)ethanol Chemical compound CCNCCO MIJDSYMOBYNHOT-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
- DETXZQGDWUJKMO-UHFFFAOYSA-N 2-hydroxymethanesulfonic acid Chemical compound OCS(O)(=O)=O DETXZQGDWUJKMO-UHFFFAOYSA-N 0.000 description 1
- 229940080296 2-naphthalenesulfonate Drugs 0.000 description 1
- WMPPDTMATNBGJN-UHFFFAOYSA-N 2-phenylethylbromide Chemical class BrCCC1=CC=CC=C1 WMPPDTMATNBGJN-UHFFFAOYSA-N 0.000 description 1
- BMYNFMYTOJXKLE-UHFFFAOYSA-N 3-azaniumyl-2-hydroxypropanoate Chemical compound NCC(O)C(O)=O BMYNFMYTOJXKLE-UHFFFAOYSA-N 0.000 description 1
- XMIIGOLPHOKFCH-UHFFFAOYSA-M 3-phenylpropionate Chemical compound [O-]C(=O)CCC1=CC=CC=C1 XMIIGOLPHOKFCH-UHFFFAOYSA-M 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-M Bicarbonate Chemical compound OC([O-])=O BVKZGUZCCUSVTD-UHFFFAOYSA-M 0.000 description 1
- 229940123208 Biguanide Drugs 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-M Butyrate Chemical compound CCCC([O-])=O FERIUCNNQQJTOY-UHFFFAOYSA-M 0.000 description 1
- FERIUCNNQQJTOY-UHFFFAOYSA-N Butyric acid Natural products CCCC(O)=O FERIUCNNQQJTOY-UHFFFAOYSA-N 0.000 description 1
- 101100505076 Caenorhabditis elegans gly-2 gene Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- QNAYBMKLOCPYGJ-UWTATZPHSA-N D-alanine Chemical compound C[C@@H](N)C(O)=O QNAYBMKLOCPYGJ-UWTATZPHSA-N 0.000 description 1
- QNAYBMKLOCPYGJ-UHFFFAOYSA-N D-alpha-Ala Natural products CC([NH3+])C([O-])=O QNAYBMKLOCPYGJ-UHFFFAOYSA-N 0.000 description 1
- 150000008574 D-amino acids Chemical group 0.000 description 1
- SUZLHDUTVMZSEV-UHFFFAOYSA-N Deoxycoleonol Natural products C12C(=O)CC(C)(C=C)OC2(C)C(OC(=O)C)C(O)C2C1(C)C(O)CCC2(C)C SUZLHDUTVMZSEV-UHFFFAOYSA-N 0.000 description 1
- 208000002249 Diabetes Complications Diseases 0.000 description 1
- 206010012655 Diabetic complications Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 239000009484 FIBS Substances 0.000 description 1
- VZCYOOQTPOCHFL-OWOJBTEDSA-N Fumaric acid Chemical compound OC(=O)\C=C\C(O)=O VZCYOOQTPOCHFL-OWOJBTEDSA-N 0.000 description 1
- 108091006027 G proteins Proteins 0.000 description 1
- 102000030782 GTP binding Human genes 0.000 description 1
- 108091000058 GTP-Binding Proteins 0.000 description 1
- 102100039997 Gastric inhibitory polypeptide receptor Human genes 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 102000051325 Glucagon Human genes 0.000 description 1
- 108060003199 Glucagon Proteins 0.000 description 1
- 108010086246 Glucagon-Like Peptide-1 Receptor Proteins 0.000 description 1
- 102100032882 Glucagon-like peptide 1 receptor Human genes 0.000 description 1
- 229940089838 Glucagon-like peptide 1 receptor agonist Drugs 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 241001622557 Hesperia Species 0.000 description 1
- 208000013016 Hypoglycemia Diseases 0.000 description 1
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
- 102000003746 Insulin Receptor Human genes 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- 206010022491 Insulin resistant diabetes Diseases 0.000 description 1
- 229940122199 Insulin secretagogue Drugs 0.000 description 1
- QUOGESRFPZDMMT-UHFFFAOYSA-N L-Homoarginine Natural products OC(=O)C(N)CCCCNC(N)=N QUOGESRFPZDMMT-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-BYPYZUCNSA-N L-Ornithine Chemical compound NCCC[C@H](N)C(O)=O AHLPHDHHMVZTML-BYPYZUCNSA-N 0.000 description 1
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 1
- ZGUNAGUHMKGQNY-ZETCQYMHSA-N L-alpha-phenylglycine zwitterion Chemical compound OC(=O)[C@@H](N)C1=CC=CC=C1 ZGUNAGUHMKGQNY-ZETCQYMHSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- RHGKLRLOHDJJDR-BYPYZUCNSA-N L-citrulline Chemical compound NC(=O)NCCC[C@H]([NH3+])C([O-])=O RHGKLRLOHDJJDR-BYPYZUCNSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- HNDVDQJCIGZPNO-YFKPBYRVSA-N L-histidine Chemical compound OC(=O)[C@@H](N)CC1=CN=CN1 HNDVDQJCIGZPNO-YFKPBYRVSA-N 0.000 description 1
- QUOGESRFPZDMMT-YFKPBYRVSA-N L-homoarginine Chemical compound OC(=O)[C@@H](N)CCCCNC(N)=N QUOGESRFPZDMMT-YFKPBYRVSA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- JVTAAEKCZFNVCJ-UHFFFAOYSA-M Lactate Chemical compound CC(O)C([O-])=O JVTAAEKCZFNVCJ-UHFFFAOYSA-M 0.000 description 1
- 108010013563 Lipoprotein Lipase Proteins 0.000 description 1
- 102100022119 Lipoprotein lipase Human genes 0.000 description 1
- AFVFQIVMOAPDHO-UHFFFAOYSA-N Methanesulfonic acid Chemical compound CS(O)(=O)=O AFVFQIVMOAPDHO-UHFFFAOYSA-N 0.000 description 1
- 241000699660 Mus musculus Species 0.000 description 1
- RHGKLRLOHDJJDR-UHFFFAOYSA-N Ndelta-carbamoyl-DL-ornithine Natural products OC(=O)C(N)CCCNC(N)=O RHGKLRLOHDJJDR-UHFFFAOYSA-N 0.000 description 1
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 1
- AHLPHDHHMVZTML-UHFFFAOYSA-N Orn-delta-NH2 Natural products NCCCC(N)C(O)=O AHLPHDHHMVZTML-UHFFFAOYSA-N 0.000 description 1
- UTJLXEIPEHZYQJ-UHFFFAOYSA-N Ornithine Natural products OC(=O)C(C)CCCN UTJLXEIPEHZYQJ-UHFFFAOYSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 235000019483 Peanut oil Nutrition 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- BELBBZDIHDAJOR-UHFFFAOYSA-N Phenolsulfonephthalein Chemical compound C1=CC(O)=CC=C1C1(C=2C=CC(O)=CC=2)C2=CC=CC=C2S(=O)(=O)O1 BELBBZDIHDAJOR-UHFFFAOYSA-N 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 108010076039 Polyproteins Proteins 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 108010076181 Proinsulin Proteins 0.000 description 1
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 description 1
- XBDQKXXYIPTUBI-UHFFFAOYSA-M Propionate Chemical compound CCC([O-])=O XBDQKXXYIPTUBI-UHFFFAOYSA-M 0.000 description 1
- 241000700157 Rattus norvegicus Species 0.000 description 1
- 108010077895 Sarcosine Proteins 0.000 description 1
- 108010071390 Serum Albumin Proteins 0.000 description 1
- 102000007562 Serum Albumin Human genes 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 229940100389 Sulfonylurea Drugs 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 1
- 206010067584 Type 1 diabetes mellitus Diseases 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000011149 active material Substances 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- WNLRTRBMVRJNCN-UHFFFAOYSA-L adipate(2-) Chemical compound [O-]C(=O)CCCCC([O-])=O WNLRTRBMVRJNCN-UHFFFAOYSA-L 0.000 description 1
- 230000003919 adipocyte function Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 150000001350 alkyl halides Chemical class 0.000 description 1
- 208000026935 allergic disease Diseases 0.000 description 1
- AFVLVVWMAFSXCK-VMPITWQZSA-N alpha-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(\C#N)=C\C1=CC=C(O)C=C1 AFVLVVWMAFSXCK-VMPITWQZSA-N 0.000 description 1
- AWUCVROLDVIAJX-UHFFFAOYSA-N alpha-glycerophosphate Natural products OCC(O)COP(O)(O)=O AWUCVROLDVIAJX-UHFFFAOYSA-N 0.000 description 1
- 239000005030 aluminium foil Substances 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 239000003708 ampul Substances 0.000 description 1
- 230000002424 anti-apoptotic effect Effects 0.000 description 1
- 230000003579 anti-obesity Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 239000011260 aqueous acid Substances 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 229940009098 aspartate Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 229940077388 benzenesulfonate Drugs 0.000 description 1
- SRSXLGNVWSONIS-UHFFFAOYSA-M benzenesulfonate Chemical compound [O-]S(=O)(=O)C1=CC=CC=C1 SRSXLGNVWSONIS-UHFFFAOYSA-M 0.000 description 1
- 229940050390 benzoate Drugs 0.000 description 1
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 229940000635 beta-alanine Drugs 0.000 description 1
- 150000004283 biguanides Chemical class 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 238000012742 biochemical analysis Methods 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 210000001124 body fluid Anatomy 0.000 description 1
- 239000010839 body fluid Substances 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- BPKIGYQJPYCAOW-FFJTTWKXSA-I calcium;potassium;disodium;(2s)-2-hydroxypropanoate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].C[C@H](O)C([O-])=O BPKIGYQJPYCAOW-FFJTTWKXSA-I 0.000 description 1
- BMLSTPRTEKLIPM-UHFFFAOYSA-I calcium;potassium;disodium;hydrogen carbonate;dichloride;dihydroxide;hydrate Chemical compound O.[OH-].[OH-].[Na+].[Na+].[Cl-].[Cl-].[K+].[Ca+2].OC([O-])=O BMLSTPRTEKLIPM-UHFFFAOYSA-I 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000006652 catabolic pathway Effects 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000013553 cell monolayer Substances 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 229940001468 citrate Drugs 0.000 description 1
- 229960002173 citrulline Drugs 0.000 description 1
- 235000013477 citrulline Nutrition 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 229960005188 collagen Drugs 0.000 description 1
- 238000004440 column chromatography Methods 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 230000021615 conjugation Effects 0.000 description 1
- 230000008094 contradictory effect Effects 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 230000001517 counterregulatory effect Effects 0.000 description 1
- 230000037029 cross reaction Effects 0.000 description 1
- WZHCOOQXZCIUNC-UHFFFAOYSA-N cyclandelate Chemical compound C1C(C)(C)CC(C)CC1OC(=O)C(O)C1=CC=CC=C1 WZHCOOQXZCIUNC-UHFFFAOYSA-N 0.000 description 1
- 125000004122 cyclic group Chemical group 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000034994 death Effects 0.000 description 1
- 125000002704 decyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000003795 desorption Methods 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000008356 dextrose and sodium chloride injection Substances 0.000 description 1
- 239000008355 dextrose injection Substances 0.000 description 1
- 125000004177 diethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- GAFRWLVTHPVQGK-UHFFFAOYSA-N dipentyl sulfate Chemical class CCCCCOS(=O)(=O)OCCCCC GAFRWLVTHPVQGK-UHFFFAOYSA-N 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 208000002173 dizziness Diseases 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 210000001198 duodenum Anatomy 0.000 description 1
- 230000004064 dysfunction Effects 0.000 description 1
- 230000000667 effect on insulin Effects 0.000 description 1
- 239000003995 emulsifying agent Substances 0.000 description 1
- 210000003890 endocrine cell Anatomy 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 108010024703 exendin (9-39) Proteins 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 230000003031 feeding effect Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012458 free base Substances 0.000 description 1
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 description 1
- 230000027119 gastric acid secretion Effects 0.000 description 1
- 108010077982 gastric inhibitory polypeptide (1-14) Proteins 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 230000014509 gene expression Effects 0.000 description 1
- WPNGPBPCQMDAAD-WRFZDFFOSA-N glp-1 (9-36) amide 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](N)CCC(O)=O)[C@@H](C)O)[C@@H](C)O)C(C)C)C1=CC=CC=C1 WPNGPBPCQMDAAD-WRFZDFFOSA-N 0.000 description 1
- MASNOZXLGMXCHN-ZLPAWPGGSA-N glucagon 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@@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 description 1
- 229960004666 glucagon Drugs 0.000 description 1
- 108700005418 glucagon-like peptide-1 (9-36)-amide Proteins 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 229940093181 glucose injection Drugs 0.000 description 1
- 230000004153 glucose metabolism Effects 0.000 description 1
- 230000004190 glucose uptake Effects 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000004116 glycogenolysis Effects 0.000 description 1
- 150000002334 glycols Chemical class 0.000 description 1
- YMAWOPBAYDPSLA-UHFFFAOYSA-N glycylglycine Chemical compound [NH3+]CC(=O)NCC([O-])=O YMAWOPBAYDPSLA-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 244000144993 groups of animals Species 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 150000004820 halides Chemical class 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- FUZZWVXGSFPDMH-UHFFFAOYSA-N hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 230000003284 homeostatic effect Effects 0.000 description 1
- 108091008039 hormone receptors Proteins 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-M hydrogensulfate Chemical compound OS([O-])(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-M 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000004679 hydroxides Chemical class 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 238000003365 immunocytochemistry Methods 0.000 description 1
- 238000003364 immunohistochemistry Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000007529 inorganic bases Chemical class 0.000 description 1
- 210000002660 insulin-secreting cell Anatomy 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 230000007794 irritation Effects 0.000 description 1
- SUMDYPCJJOFFON-UHFFFAOYSA-N isethionic acid Chemical compound OCCS(O)(=O)=O SUMDYPCJJOFFON-UHFFFAOYSA-N 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- JJWLVOIRVHMVIS-UHFFFAOYSA-N isopropylamine Chemical compound CC(C)N JJWLVOIRVHMVIS-UHFFFAOYSA-N 0.000 description 1
- 210000001630 jejunum Anatomy 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 210000003292 kidney cell Anatomy 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 230000004130 lipolysis Effects 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 1
- 239000006193 liquid solution Substances 0.000 description 1
- 239000006194 liquid suspension Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 150000004668 long chain fatty acids Chemical class 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000011976 maleic acid Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- XZWYZXLIPXDOLR-UHFFFAOYSA-N metformin Chemical compound CN(C)C(=N)NC(N)=N XZWYZXLIPXDOLR-UHFFFAOYSA-N 0.000 description 1
- 229960003105 metformin Drugs 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 230000000394 mitotic effect Effects 0.000 description 1
- 102000035118 modified proteins Human genes 0.000 description 1
- 108091005573 modified proteins Proteins 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 210000004898 n-terminal fragment Anatomy 0.000 description 1
- KVBGVZZKJNLNJU-UHFFFAOYSA-M naphthalene-2-sulfonate Chemical compound C1=CC=CC2=CC(S(=O)(=O)[O-])=CC=C21 KVBGVZZKJNLNJU-UHFFFAOYSA-M 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 235000015816 nutrient absorption Nutrition 0.000 description 1
- 235000021062 nutrient metabolism Nutrition 0.000 description 1
- 238000001543 one-way ANOVA Methods 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- 229960003104 ornithine Drugs 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 235000006408 oxalic acid Nutrition 0.000 description 1
- 239000006179 pH buffering agent Substances 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000007911 parenteral administration Methods 0.000 description 1
- 230000008506 pathogenesis Effects 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 239000000312 peanut oil Substances 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- JRKICGRDRMAZLK-UHFFFAOYSA-L peroxydisulfate Chemical compound [O-]S(=O)(=O)OOS([O-])(=O)=O JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 description 1
- 239000003208 petroleum Substances 0.000 description 1
- 239000000825 pharmaceutical preparation Substances 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000008196 pharmacological composition Substances 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229960003531 phenolsulfonphthalein Drugs 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 150000003904 phospholipids Chemical class 0.000 description 1
- 150000003016 phosphoric acids Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229940075930 picrate Drugs 0.000 description 1
- OXNIZHLAWKMVMX-UHFFFAOYSA-M picrate anion Chemical compound [O-]C1=C([N+]([O-])=O)C=C([N+]([O-])=O)C=C1[N+]([O-])=O OXNIZHLAWKMVMX-UHFFFAOYSA-M 0.000 description 1
- 229950010765 pivalate Drugs 0.000 description 1
- IUGYQRQAERSCNH-UHFFFAOYSA-N pivalic acid Chemical compound CC(C)(C)C(O)=O IUGYQRQAERSCNH-UHFFFAOYSA-N 0.000 description 1
- 229920001308 poly(aminoacid) Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 230000029537 positive regulation of insulin secretion Effects 0.000 description 1
- 230000000291 postprandial effect Effects 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 229940124606 potential therapeutic agent Drugs 0.000 description 1
- 230000002028 premature Effects 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- MFDFERRIHVXMIY-UHFFFAOYSA-N procaine Chemical compound CCN(CC)CCOC(=O)C1=CC=C(N)C=C1 MFDFERRIHVXMIY-UHFFFAOYSA-N 0.000 description 1
- 229960004919 procaine Drugs 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 description 1
- 125000004805 propylene group Chemical group [H]C([H])([H])C([H])([*:1])C([H])([H])[*:2] 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 230000002797 proteolythic effect Effects 0.000 description 1
- 239000003488 releasing hormone Substances 0.000 description 1
- 230000013878 renal filtration Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000002390 rotary evaporation Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229940043230 sarcosine Drugs 0.000 description 1
- 230000000580 secretagogue effect Effects 0.000 description 1
- 239000008159 sesame oil Substances 0.000 description 1
- 235000011803 sesame oil Nutrition 0.000 description 1
- 235000021309 simple sugar Nutrition 0.000 description 1
- AWUCVROLDVIAJX-GSVOUGTGSA-N sn-glycerol 3-phosphate Chemical compound OC[C@@H](O)COP(O)(O)=O AWUCVROLDVIAJX-GSVOUGTGSA-N 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000008354 sodium chloride injection Substances 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000003549 soybean oil Substances 0.000 description 1
- 235000012424 soybean oil Nutrition 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 239000000021 stimulant Substances 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 239000001384 succinic acid Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 239000001117 sulphuric acid Substances 0.000 description 1
- 235000011149 sulphuric acid Nutrition 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000002562 thickening agent Substances 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 238000011830 transgenic mouse model Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- ZDPHROOEEOARMN-UHFFFAOYSA-N undecanoic acid Chemical compound CCCCCCCCCCC(O)=O ZDPHROOEEOARMN-UHFFFAOYSA-N 0.000 description 1
- 238000012762 unpaired Student’s t-test Methods 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 229920002554 vinyl polymer Polymers 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 238000004018 waxing Methods 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
- 238000011684 zucker rat (obese) Methods 0.000 description 1
Classifications
-
- 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
-
- 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
-
- 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
- A61P3/06—Antihyperlipidemics
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/12—Antihypertensives
Abstract
The present invention provides peptide analogues which are antagonists of gastric inhibitory peptide (GIP). The peptides, based on GIP 1-42 include substitutions and/or modifications which have enhanced resistance to degradation by the enzyme dipeptidyl peptidase IV (DPP IV). The invention also provides a process of N terminally modifying GIP and the use of the peptide analogues for treatment of diabetes.
Description
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional volumes please contact the Canadian Patent Office.
PEPTIDE ANALOGUES OF GIP FOR TREATMENT OF DIABETES, INSULIN RESISTANCE AND OBESITY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.K. Application No. GB 0404124.0, filed on 25 February 2004.
FIELD OF THE INVENTION
The present invention relates to the release of insulin and the control of blood glucose concentration. More particularly the invention relates to antagonists of gastric inhibitory peptide (GIP) as pharmaceutical preparations for treatment of type 2 diabetes.
BACKGROUND
Obesity and diabetes are predicted to reach epidemic proportions throughout the world in the next 20 years and current treatments do not restore normal insulin sensitivity or glucose homeostasis, therein resulting in debilitating diabetic complications and premature death.
Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1(7-36)amide (truncated GLP-1; tGLP-1) are two important insulin-releasing hormones secreted from endocrine cells in the intestinal tract in response to feeding. Together with autonomic nerves they play a vital supporting role to the pancreatic islets in the control of blood glucose homeostasis and nutrient metabolism.
GIP is released from intestinal endocrine K-cells into the bloodstream following ingestion of carbohydrate, protein and particularly fat (Meier, LI.
et aL, 2002, Regul. Pept. 107:1-13). GIP was initially discovered through its ability to inhibit gastric acid secretion (Brown, J.C. et al. 1969, Can. J. PhysioL
PharmacoL
47:113-114) but its major physiological role is now generally believed to be that of an incretin hormone that targets pancreatic islets to enhance insulin secretion and help reduce postprandial hyperglycemia (Creutzfeldt, W., 2001, Exp. Clin.
EndocrinoL
Diabetes 109:S288-S303). GIP acts through binding to specific G-protein coupled GIP receptors located on pancreatic beta-cells (Wheeler, M.B. et aL, 1995, Endocrinology 136:4629-4639). Like its sister incretin hormone, glucagon-like peptide-1 (GLP-1), this ability to stimulate insulin secretion plus other potentially beneficial actions on pancreatic beta-cell growth and differentiation have led to much interest in using GIP or GLP-1 receptor agonists in the treatment of type 2 diabetes (Creutzfeldt, W., 2001, Exp. Clin. Endocrinol. Diabetes 109:S288-S303; Holz, G.G.
et al., 2003, Cum. Med. Chem. 10:2471-2483).
Since GIP functions as a potent and natural stimulator of insulin secretion released from the intestine by feeding, it is widely expected that antagonists opposing GIP action will block the insulin-releasing actions of GIP and impair both oral glucose tolerance and the glycemic response to nutrient ingestion. In fact, all studies published to date indicate that GIP is a key physiological component of the enteroinsular axis and that functional ablation of GIP leads to impaired glucose homeostasis moving the metabolic characteristic towards a type 2 diabetes phenotype (Gault, V.A. et al., 2002, Biochem. Biophys. Res. Commun. 290:1420-1426).
Dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) has been identified as a key enzyme responsible for inactivation of GIP and tGLP-1 in serum. This occurs through the rapid removal of the N-terminal dipeptides Tyr'-Ala 2 and His7-A1a8 giving rise to the main metabolites GIP(3-42) and GLP-1(9-36)amide, respectively.
These truncated peptides are reported to lack biological activity or to even serve as antagonists at GIP or tGLP-1 receptors. The resulting biological half-lives of these incretin hormones in vivo are therefore very short, estimated to be no longer than 5 minutes. DPP IV is completely inhibited in serum by the addition of diprotin A
(DPA, 0.1 mmo1/1).
In situations of normal glucose regulation and pancreatic B-cell sensitivity, this short duration of action is advantageous in facilitating momentary adjustments to homeostatic control. However, the current goal of a possible therapeutic role of incretin hormones, particularly tGLP-1 in non-insulin dependent diabetes (NIDDM) therapy is frustrated by a number of factors in addition to finding a convenient route of administration. Most notable of these are rapid peptide degradation and rapid absorption (peak concentrations are reached in 20 minutes) and the resulting need for both high dosage and precise timing with meals. Recent therapeutic strategies have focused on precipitated preparations to delay peptide absorption and inhibition of GLP-1 degradation using specific inhibitors of DPP IV. A possible therapeutic role is also suggested by the observation that a specific inhibitor of DPP IV, isoleucine thiazolidide, lowered blood glucose and enhanced insulin secretion in glucose-treated diabetic obese Zucker rats presumably by protecting against catabolism of the incretin hormones tGLP-1 and GIP.
Studies have indicated that tGLP-1 infusion restores pancreatic B-cell sensitivity, insulin secretory oscillations and improved glycemic control in various groups of patients with impaired glucose tolerance (IGT) or NIDDM. Longer term studies also show significant benefits of tGLP-1 injections in NIDDM and possibly IDDM therapy, providing a major incentive to develop an orally effective or long-acting tGLP-1 analogue. Several attempts have been made to produce structurally modified analogues of tGLP-1 which are resistant to DPP IV degradation. A
significant extension of serum half-life is observed with His7-glucitol tGLP-1 and tGLP-1 analogues substituted at position 8 with Gly, Aib (amino isobutyric acid), Ser or Thr. However, these structural modifications seem to impair receptor binding and insulinotrophic activity thereby compromising part of the benefits of protection from proteolytic degradation. In recent studies using His7-glucitol tGLP-1, resistance to DPP IV and serum degradation was accompanied by severe loss of insulin releasing activity.
GIP shares not only the same degradation pathway as tGLP-1 but many similar physiological actions, including stimulation of insulin and somatostatin secretion, and the enhancement of glucose disposal. These actions are viewed as key aspects in the antihyperglycemic properties of tGLP-1, and there is therefore good expectation that GIP may have similar potential as NIDDM therapy. Indeed, compensation by GIP is held to explain the modest disturbances of glucose homeostasis observed in tGLP-1 knockout mice. Apart from early studies, the anti-diabetic potential of GIP has not been explored and tGLP-1 may seem more attractive since it is viewed by some as a more potent insulin secretagogue when infused at so called physiological concentrations estimated by radioimmunoassay (RIA).
There is therefore a need for a diabetes treatment that includes an analogue of GIP which can cause release of insulin, yet also be resistant to rapid degradation by DPP IV.
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional volumes please contact the Canadian Patent Office.
PEPTIDE ANALOGUES OF GIP FOR TREATMENT OF DIABETES, INSULIN RESISTANCE AND OBESITY
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.K. Application No. GB 0404124.0, filed on 25 February 2004.
FIELD OF THE INVENTION
The present invention relates to the release of insulin and the control of blood glucose concentration. More particularly the invention relates to antagonists of gastric inhibitory peptide (GIP) as pharmaceutical preparations for treatment of type 2 diabetes.
BACKGROUND
Obesity and diabetes are predicted to reach epidemic proportions throughout the world in the next 20 years and current treatments do not restore normal insulin sensitivity or glucose homeostasis, therein resulting in debilitating diabetic complications and premature death.
Gastric inhibitory polypeptide (GIP) and glucagon-like peptide-1(7-36)amide (truncated GLP-1; tGLP-1) are two important insulin-releasing hormones secreted from endocrine cells in the intestinal tract in response to feeding. Together with autonomic nerves they play a vital supporting role to the pancreatic islets in the control of blood glucose homeostasis and nutrient metabolism.
GIP is released from intestinal endocrine K-cells into the bloodstream following ingestion of carbohydrate, protein and particularly fat (Meier, LI.
et aL, 2002, Regul. Pept. 107:1-13). GIP was initially discovered through its ability to inhibit gastric acid secretion (Brown, J.C. et al. 1969, Can. J. PhysioL
PharmacoL
47:113-114) but its major physiological role is now generally believed to be that of an incretin hormone that targets pancreatic islets to enhance insulin secretion and help reduce postprandial hyperglycemia (Creutzfeldt, W., 2001, Exp. Clin.
EndocrinoL
Diabetes 109:S288-S303). GIP acts through binding to specific G-protein coupled GIP receptors located on pancreatic beta-cells (Wheeler, M.B. et aL, 1995, Endocrinology 136:4629-4639). Like its sister incretin hormone, glucagon-like peptide-1 (GLP-1), this ability to stimulate insulin secretion plus other potentially beneficial actions on pancreatic beta-cell growth and differentiation have led to much interest in using GIP or GLP-1 receptor agonists in the treatment of type 2 diabetes (Creutzfeldt, W., 2001, Exp. Clin. Endocrinol. Diabetes 109:S288-S303; Holz, G.G.
et al., 2003, Cum. Med. Chem. 10:2471-2483).
Since GIP functions as a potent and natural stimulator of insulin secretion released from the intestine by feeding, it is widely expected that antagonists opposing GIP action will block the insulin-releasing actions of GIP and impair both oral glucose tolerance and the glycemic response to nutrient ingestion. In fact, all studies published to date indicate that GIP is a key physiological component of the enteroinsular axis and that functional ablation of GIP leads to impaired glucose homeostasis moving the metabolic characteristic towards a type 2 diabetes phenotype (Gault, V.A. et al., 2002, Biochem. Biophys. Res. Commun. 290:1420-1426).
Dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5) has been identified as a key enzyme responsible for inactivation of GIP and tGLP-1 in serum. This occurs through the rapid removal of the N-terminal dipeptides Tyr'-Ala 2 and His7-A1a8 giving rise to the main metabolites GIP(3-42) and GLP-1(9-36)amide, respectively.
These truncated peptides are reported to lack biological activity or to even serve as antagonists at GIP or tGLP-1 receptors. The resulting biological half-lives of these incretin hormones in vivo are therefore very short, estimated to be no longer than 5 minutes. DPP IV is completely inhibited in serum by the addition of diprotin A
(DPA, 0.1 mmo1/1).
In situations of normal glucose regulation and pancreatic B-cell sensitivity, this short duration of action is advantageous in facilitating momentary adjustments to homeostatic control. However, the current goal of a possible therapeutic role of incretin hormones, particularly tGLP-1 in non-insulin dependent diabetes (NIDDM) therapy is frustrated by a number of factors in addition to finding a convenient route of administration. Most notable of these are rapid peptide degradation and rapid absorption (peak concentrations are reached in 20 minutes) and the resulting need for both high dosage and precise timing with meals. Recent therapeutic strategies have focused on precipitated preparations to delay peptide absorption and inhibition of GLP-1 degradation using specific inhibitors of DPP IV. A possible therapeutic role is also suggested by the observation that a specific inhibitor of DPP IV, isoleucine thiazolidide, lowered blood glucose and enhanced insulin secretion in glucose-treated diabetic obese Zucker rats presumably by protecting against catabolism of the incretin hormones tGLP-1 and GIP.
Studies have indicated that tGLP-1 infusion restores pancreatic B-cell sensitivity, insulin secretory oscillations and improved glycemic control in various groups of patients with impaired glucose tolerance (IGT) or NIDDM. Longer term studies also show significant benefits of tGLP-1 injections in NIDDM and possibly IDDM therapy, providing a major incentive to develop an orally effective or long-acting tGLP-1 analogue. Several attempts have been made to produce structurally modified analogues of tGLP-1 which are resistant to DPP IV degradation. A
significant extension of serum half-life is observed with His7-glucitol tGLP-1 and tGLP-1 analogues substituted at position 8 with Gly, Aib (amino isobutyric acid), Ser or Thr. However, these structural modifications seem to impair receptor binding and insulinotrophic activity thereby compromising part of the benefits of protection from proteolytic degradation. In recent studies using His7-glucitol tGLP-1, resistance to DPP IV and serum degradation was accompanied by severe loss of insulin releasing activity.
GIP shares not only the same degradation pathway as tGLP-1 but many similar physiological actions, including stimulation of insulin and somatostatin secretion, and the enhancement of glucose disposal. These actions are viewed as key aspects in the antihyperglycemic properties of tGLP-1, and there is therefore good expectation that GIP may have similar potential as NIDDM therapy. Indeed, compensation by GIP is held to explain the modest disturbances of glucose homeostasis observed in tGLP-1 knockout mice. Apart from early studies, the anti-diabetic potential of GIP has not been explored and tGLP-1 may seem more attractive since it is viewed by some as a more potent insulin secretagogue when infused at so called physiological concentrations estimated by radioimmunoassay (RIA).
There is therefore a need for a diabetes treatment that includes an analogue of GIP which can cause release of insulin, yet also be resistant to rapid degradation by DPP IV.
SUMMARY OF THE INVENTION
Disclosed herein are GIP antagonist peptides which are resistant to rapid degradation by DPP IV.
The invention includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), which includes at least 12 amino acid residues from the N-terminal end of GIP(3-42).
The invention also includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), which includes at least 12 amino acid residues from the N-terminal end of GIP(1-42) and having an amino acid substitution at G1u3.
The amino acid substituted at G1u3 can be selected from the group consisting of: proline, hydroxyproline, lysine, tyrosine, phenylalanine and tryptophan.
Specifically, a proline can be substituted for G1u3. The peptide analogue can further include modification by fatty acid addition at an epsilon amino group of at least one lysine residue. The lysine residue can be Lys16, or Lys37.
The peptide analogue of GIP(1-42) (SEQ ID NO:1) can include at least 12 amino acid residues from the N-terminal end of GIP(1-42), and an amino acid modification at amino acid residues 1, 2 or 3. The N-terminal amino acid residue can be acetylated. It can further comprising modification by fatty acid addition at an epsilon amino group of at least one lysine residue. The modification can be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-16, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue. The lysine residue can be Lys16, or Lys37.
The invention also includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), wherein the analogue comprises a base peptide consisting of one of the following:
GIP(1-12), GIP(1-13), GIP(1-14), GIP(1-15), GIP(1-16), GIP(1-17), GIP(1-18), GIP(1-19), GIP(1-20), GIP(1-21), GIP(1-22), GIP(1-23), GIP(1-24), GIP(1-25), GIP(1-26), GIP(1-27), GIP(1-28), GIP(1-29), GIP(1-30), GIP(1-31), GIP(1-32), GIP(1-33), GIP(1-34), GIP(1-35), GIP(1-36), GIP(1-37), GIP(1-38), GIP(1-39), GIP(1-40), GIP(1-41) and GIP(1-42), where the base peptide possesses one or more of the following modifications: (1) an amino acid substitution at Glu3; (2) a modification by fatty acid addition at an epsilon amino group of at least one lysine residue; and (3) a modification by N-terminal acetylation. Such a peptide analogue can have a proline substituted for Glu3. It can also have a modification in the form of a C-16 palmitate group linked to the epsilon amino group of a lysine residue.
The modification can be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-16, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue. The lysine residue can be Lys16, or Lys37.
The invention further includes a peptide analogue of GIP(1-42) (SEQ ID
NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(3-42), wherein the peptide analogue is resistant to degradation by enzyme DPP IV
when compared to naturally-occurring GIP.
Also included is a peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(1-42) and having an amino acid substitution at Glu3, wherein the peptide analogue is resistant to degradation by enzyme DPP IV when compared to naturally-occurring GIP.
In addition, the invention includes a peptide analogue of GIP(1-42) (SEQ ID
NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(3-42), wherein the peptide analogue modulates insulin secretion.
The invention also includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(1-42) and having an amino acid substitution at G1u3, wherein the peptide analogue modulates insulin secretion.
The invention also includes use of any of the analogues in the preparation of a medicament for the treatment of obesity, insulin resistance, insulin resistant metabolic syndrome (Syndrome X) or type 2 diabetes.
The invention also includes a pharmaceutical composition including the peptide analogues. The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. The peptide analogue can be in the form of a pharmaceutically acceptable salt, or a pharmaceutically acceptable acid addition salt.
In a further aspect, the invention includes a method of treating insulin resistance, obesity, or type 2 diabetes, where the method comprises administering to a mammal in need of such treatment a therapeutically effective amount of the pharmaceutical composition.
According to the present invention there is provided an effective peptide analogue of the biologically active GIP(1-42) which has improved characteristics for treatment of Type 2 diabetes wherein the analogue comprises at least 15 amino acid residues from the N terminus of GIP(1-42) and has at least one amino acid substitution or modification at position 1-3 and not including Tyr' glucitol GIP(1-42).
The structures of human and porcine GIP(1-42) are shown below. The porcine peptide differs by. just two amino acid substitutions at positions 18 and 34.
The analogue may include modification by fatty acid addition at an epsilon amino group of at least one lysine residue.
The invention includes Tyri glucitol GIP(1-42) having fatty acid addition at an epsilon amino group of at least one lysine residue.
Analogues of GIP(1-42) may have an enhanced capacity to stimulate insulin secretion, enhance glucose disposal, delay glucose absorption or may exhibit enhanced stability in plasma as compared to native GIP. They also may have enhanced resistance to degradation.
Any of these properties will enhance the potency of the analogue as a therapeutic agent.
Analogues having D-amino acid substitutions in the 1, 2 and 3 positions and/or N-glycated, N-alkylated, N-acetylated or N-acylated amino acids in the position are resistant to degradation in vivo.
Various amino acid substitutions at second and third amino terminal residues are included, such as GIP(1-42)G1y2, GIP(1-42)Ser2, GIP(1-42)Abu2, GIP(1-42)Aib2, GIP(1-42)D-A1a2, GIP(1-42)Sar2, and GIP(1-42)Pro3.
Amino-terminally modified GIP analogues include N-glycated GIP(1-42), N-alkylated GIP(1-42), N-acetylated GIP(1-42), N-acetyl-GIP(1-42) and N-isopropyl GIP(1-42).
Other stabilized analogues include those with a peptide isostere bond between amino terminal residues at position 2 and 3. These analogues may be resistant to the plasma enzyme dipeptidyl-peptidase IV (DPP IV) which is largely responsible for inactivation of GIP by removal of the amino-terminal dipeptide TyrI-A1a2.
In particular embodiments, the invention provides a peptide which is more potent than human or porcine GIP in moderating blood glucose excursions, said peptide consisting of GIP(1-42) or N-terminal fragments of GIP(1-42) consisting of up to between 15 to 30 amino acid residues from the N-terminus (i.e., GIP(1-15) GIP(1-3)) with one or more modifications selected from the group consisting of:
(a) substitution of Ala2 by Gly;
Disclosed herein are GIP antagonist peptides which are resistant to rapid degradation by DPP IV.
The invention includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), which includes at least 12 amino acid residues from the N-terminal end of GIP(3-42).
The invention also includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), which includes at least 12 amino acid residues from the N-terminal end of GIP(1-42) and having an amino acid substitution at G1u3.
The amino acid substituted at G1u3 can be selected from the group consisting of: proline, hydroxyproline, lysine, tyrosine, phenylalanine and tryptophan.
Specifically, a proline can be substituted for G1u3. The peptide analogue can further include modification by fatty acid addition at an epsilon amino group of at least one lysine residue. The lysine residue can be Lys16, or Lys37.
The peptide analogue of GIP(1-42) (SEQ ID NO:1) can include at least 12 amino acid residues from the N-terminal end of GIP(1-42), and an amino acid modification at amino acid residues 1, 2 or 3. The N-terminal amino acid residue can be acetylated. It can further comprising modification by fatty acid addition at an epsilon amino group of at least one lysine residue. The modification can be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-16, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue. The lysine residue can be Lys16, or Lys37.
The invention also includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), wherein the analogue comprises a base peptide consisting of one of the following:
GIP(1-12), GIP(1-13), GIP(1-14), GIP(1-15), GIP(1-16), GIP(1-17), GIP(1-18), GIP(1-19), GIP(1-20), GIP(1-21), GIP(1-22), GIP(1-23), GIP(1-24), GIP(1-25), GIP(1-26), GIP(1-27), GIP(1-28), GIP(1-29), GIP(1-30), GIP(1-31), GIP(1-32), GIP(1-33), GIP(1-34), GIP(1-35), GIP(1-36), GIP(1-37), GIP(1-38), GIP(1-39), GIP(1-40), GIP(1-41) and GIP(1-42), where the base peptide possesses one or more of the following modifications: (1) an amino acid substitution at Glu3; (2) a modification by fatty acid addition at an epsilon amino group of at least one lysine residue; and (3) a modification by N-terminal acetylation. Such a peptide analogue can have a proline substituted for Glu3. It can also have a modification in the form of a C-16 palmitate group linked to the epsilon amino group of a lysine residue.
The modification can be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-16, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue. The lysine residue can be Lys16, or Lys37.
The invention further includes a peptide analogue of GIP(1-42) (SEQ ID
NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(3-42), wherein the peptide analogue is resistant to degradation by enzyme DPP IV
when compared to naturally-occurring GIP.
Also included is a peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(1-42) and having an amino acid substitution at Glu3, wherein the peptide analogue is resistant to degradation by enzyme DPP IV when compared to naturally-occurring GIP.
In addition, the invention includes a peptide analogue of GIP(1-42) (SEQ ID
NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(3-42), wherein the peptide analogue modulates insulin secretion.
The invention also includes a peptide analogue of GIP(1-42) (SEQ ID NO:1), comprising at least 12 amino acid residues from the N-terminal end of GIP(1-42) and having an amino acid substitution at G1u3, wherein the peptide analogue modulates insulin secretion.
The invention also includes use of any of the analogues in the preparation of a medicament for the treatment of obesity, insulin resistance, insulin resistant metabolic syndrome (Syndrome X) or type 2 diabetes.
The invention also includes a pharmaceutical composition including the peptide analogues. The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. The peptide analogue can be in the form of a pharmaceutically acceptable salt, or a pharmaceutically acceptable acid addition salt.
In a further aspect, the invention includes a method of treating insulin resistance, obesity, or type 2 diabetes, where the method comprises administering to a mammal in need of such treatment a therapeutically effective amount of the pharmaceutical composition.
According to the present invention there is provided an effective peptide analogue of the biologically active GIP(1-42) which has improved characteristics for treatment of Type 2 diabetes wherein the analogue comprises at least 15 amino acid residues from the N terminus of GIP(1-42) and has at least one amino acid substitution or modification at position 1-3 and not including Tyr' glucitol GIP(1-42).
The structures of human and porcine GIP(1-42) are shown below. The porcine peptide differs by. just two amino acid substitutions at positions 18 and 34.
The analogue may include modification by fatty acid addition at an epsilon amino group of at least one lysine residue.
The invention includes Tyri glucitol GIP(1-42) having fatty acid addition at an epsilon amino group of at least one lysine residue.
Analogues of GIP(1-42) may have an enhanced capacity to stimulate insulin secretion, enhance glucose disposal, delay glucose absorption or may exhibit enhanced stability in plasma as compared to native GIP. They also may have enhanced resistance to degradation.
Any of these properties will enhance the potency of the analogue as a therapeutic agent.
Analogues having D-amino acid substitutions in the 1, 2 and 3 positions and/or N-glycated, N-alkylated, N-acetylated or N-acylated amino acids in the position are resistant to degradation in vivo.
Various amino acid substitutions at second and third amino terminal residues are included, such as GIP(1-42)G1y2, GIP(1-42)Ser2, GIP(1-42)Abu2, GIP(1-42)Aib2, GIP(1-42)D-A1a2, GIP(1-42)Sar2, and GIP(1-42)Pro3.
Amino-terminally modified GIP analogues include N-glycated GIP(1-42), N-alkylated GIP(1-42), N-acetylated GIP(1-42), N-acetyl-GIP(1-42) and N-isopropyl GIP(1-42).
Other stabilized analogues include those with a peptide isostere bond between amino terminal residues at position 2 and 3. These analogues may be resistant to the plasma enzyme dipeptidyl-peptidase IV (DPP IV) which is largely responsible for inactivation of GIP by removal of the amino-terminal dipeptide TyrI-A1a2.
In particular embodiments, the invention provides a peptide which is more potent than human or porcine GIP in moderating blood glucose excursions, said peptide consisting of GIP(1-42) or N-terminal fragments of GIP(1-42) consisting of up to between 15 to 30 amino acid residues from the N-terminus (i.e., GIP(1-15) GIP(1-3)) with one or more modifications selected from the group consisting of:
(a) substitution of Ala2 by Gly;
(b) substitution of Ala2 by Ser;
(c) substitution of Ala2 by Abu;
(d) substitution of Ala2 by Aib;
(e) substitution of Ala2 by D-Ala;
(f) substitution of Ala2 by Sar (sarcosine);
(g) substitution of G1u3 by Pro;
(h) modification of Tyr1 by acetylation;
(i) modification of Tyr1 by acylation;
(j) modification of Tyr1 by alkylation;
(k) modification of Tyr1 by glycation;
(1) conversion of Ala2 - G1u3 bond to a psi [CH2NH] bond;
(m) conversion of Ala2-G1u3 bond to a stable peptide isotere bond; and (n) (n-isopropyl-H) 1GIP.
The invention also provides the use of Tyr1-glucitol GIP in the preparation of a medicament for the treatment of diabetes.
The invention further provides improved pharmaceutical compositions including analogues of GIP with improved pharmacological properties.
Other possible analogues include certain commonly encountered amino acids, which are not encoded by the genetic code, for example, beta-alanine (beta-ala), or other omega-amino acids, such as 3-amino propionic, 4-amino butyric and so forth, ornithine (Orn), citrulline (Cit), homoarginine (Har), t-butylalanine (t-BuA), t-butylglycine (t-BUG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide (MSO), substitution of the D form of a neutral or acidic amino acid or the D
form of tyrosine for tyrosine.
According to the present invention there is also provided a pharmaceutical composition useful in the treatment of diabetes type II which comprises an effective amount of the peptide as described herein, in admixture with a pharmaceutically acceptable excipient.
The invention also provides a method of N-terminally modifying GIP or analogues thereof the method comprising the steps of synthesizing the peptide from the C terminal to the penultimate N terminal amino acid, adding tyrosine to a bubbler system as a F-moc protected Tyr(tBu)-Wang resin, deprotecting the N-terminus of the tyrosine and reacting with the modifying agent, allowing the reaction to proceed to completion, cleaving the modified tyrosine from the Wang resin and adding the modified tyrosine to the peptide synthesis reaction.
Preferably the agent is glucose, acetic anhydride or pyroglutamic acid.
The invention will now be demonstrated with reference to the following non-limiting examples and the accompanying figures wherein.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la illustrates degradation of GIP by DPP IV.
Fig. lb illustrates degradation of GIP and Tyr' glucitol GIP by DPP IV.
Fig. 2a illustrates degradation of GIP human plasma.
Fig. 2b illustrates degradation of GIP and Tyr' glucitol GIP by human plasma.
Fig. 3 illustrates electrospray ionization mass spectrometry of GIP, Tyri-glucitol GIP and the major degradation fragment GIP(3-42).
Fig. 4 shows the effects of GIP and glycated GIP on plasma glucose homeostasis.
Fig. 5 shows effects of GIP on plasma insulin responses.
Fig. 6 illustrates DPP-IV degradation of GIP (1-42).
Fig. 7 illustrates DPP-IV degradation of GIP (Abu2).
Fig. 8 illustrates DPP-IV degradation of GIP (Sar2).
Fig. 9 illustrates DPP-IV degradation of GIP (Ser2).
Fig. 10 illustrates DPP-IV degradation of N-Acetyl-GIP.
Fig. 11 illustrates DPP-IV degradation of glycated GIP.
Fig. 12 illustrates human plasma degradation of GIP.
Fig. 13 illustrates human plasma degradation of GIP (Abu2).
Fig. 14 illustrates human plasma degradation of GIP (Sar2).
Fig. 15 illustrates human plasma degradation of GIP (Ser2).
Fig. 16 illustrates human plasma degradation of glycated GIP.
Fig. 17 shows the effects of various concentrations of GIP 1-42 and GIP
(Abu2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 18 shows the effects of various concentrations of GIP 1-42 and GIP
(Abu2) on insulin release from BRIN-BD 11 cells incubated at 16.7mM glucose.
Fig. 19 shows the effects of various concentrations of GIP 1-42 and GIP (Sar2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 20 shows the effects of various concentrations of GIP 1-42 and GIP (Sar2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 21 shows the effects of various concentrations of GIP 1-42 and GIP (Ser2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 22 shows the effects of various concentrations of GIP 1-42 and GIP (Ser2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 23 shows the effects of various concentrations of GIP 1-42 and N-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 24 shows the effects of various concentrations of GIP 1-42 and N-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 25 shows the effects of various concentrations of GIP 1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 26 shows the effects of various concentrations of GIP 1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 27 shows the effects of various concentrations of GIP 1-42 and GIP
(G1y2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 28 shows the effects of various concentrations of GIP 1-42 and GIP
(G1y2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 29 shows the effects of various concentrations of GIP 1-42 and GIP (Pro3) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 30 shows the effects of various concentrations of CIP 1-42 and GIP (Pro3) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 31a shows the primary structure of human gastric inhibitory polypeptide (GIP) (SEQ ID NO:1), and Fig. 31b shows the primary structure of porcine gastric inhibitory polypeptide (GIP) (SEQ ID NO:2).
Figs. 32A and 32B are a line graph and a bar graph, respectively, showing the effects of (Pro3)GIP on GIP-stimulated cyclic AMP generation and insulin secretion in vitro.
Figs. 33A - 33F are a set of six bar graphs showing the effects of Glu3-substituted forms of GIP and GIP(3-42) on GIP-stimulated insulin secretion in vitro.
(c) substitution of Ala2 by Abu;
(d) substitution of Ala2 by Aib;
(e) substitution of Ala2 by D-Ala;
(f) substitution of Ala2 by Sar (sarcosine);
(g) substitution of G1u3 by Pro;
(h) modification of Tyr1 by acetylation;
(i) modification of Tyr1 by acylation;
(j) modification of Tyr1 by alkylation;
(k) modification of Tyr1 by glycation;
(1) conversion of Ala2 - G1u3 bond to a psi [CH2NH] bond;
(m) conversion of Ala2-G1u3 bond to a stable peptide isotere bond; and (n) (n-isopropyl-H) 1GIP.
The invention also provides the use of Tyr1-glucitol GIP in the preparation of a medicament for the treatment of diabetes.
The invention further provides improved pharmaceutical compositions including analogues of GIP with improved pharmacological properties.
Other possible analogues include certain commonly encountered amino acids, which are not encoded by the genetic code, for example, beta-alanine (beta-ala), or other omega-amino acids, such as 3-amino propionic, 4-amino butyric and so forth, ornithine (Orn), citrulline (Cit), homoarginine (Har), t-butylalanine (t-BuA), t-butylglycine (t-BUG), N-methylisoleucine (N-MeIle), phenylglycine (Phg), and cyclohexylalanine (Cha), norleucine (Nle), cysteic acid (Cya) and methionine sulfoxide (MSO), substitution of the D form of a neutral or acidic amino acid or the D
form of tyrosine for tyrosine.
According to the present invention there is also provided a pharmaceutical composition useful in the treatment of diabetes type II which comprises an effective amount of the peptide as described herein, in admixture with a pharmaceutically acceptable excipient.
The invention also provides a method of N-terminally modifying GIP or analogues thereof the method comprising the steps of synthesizing the peptide from the C terminal to the penultimate N terminal amino acid, adding tyrosine to a bubbler system as a F-moc protected Tyr(tBu)-Wang resin, deprotecting the N-terminus of the tyrosine and reacting with the modifying agent, allowing the reaction to proceed to completion, cleaving the modified tyrosine from the Wang resin and adding the modified tyrosine to the peptide synthesis reaction.
Preferably the agent is glucose, acetic anhydride or pyroglutamic acid.
The invention will now be demonstrated with reference to the following non-limiting examples and the accompanying figures wherein.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. la illustrates degradation of GIP by DPP IV.
Fig. lb illustrates degradation of GIP and Tyr' glucitol GIP by DPP IV.
Fig. 2a illustrates degradation of GIP human plasma.
Fig. 2b illustrates degradation of GIP and Tyr' glucitol GIP by human plasma.
Fig. 3 illustrates electrospray ionization mass spectrometry of GIP, Tyri-glucitol GIP and the major degradation fragment GIP(3-42).
Fig. 4 shows the effects of GIP and glycated GIP on plasma glucose homeostasis.
Fig. 5 shows effects of GIP on plasma insulin responses.
Fig. 6 illustrates DPP-IV degradation of GIP (1-42).
Fig. 7 illustrates DPP-IV degradation of GIP (Abu2).
Fig. 8 illustrates DPP-IV degradation of GIP (Sar2).
Fig. 9 illustrates DPP-IV degradation of GIP (Ser2).
Fig. 10 illustrates DPP-IV degradation of N-Acetyl-GIP.
Fig. 11 illustrates DPP-IV degradation of glycated GIP.
Fig. 12 illustrates human plasma degradation of GIP.
Fig. 13 illustrates human plasma degradation of GIP (Abu2).
Fig. 14 illustrates human plasma degradation of GIP (Sar2).
Fig. 15 illustrates human plasma degradation of GIP (Ser2).
Fig. 16 illustrates human plasma degradation of glycated GIP.
Fig. 17 shows the effects of various concentrations of GIP 1-42 and GIP
(Abu2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 18 shows the effects of various concentrations of GIP 1-42 and GIP
(Abu2) on insulin release from BRIN-BD 11 cells incubated at 16.7mM glucose.
Fig. 19 shows the effects of various concentrations of GIP 1-42 and GIP (Sar2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 20 shows the effects of various concentrations of GIP 1-42 and GIP (Sar2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 21 shows the effects of various concentrations of GIP 1-42 and GIP (Ser2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 22 shows the effects of various concentrations of GIP 1-42 and GIP (Ser2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 23 shows the effects of various concentrations of GIP 1-42 and N-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 24 shows the effects of various concentrations of GIP 1-42 and N-Acetyl-GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 25 shows the effects of various concentrations of GIP 1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 26 shows the effects of various concentrations of GIP 1-42 and glycated GIP 1-42 on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 27 shows the effects of various concentrations of GIP 1-42 and GIP
(G1y2) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 28 shows the effects of various concentrations of GIP 1-42 and GIP
(G1y2) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 29 shows the effects of various concentrations of GIP 1-42 and GIP (Pro3) on insulin release from BRIN-BD11 cells incubated at 5.6mM glucose.
Fig. 30 shows the effects of various concentrations of CIP 1-42 and GIP (Pro3) on insulin release from BRIN-BD11 cells incubated at 16.7mM glucose.
Fig. 31a shows the primary structure of human gastric inhibitory polypeptide (GIP) (SEQ ID NO:1), and Fig. 31b shows the primary structure of porcine gastric inhibitory polypeptide (GIP) (SEQ ID NO:2).
Figs. 32A and 32B are a line graph and a bar graph, respectively, showing the effects of (Pro3)GIP on GIP-stimulated cyclic AMP generation and insulin secretion in vitro.
Figs. 33A - 33F are a set of six bar graphs showing the effects of Glu3-substituted forms of GIP and GIP(3-42) on GIP-stimulated insulin secretion in vitro.
Figs. 34A through 34D are a set of two line graphs (Figs. 34A, 34C) and two bar graphs (Figs. 34B, 34D) showing that acute administration of (Pro3)GIP
completely antagonises the actions of GIP on glucose tolerance (Figs. 34A, 34B) and plasma insulin (Figs. 34C, 34D) responses in obese diabetic ob/ob mice.
Figs. 35A through 35D are a set of two line graphs (Figs. 35A, 35C) and two bar graphs (Figs. 35B, 35D) showing that acute administration of (Pro3)GIP
impairs physiological meal-stimulated insulin release and worsens glycemic excursion in obese diabetic ob/ob mice.
Figs. 36A and 36B are a set of two bar graphs showing that chronic administration of (Pro3)GIP for 11 days decreases plasma glucose and insulin concentrations of obese diabetic ob/ob mice.
Figs. 37A through 37C are a set of three bar graphs showing that chronic administration of (Pro3)GIP for 11 days decreases glycated HbAic, pancreatic insulin content and associated islet hypertrophy of obese diabetic ob/ob mice.
Figs. 38A through 38D are a set of two line graphs (Figs. 38A, 38C) and two bar graphs (Figs. 38B, 38D) showing that chronic administration of (Pro3)GIP
for 11 days improves glucose tolerance of obese diabetic ob/ob mice without change of circulating insulin.
Fig. 39 is a line graph showing that chronic administration of (Pro3)GIP for days improves insulin sensitivity in obese diabetic ob/ob mice.
Fig. 40 is a line graph showing that the beneficial effects of chronic administration of (Pro3)GIP for 11 days in obese diabetic ob/ob mice are reversed 9 days after cessation of treatment.
Figs. 41A and 41B are a set of two line graphs showing that chronic administration of (Pro3)GIP for 11 days causes glucose intolerance in normal mice with reversal by 9 days after cessation of treatment.
Figs. 42A through 42D are a set of two line graphs (Figs. 42A, 42C) and two bar graphs (Figs. 42B, 42D) showing the effects of (Pro3)GIP on plasma glucose and insulin response to native GIP 4 hours after administration.
Figs. 43A through 43D are a set of two line graphs and two bar graphs showing the effects of daily (Pro3)GIP administration on food intake (Fig.
43A), body weight (Fig. 43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D) concentrations in ob/ob mice.
completely antagonises the actions of GIP on glucose tolerance (Figs. 34A, 34B) and plasma insulin (Figs. 34C, 34D) responses in obese diabetic ob/ob mice.
Figs. 35A through 35D are a set of two line graphs (Figs. 35A, 35C) and two bar graphs (Figs. 35B, 35D) showing that acute administration of (Pro3)GIP
impairs physiological meal-stimulated insulin release and worsens glycemic excursion in obese diabetic ob/ob mice.
Figs. 36A and 36B are a set of two bar graphs showing that chronic administration of (Pro3)GIP for 11 days decreases plasma glucose and insulin concentrations of obese diabetic ob/ob mice.
Figs. 37A through 37C are a set of three bar graphs showing that chronic administration of (Pro3)GIP for 11 days decreases glycated HbAic, pancreatic insulin content and associated islet hypertrophy of obese diabetic ob/ob mice.
Figs. 38A through 38D are a set of two line graphs (Figs. 38A, 38C) and two bar graphs (Figs. 38B, 38D) showing that chronic administration of (Pro3)GIP
for 11 days improves glucose tolerance of obese diabetic ob/ob mice without change of circulating insulin.
Fig. 39 is a line graph showing that chronic administration of (Pro3)GIP for days improves insulin sensitivity in obese diabetic ob/ob mice.
Fig. 40 is a line graph showing that the beneficial effects of chronic administration of (Pro3)GIP for 11 days in obese diabetic ob/ob mice are reversed 9 days after cessation of treatment.
Figs. 41A and 41B are a set of two line graphs showing that chronic administration of (Pro3)GIP for 11 days causes glucose intolerance in normal mice with reversal by 9 days after cessation of treatment.
Figs. 42A through 42D are a set of two line graphs (Figs. 42A, 42C) and two bar graphs (Figs. 42B, 42D) showing the effects of (Pro3)GIP on plasma glucose and insulin response to native GIP 4 hours after administration.
Figs. 43A through 43D are a set of two line graphs and two bar graphs showing the effects of daily (Pro3)GIP administration on food intake (Fig.
43A), body weight (Fig. 43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D) concentrations in ob/ob mice.
Figs. 44A through 44D are a set of four line graphs with inset bar graphs showing the effects of daily (Pro3)GIP administration on glucose tolerance and plasma insulin response to glucose in ob/ob mice.
Figs. 45A through 45D are a set of two line graphs (Figs. 45A, 45C) and two bar graphs (Figs. 45B, 45D) showing the effects of daily (Pro3)GIP
administration (A; black bars) or saline (o; white bars) on glucose (Figs. 45A, 45B) and insulin (Figs. 45C, 45D) responses to feeding in ob/ob mice fasted for 18 hours.
Figs. 46A through 46D are a set of two line graphs (Figs. 46A, 46C) and two bar graphs (Figs. 46B, 46D) showing the effects of daily (Pro3)GIP
administration on insulin sensitivity in ob/ob mice.
Figs. 47A through 47D are a set of four bar graphs showing the effects of daily (Pro3)GIP administration on pancreatic weight (Fig. 47A), insulin content (Fig. 47B), islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice.
Figs. 48A through 48F are a set of two bar graphs (Figs. 48A, 48D) and four photomicrographs (Figs. 48B, 48C, 48E, 48F), showing the effects of daily (Pro3)GIP
administration on islet size and morphology in ob/ob)mice.
Fig. 49 is an illustration of how the GIP receptor ("GIP-R") antagonist, (Pro3)GIP, counters beta cell hyperplasia, hyperinsulinemia and insulin resistance lead to improved glucose intolerance and diabetes control.
Figs. 50A and 50B are a line graph and a bar graph, respectively, showing intracellular cyclic AMP production (Fig. 50A) by GIP (A) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL16) (o) and N-AcGIP(LysPAL37) (9), and insulin-releasing activity of glucose (5.6 mmo/1 glucose; white bars), GIP (lined bars) and fatty acid derivatised GIP analogues (Fig. 50B) N-AcGIP(LysPAL16) (grey bars) and N-AcGIP(LysPAL37) (black bars) in the clonal pancreatic beta cell line, BRIN-BD11.
Figs. 51A through 51D are a set of two line graphs (Figs. 51A, 51C) and two bar graphs (Figs. 51B, 51D) showing glucose lowering effects (Figs. 51A, 51B) and insulin-releasing activity (Figs. 51C, 51D) of GIP and fatty acid derivatised GIP
analogues in 18 hour-fasted ob/ob mice.
Figs. 52A and 52B are a pair of bar graphs showing dose-dependent effects of GIP and N-AcGIP(LysPAL37) in ob/ob mice fasted for 18 hours.
Figs. 53A through 53E are a set of graphs showing the effects of daily N-AcGIP(LysP AL37) (9; black bars) administration on food intake (Fig. 53A), body weight (Fig. 53B), plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated hemoglobin N-AcGIP(LysPAL37) (12.5 nmoles/kg/day) (Fig. 53E).
Figs. 54A through 54D are a set of two line graphs (Figs. 54A, 54C) and two bar graphs (Figs. 54B, 54D) showing the effects of daily N-AcGIP(LysPAL37) administration on glucose tolerance (Figs. 54A, 54B) and plasma insulin response (Figs. 54C, 54D) to glucose.
Figs. 55A through 55D are a line graph and three bar graphs showing the effects of daily N-AcGIP(LysPAL37) administration on insulin sensitivity (Figs. 55A, 55B) and pancreatic weight (Fig. 55C) and insulin content (Fig. 55D).
Figs. 56A through 56D are a set of two line graphs (Figs. 56A, 56C) and two bar graphs (Figs. 56B, 56D) showing the retention of glucose lowering (Figs.
56A, 56B) and insulin releasing (Figs. 56C, 56D) activity of N-AcGIP(Ly5PAL37) and native GIP after daily injection for 14 days.
Figs. 57A through 57D are a set of two line graphs (Figs. 57A, 57C) and two bar graphs (Figs. 57B, 57D) showing the acute glucose lowering (Figs. 57A, 57B) and insulin releasing (Figs. 57C, 57D) effects of N-AcGIP(LysPAL37) after 14 daily injections of either N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; fi; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; ID; white bars).
DETAILED DESCRIPTION
The peptide analogues disclosed herein display resistance to degradation by the enzyme DPP IV. These analogues include those with alterations at residues 1, 2 and/or 3 of the native GIP(1-42) peptide, where the alterations interfere with or delay cleavage by DPP IV. The alterations can include chemical modification of one or more of the first three amino acids, such as by acylation, acetylation, alkylation, glycation, conversion of a bond between two amino acids, such as to a psi-[CH2NFI]
bond, or to a stable isotere bond, or addition of an isopropyl group. The alterations can also include amino acid substitutions at the 1, 2, and/or 3 position, to either a different naturally-occurring amino acid, or an amino acid not encoded by the genetic code. Other alterations are also possible, and the object is to prevent cleavage of the peptide by DPP IV, yet still allow for insulin secretion. This may be accomplished by alterations at other regions of the peptide, such as by alterations that alter the three-dimensional structure to prevent DPP IV cleavage, yet still allow insulin secretion.
Figs. 45A through 45D are a set of two line graphs (Figs. 45A, 45C) and two bar graphs (Figs. 45B, 45D) showing the effects of daily (Pro3)GIP
administration (A; black bars) or saline (o; white bars) on glucose (Figs. 45A, 45B) and insulin (Figs. 45C, 45D) responses to feeding in ob/ob mice fasted for 18 hours.
Figs. 46A through 46D are a set of two line graphs (Figs. 46A, 46C) and two bar graphs (Figs. 46B, 46D) showing the effects of daily (Pro3)GIP
administration on insulin sensitivity in ob/ob mice.
Figs. 47A through 47D are a set of four bar graphs showing the effects of daily (Pro3)GIP administration on pancreatic weight (Fig. 47A), insulin content (Fig. 47B), islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice.
Figs. 48A through 48F are a set of two bar graphs (Figs. 48A, 48D) and four photomicrographs (Figs. 48B, 48C, 48E, 48F), showing the effects of daily (Pro3)GIP
administration on islet size and morphology in ob/ob)mice.
Fig. 49 is an illustration of how the GIP receptor ("GIP-R") antagonist, (Pro3)GIP, counters beta cell hyperplasia, hyperinsulinemia and insulin resistance lead to improved glucose intolerance and diabetes control.
Figs. 50A and 50B are a line graph and a bar graph, respectively, showing intracellular cyclic AMP production (Fig. 50A) by GIP (A) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL16) (o) and N-AcGIP(LysPAL37) (9), and insulin-releasing activity of glucose (5.6 mmo/1 glucose; white bars), GIP (lined bars) and fatty acid derivatised GIP analogues (Fig. 50B) N-AcGIP(LysPAL16) (grey bars) and N-AcGIP(LysPAL37) (black bars) in the clonal pancreatic beta cell line, BRIN-BD11.
Figs. 51A through 51D are a set of two line graphs (Figs. 51A, 51C) and two bar graphs (Figs. 51B, 51D) showing glucose lowering effects (Figs. 51A, 51B) and insulin-releasing activity (Figs. 51C, 51D) of GIP and fatty acid derivatised GIP
analogues in 18 hour-fasted ob/ob mice.
Figs. 52A and 52B are a pair of bar graphs showing dose-dependent effects of GIP and N-AcGIP(LysPAL37) in ob/ob mice fasted for 18 hours.
Figs. 53A through 53E are a set of graphs showing the effects of daily N-AcGIP(LysP AL37) (9; black bars) administration on food intake (Fig. 53A), body weight (Fig. 53B), plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated hemoglobin N-AcGIP(LysPAL37) (12.5 nmoles/kg/day) (Fig. 53E).
Figs. 54A through 54D are a set of two line graphs (Figs. 54A, 54C) and two bar graphs (Figs. 54B, 54D) showing the effects of daily N-AcGIP(LysPAL37) administration on glucose tolerance (Figs. 54A, 54B) and plasma insulin response (Figs. 54C, 54D) to glucose.
Figs. 55A through 55D are a line graph and three bar graphs showing the effects of daily N-AcGIP(LysPAL37) administration on insulin sensitivity (Figs. 55A, 55B) and pancreatic weight (Fig. 55C) and insulin content (Fig. 55D).
Figs. 56A through 56D are a set of two line graphs (Figs. 56A, 56C) and two bar graphs (Figs. 56B, 56D) showing the retention of glucose lowering (Figs.
56A, 56B) and insulin releasing (Figs. 56C, 56D) activity of N-AcGIP(Ly5PAL37) and native GIP after daily injection for 14 days.
Figs. 57A through 57D are a set of two line graphs (Figs. 57A, 57C) and two bar graphs (Figs. 57B, 57D) showing the acute glucose lowering (Figs. 57A, 57B) and insulin releasing (Figs. 57C, 57D) effects of N-AcGIP(LysPAL37) after 14 daily injections of either N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; fi; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; ID; white bars).
DETAILED DESCRIPTION
The peptide analogues disclosed herein display resistance to degradation by the enzyme DPP IV. These analogues include those with alterations at residues 1, 2 and/or 3 of the native GIP(1-42) peptide, where the alterations interfere with or delay cleavage by DPP IV. The alterations can include chemical modification of one or more of the first three amino acids, such as by acylation, acetylation, alkylation, glycation, conversion of a bond between two amino acids, such as to a psi-[CH2NFI]
bond, or to a stable isotere bond, or addition of an isopropyl group. The alterations can also include amino acid substitutions at the 1, 2, and/or 3 position, to either a different naturally-occurring amino acid, or an amino acid not encoded by the genetic code. Other alterations are also possible, and the object is to prevent cleavage of the peptide by DPP IV, yet still allow for insulin secretion. This may be accomplished by alterations at other regions of the peptide, such as by alterations that alter the three-dimensional structure to prevent DPP IV cleavage, yet still allow insulin secretion.
Preferred alterations include chemical modifications of residues 1, 2, and 3, amino acid substitutions at residues 1, 2, and 3, and chemical modifications of lysine residues throughout the protein.
Particularly preferred alterations include acetylation of Tyr1 and linkage of a C-16 palmitate group to the epsilon amino group of a lysine residue (especially Lys16 or Lys37), or substitution of G1u3, especially by proline. The modification can also be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue.
It has been found that longer-term, as opposed to acute, GIP receptor antagonism using Glu3-substituted forms of GIP, such as (Pro3)GIP, improve obesity-related insulin resistance and associated glucose intolerance. This has uncovered an unexpected approach to the therapy of obesity, insulin resistance and type 2 diabetes.
As described in Example 1 below, an N-terminally modified version of the GIP protein was prepared, as were analogues of the modified protein. The protein and its analogues were then evaluated in Example 2 for their antihyperglycemic and insulin-releasing properties in vivo, and were found to exhibit a substantial resistance to amino peptidase degradation and increased glucose lowering activity relative to native GIP.
The 42 amino acid GIP is an important incretin hormone released into the circulation from endocrine K-cells of the duodenum and jejunum following ingestion of food. The high degree of structural conservation of GIP among species supports the view that this peptide plays an important role in metabolism. Secretion of GIP is stimulated directly by actively transported nutrients in the gut lumen without a notable input from autonomic nerves. The most important stimulants of GIP release are simple sugars and unsaturated long chain fatty acids, with amino acids exerting weaker effects. As with tGLP-1, the insulin-releasing effect of GIP is strictly glucose-dependent. This affords protection against hypoglycemia and thereby fulfills one of the most desirable features of any current or potentially new antidiabetic drug.
The present results demonstrate for the first time that Tyrl-glucitol GIP
displays profound resistance to serum and DPP IV degradation. Using ESI-MS the present study showed that native GIP was rapidly cleaved in vitro to a major 4748.4 Da degradation product corresponding to GIP(3-42), which confirmed previous findings using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Serum degradation was completely inhibited by diprotin A (Ile-Pro-Ile), a specific competitive inhibitor of DPP IV, confirming this as the main enzyme for GIP inactivation in vivo. In contrast, Tyr'-glucitol GIP remained almost completely intact after incubation with serum or DPP IV for up to 12 hours.
This indicates that glycation of GIP at the amino-terminal Tyri residue masks the potential cleavage site from DPP IV and prevents removal of the Tyr'-Ala2 dipeptide from the N-terminus preventing the formation of GIP(3-42).
Consistent with in vitro protection against DPP IV, administration of Tyrl-glucitol GIP significantly enhanced the antihyperglycemic activity and insulin-releasing action of the peptide when administered with glucose to rats. Native GIP
enhanced insulin release and reduced the glycemic excursion as observed in many previous studies. However, amino-terminal glycation of GIP increased the insulin-releasing and antihyperglycemic actions of the peptide by 62% and 38%
respectively, as estimated from insulin area under the curve (AUC) measurements. Detailed kinetic analysis is difficult due to necessary limitation of sampling times, but the greater insulin concentrations following Tyr'-glucitol GIP as opposed to GIP at 30 minutes post-injection is indicative of a longer half-life. The glycemic rise was modest in both peptide-treated groups and glucose concentrations following injection of Tyrl-glucitol GIP were consistently lower than after GIP. Since the insulinotropic actions of GIP
are glucose-dependent, it is likely that the relative insulin-releasing potency of Tyrl-glucitol GIP is greatly underestimated in the present in vivo experiments.
In vitro studies in the laboratory of the present inventors using glucose-responsive clonal B-cells showed that the insulin-releasing potency of Tyriglucitol GIP was several orders of magnitude greater than GIP and that its effectiveness was more sensitive to change of glucose concentrations within the physiological range.
Together with the present in vivo observations, this suggests that N-terminal glycation of GIP confers resistance to DPP IV degradation whilst enhancing receptor binding and insulin secretory effects on the B-cell. These attributes of Tyri-glucitol GIP are fully expressed in vivo where DPP IV resistance impedes degradation of the peptide to GIP(3-42), thereby prolonging the half-life and enhancing effective concentrations of the intact biologically active peptide. It is thus possible that glycated GIP is enhancing insulin secretion in vivo both by enhanced potency at the receptor as well as improving DPP IV resistance. Thus numerous studies have shown that GIP (3-42) and other N-terminally modified fragments, including GIP(4-42), and GIP(17-42) are either weakly effective or inactive in stimulating insulin release.
Furthermore, evidence exists that N-terminal deletions of GIP result in receptor antagonist properties in GIP receptor transfected Chinese hamster kidney cells [9], suggesting that inhibition of GIP catabolism would also reduce the possible feedback antagonism at the receptor level by the truncated GIP(3-42).
In addition to its insulinotopic actions, a number of other potentially important extrapancreatic actions of GIP may contribute to the enhanced antihyperglycemic activity and other beneficial metabolic effects of Tyrl-glucitol GIP. These include the stimulation of glucose uptake in adipocytes, increased synthesis of fatty acids and activation of lipoprotein lipase in adipose tissue. GIP also promotes plasma triglyceride clearance in response to oral fat loading. In liver, GIP has been shown to enhance insulin-dependent inhibition of glycogenolysis. GIP also reduces both glucagon-stimulated lipolysis in adipose tissue as well as hepatic glucose production.
Finally, recent findings indicate that GIP has a potent effect on glucose uptake and metabolism in mouse isolated diaphragm muscle. This latter action may be shared with tGLP-1 and both peptides have additional benefits of stimulating somatostatin secretion and slowing down gastric emptying and nutrient absorption.
This study demonstrates that the glycation of GIP at the aminotenninal Tyr' residue limits GIP catabolism through impairment of the proteolytic actions of serum peptidases and thus prolongs its half-life in vivo. This effect is accompanied by enhanced antihyperglycemic activity and raised insulin concentrations in vivo, suggesting that such DPP IV resistant analogues are potentially useful therapeutic agents for NIDDM. Tyrl-glucitol GIP appears to be particularly interesting in this regard since such amino-terminal modification of GIP enhances rather than impairs glucose-dependent insulinotropic potency as was observed recently for tGLP-1.
As shown in Table 1 in Example 3, glycated GIP, acetylated GIP, GIP(Ser2) are GIP(Abu2) more resistant than native GIP to in vitro degradation with DPP
IV.
From these data GIP(Sar2) appears to be less resistant. As shown in Table 2, all analogues tested exhibited resistance to plasma degradation, including GIP(Sar2) which from DPP IV data appeared least resistant of the peptides tested. DPA
substantially inhibited degradation of GIP and all analogues tested with complete abolition of degradation in the cases of GIP(Abu2), GIP(Ser2) and glycated GIP. This indicates that DPP IV is a key factor in the in vivo degradation of GIP.
As shown in Figs. 17-30, the glycated GIP analogue exhibited a considerably greater insulinotropic response relative to native GIP. N-terminal acetylated GIP
exhibited a similar pattern and the GIP(Ser2) analogue also evoked a strong response.
From these tests, GIP(Gly2) and GIP(Pro3) appeared to the least potent analogues in terms of insulin release. Other stable analogues tested, namely GIP(Abu2) and GIP(Sar2), exhibited a complex pattern of responsiveness dependent on glucose concentration and dose employed. Thus, very low concentrations were extremely potent under hyperglycemic conditions (16.7 mM glucose). This suggests that even these analogues may prove therapeutically useful in the treatment of type 2 diabetes where insulinotropic capacity combined with in vivo degradation dictates peptide potency.
A major limitation to the possible therapeutic use of both GIP and GLP-1 as insulin-releasing agents for the treatment of diabetes is their rapid degradation in vivo by dipeptidylpeptidase-IV (DPP-IV; EC 3.4.14.5). This enzyme rapidly removes the amino-terminal dipeptide from the two peptides producing GIP(3-42) and GLP-1(9-36), which lack biological activity (Gault, V.A. et al., 2002, Biochem.
Biophys. Res.
Commun. 290:1420-1426). In searching for stable amino-terminally modified forms of GIP and GLP-1, it was discovered that a novel synthetic GIP analogue with a single proline substitution at position 3 close to the cleavage site, (Pro3)GIP, functioned as a potent GIP receptor antagonist.
As shown in Example 4, below, (Pro3)GIP, other G1u3-substituted forms of GIP and GIP(3-42) are potent GIP receptor antagonists both in vitro and in vivo.
Experiments evaluating the effects of chronic GIP receptor antagonism in normal mice using (Pro3)GIP demonstrated a substantial but reversible deterioration of glucose tolerance. This is entirely consistent with the widely recognised physiological role of GIP as an important insulin-releasing intestinal hormone involved in the regulation of glucose disposal following feeding (Meier, J.J.
et aL, 2002, ReguL P ept. 107:1-13).
Most notably, and in complete contrast to normal mice, the experiments disclosed herein show that chronic (Pro3)GIP administration to obese diabetic ob/ob mice for 11 days does not worsen glucose intolerance and diabetes status at all.
Particularly preferred alterations include acetylation of Tyr1 and linkage of a C-16 palmitate group to the epsilon amino group of a lysine residue (especially Lys16 or Lys37), or substitution of G1u3, especially by proline. The modification can also be the linking of, e.g., a C-8, a C-10, a C-12, a C-14, a C-18 or a C-20 palmitate group to the epsilon amino group of a lysine residue.
It has been found that longer-term, as opposed to acute, GIP receptor antagonism using Glu3-substituted forms of GIP, such as (Pro3)GIP, improve obesity-related insulin resistance and associated glucose intolerance. This has uncovered an unexpected approach to the therapy of obesity, insulin resistance and type 2 diabetes.
As described in Example 1 below, an N-terminally modified version of the GIP protein was prepared, as were analogues of the modified protein. The protein and its analogues were then evaluated in Example 2 for their antihyperglycemic and insulin-releasing properties in vivo, and were found to exhibit a substantial resistance to amino peptidase degradation and increased glucose lowering activity relative to native GIP.
The 42 amino acid GIP is an important incretin hormone released into the circulation from endocrine K-cells of the duodenum and jejunum following ingestion of food. The high degree of structural conservation of GIP among species supports the view that this peptide plays an important role in metabolism. Secretion of GIP is stimulated directly by actively transported nutrients in the gut lumen without a notable input from autonomic nerves. The most important stimulants of GIP release are simple sugars and unsaturated long chain fatty acids, with amino acids exerting weaker effects. As with tGLP-1, the insulin-releasing effect of GIP is strictly glucose-dependent. This affords protection against hypoglycemia and thereby fulfills one of the most desirable features of any current or potentially new antidiabetic drug.
The present results demonstrate for the first time that Tyrl-glucitol GIP
displays profound resistance to serum and DPP IV degradation. Using ESI-MS the present study showed that native GIP was rapidly cleaved in vitro to a major 4748.4 Da degradation product corresponding to GIP(3-42), which confirmed previous findings using matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Serum degradation was completely inhibited by diprotin A (Ile-Pro-Ile), a specific competitive inhibitor of DPP IV, confirming this as the main enzyme for GIP inactivation in vivo. In contrast, Tyr'-glucitol GIP remained almost completely intact after incubation with serum or DPP IV for up to 12 hours.
This indicates that glycation of GIP at the amino-terminal Tyri residue masks the potential cleavage site from DPP IV and prevents removal of the Tyr'-Ala2 dipeptide from the N-terminus preventing the formation of GIP(3-42).
Consistent with in vitro protection against DPP IV, administration of Tyrl-glucitol GIP significantly enhanced the antihyperglycemic activity and insulin-releasing action of the peptide when administered with glucose to rats. Native GIP
enhanced insulin release and reduced the glycemic excursion as observed in many previous studies. However, amino-terminal glycation of GIP increased the insulin-releasing and antihyperglycemic actions of the peptide by 62% and 38%
respectively, as estimated from insulin area under the curve (AUC) measurements. Detailed kinetic analysis is difficult due to necessary limitation of sampling times, but the greater insulin concentrations following Tyr'-glucitol GIP as opposed to GIP at 30 minutes post-injection is indicative of a longer half-life. The glycemic rise was modest in both peptide-treated groups and glucose concentrations following injection of Tyrl-glucitol GIP were consistently lower than after GIP. Since the insulinotropic actions of GIP
are glucose-dependent, it is likely that the relative insulin-releasing potency of Tyrl-glucitol GIP is greatly underestimated in the present in vivo experiments.
In vitro studies in the laboratory of the present inventors using glucose-responsive clonal B-cells showed that the insulin-releasing potency of Tyriglucitol GIP was several orders of magnitude greater than GIP and that its effectiveness was more sensitive to change of glucose concentrations within the physiological range.
Together with the present in vivo observations, this suggests that N-terminal glycation of GIP confers resistance to DPP IV degradation whilst enhancing receptor binding and insulin secretory effects on the B-cell. These attributes of Tyri-glucitol GIP are fully expressed in vivo where DPP IV resistance impedes degradation of the peptide to GIP(3-42), thereby prolonging the half-life and enhancing effective concentrations of the intact biologically active peptide. It is thus possible that glycated GIP is enhancing insulin secretion in vivo both by enhanced potency at the receptor as well as improving DPP IV resistance. Thus numerous studies have shown that GIP (3-42) and other N-terminally modified fragments, including GIP(4-42), and GIP(17-42) are either weakly effective or inactive in stimulating insulin release.
Furthermore, evidence exists that N-terminal deletions of GIP result in receptor antagonist properties in GIP receptor transfected Chinese hamster kidney cells [9], suggesting that inhibition of GIP catabolism would also reduce the possible feedback antagonism at the receptor level by the truncated GIP(3-42).
In addition to its insulinotopic actions, a number of other potentially important extrapancreatic actions of GIP may contribute to the enhanced antihyperglycemic activity and other beneficial metabolic effects of Tyrl-glucitol GIP. These include the stimulation of glucose uptake in adipocytes, increased synthesis of fatty acids and activation of lipoprotein lipase in adipose tissue. GIP also promotes plasma triglyceride clearance in response to oral fat loading. In liver, GIP has been shown to enhance insulin-dependent inhibition of glycogenolysis. GIP also reduces both glucagon-stimulated lipolysis in adipose tissue as well as hepatic glucose production.
Finally, recent findings indicate that GIP has a potent effect on glucose uptake and metabolism in mouse isolated diaphragm muscle. This latter action may be shared with tGLP-1 and both peptides have additional benefits of stimulating somatostatin secretion and slowing down gastric emptying and nutrient absorption.
This study demonstrates that the glycation of GIP at the aminotenninal Tyr' residue limits GIP catabolism through impairment of the proteolytic actions of serum peptidases and thus prolongs its half-life in vivo. This effect is accompanied by enhanced antihyperglycemic activity and raised insulin concentrations in vivo, suggesting that such DPP IV resistant analogues are potentially useful therapeutic agents for NIDDM. Tyrl-glucitol GIP appears to be particularly interesting in this regard since such amino-terminal modification of GIP enhances rather than impairs glucose-dependent insulinotropic potency as was observed recently for tGLP-1.
As shown in Table 1 in Example 3, glycated GIP, acetylated GIP, GIP(Ser2) are GIP(Abu2) more resistant than native GIP to in vitro degradation with DPP
IV.
From these data GIP(Sar2) appears to be less resistant. As shown in Table 2, all analogues tested exhibited resistance to plasma degradation, including GIP(Sar2) which from DPP IV data appeared least resistant of the peptides tested. DPA
substantially inhibited degradation of GIP and all analogues tested with complete abolition of degradation in the cases of GIP(Abu2), GIP(Ser2) and glycated GIP. This indicates that DPP IV is a key factor in the in vivo degradation of GIP.
As shown in Figs. 17-30, the glycated GIP analogue exhibited a considerably greater insulinotropic response relative to native GIP. N-terminal acetylated GIP
exhibited a similar pattern and the GIP(Ser2) analogue also evoked a strong response.
From these tests, GIP(Gly2) and GIP(Pro3) appeared to the least potent analogues in terms of insulin release. Other stable analogues tested, namely GIP(Abu2) and GIP(Sar2), exhibited a complex pattern of responsiveness dependent on glucose concentration and dose employed. Thus, very low concentrations were extremely potent under hyperglycemic conditions (16.7 mM glucose). This suggests that even these analogues may prove therapeutically useful in the treatment of type 2 diabetes where insulinotropic capacity combined with in vivo degradation dictates peptide potency.
A major limitation to the possible therapeutic use of both GIP and GLP-1 as insulin-releasing agents for the treatment of diabetes is their rapid degradation in vivo by dipeptidylpeptidase-IV (DPP-IV; EC 3.4.14.5). This enzyme rapidly removes the amino-terminal dipeptide from the two peptides producing GIP(3-42) and GLP-1(9-36), which lack biological activity (Gault, V.A. et al., 2002, Biochem.
Biophys. Res.
Commun. 290:1420-1426). In searching for stable amino-terminally modified forms of GIP and GLP-1, it was discovered that a novel synthetic GIP analogue with a single proline substitution at position 3 close to the cleavage site, (Pro3)GIP, functioned as a potent GIP receptor antagonist.
As shown in Example 4, below, (Pro3)GIP, other G1u3-substituted forms of GIP and GIP(3-42) are potent GIP receptor antagonists both in vitro and in vivo.
Experiments evaluating the effects of chronic GIP receptor antagonism in normal mice using (Pro3)GIP demonstrated a substantial but reversible deterioration of glucose tolerance. This is entirely consistent with the widely recognised physiological role of GIP as an important insulin-releasing intestinal hormone involved in the regulation of glucose disposal following feeding (Meier, J.J.
et aL, 2002, ReguL P ept. 107:1-13).
Most notably, and in complete contrast to normal mice, the experiments disclosed herein show that chronic (Pro3)GIP administration to obese diabetic ob/ob mice for 11 days does not worsen glucose intolerance and diabetes status at all.
Surprisingly, GIP receptor antagonism in this obese insulin resistant model was associated with highly substantial improvements of glycated HbAic, plasma glucose and insulin concentrations, glucose tolerance and insulin sensitivity.
Pancreatic insulin content was also decreased and the characteristic islet hypertrophy of the obese mutant was partially reversed. These latter observations indicate a decreased secretory demand for endogenous insulin following (Pro3)GIP as a result of improved =
insulin resistance.
Indeed, insulin sensitivity tests conducted in ob/ob mice 11 days into (Pro3)GIP treatment revealed a substantial improvement in tissue insulin insensitivity, which more than compensated for the functional ablation of the insulin-releasing GIP
component of the enteroinsular axis. The exact mechanism responsible for this effect on insulin sensitivity is unknown but ablation of direct action of circulating GIP on adipose tissue metabolism is a likely candidate. Also noteworthy was the fact that all these beneficial actions of (Pro3)GIP in obese diabetic ob/ob mice were reversed within 9 days cessation of treatment.
These results clearly indicate that (Pro3)GIP and other analogues based on G1u3-substituted or N-terminally truncated forms of the gastrointestinal hormone GIP
can offer an important therapeutic means of alleviating insulin resistance for the treatment of obesity, the so-called insulin resistant (metabolic) syndrome and type 2 diabetes in humans.
Some studies have attempted to enhance incretin action using DPP IV
inhibitors or stable analogs of GLP-1 and GIP for the treatment of type 2 diabetes (Green, B.D. et al., 2004, Curr. Pharm. Des. 10:In Press; Drucker, D.J. et al., Diabetes Care 10:2929-2940). Such an approach is reliant on the possibility that incretin action is defective in diabetes and that the underlying defects responsible for metabolic disarray might be over-ridden by exogenous GLP-1 or GIP
administration.
There is some evidence for a beneficial and possibly therapeutic role of both and GIP analogs in diabetes (Meier, J.J. et al., 2002, Regul. Pept. 107:1-13;
Gault, V.A. et al., 2003, Biochem Biophys Res Commun 308:207-213; Holst, J.J. et al., 2004, Am. J. PhysioL EndocrinoL Metab. 287:E199-E206; Green, B.D. et aL, 2004, Curr.
Pharm. Des. 10:In Press; Drucker, D.J. et al., Diabetes Care 10:2929-2940).
Nevertheless, understanding of the possible involvement of incretin hormones in the pathophysiology of diabetes is lacking, partly due to cross-reaction of classical GLP-1 and GIP radioimmunoassays with the predominant DPP IV-generated truncated peptide forms, GLP-1(9-36) and GIP(3-42), which circulate at particularly high concentrations (Meier, J.J. etal., 2002, ReguL Pept. 107:1-13). Some clinical studies seems to suggest existence of a defect in the secretion of GLP-1 and a defect in the action of GIP in type 2 diabetes (Hoist, J.J. et al., 2004, Am. J. PhysioL
EndocrinoL
Metab. 287:E199-E206). However, the basis for such a conclusion is not impressive given the many previous contradictory human studies (Morgan, L.M., "Insulin secretion and the enteroinsular axis," In: Nutrient regulation of insulin secretion, Flatt, P.R., ed.; London, Portland Press, 1992, p. 1-22), and the likelihood that the reported insensitivity of pancreatic beta cells to GIP (Vilsboll, T. etal., 2002, Diabetologia 45:1111-1119) may reflect a generalized secretory dysfunction rather than a specific cellular defect (Meier, J.J. etal., 2003, Metabolism 52:1579-1585).
Indeed, the insulin secretory response to all secretagogues, including GLP-1 is compromised in type 2 diabetes (Kjems, L.L. etal., 2003, Diabetes 52:380-386;
Flatt, P.R. etal., "Defective insulin secretion in diabetes and insulinoma," in Nutrient regulation of insulin secretion, Flatt P.R., ed. London, Portland Press, 1992, p. 341-386). Thus the proposed use of GLP-1 and GIP for diabetes therapy is reliant on peptide engineering to provide analogs of incretin hormones with improved potency due to DPP IV resistance, decreased renal clearance and/or enhanced GIP
receptor and post-receptor activity (Gault, V.A. etal., 2003, Biochem Biophys Res Commun 308:207-213).
Although no single animal model can match the complex etiology of type 2 diabetes in man, studies of the ob/ob syndrome in mice have highlighted notable abnormalities of GIP in relation to the interplay between hyperphagia, hyperinsulinemia and the metabolic demise associated with progressive obesity-diabetes (Flatt, P.R. etal., 1983, Diabetes 32:433-435; Flatt, P.R. etal., 1984, J.
Endocrinol. 101:249-256; Bailey, C.J. et al., 1986, Acta EndocrinoL (Copenh) 112:224-229). These animals constitute a model of non-insulin dependent diabetes associated with gross obesity and severe insulin resistance, driven by leptin deficiency (Bailey, C.J. etal., "Animal syndromes resembling type 2 diabetes," in Textbook of Diabetes, 3rd ed. Pickup J.C. & Williams G., eds. Oxford, Blackwell Science Ltd., 2003, p. 25.1-25.30). Furthermore, recent research suggests an interaction between leptin and the enteroinsular axis (Anini, Y. etal., 2003, Diabetes 52:252-259) and that over-stimulation of the GIP receptor ("GIP-R") on adipocytes appears to be an important contributor to fat deposition in ob/ob mice (Miyawaki, K. et al., 2002, Nat.
Med. 8:738-742).
As shown in Example 5, below, daily injections of the stable and specific GIP-S R antagonist, (Pro3)GIP can be used to chemically ablate the GIP-R and evaluate the role of endogenous circulating GIP in obesity-diabetes as manifested in ob/ob mice.
The results reveal a cardinal role for GIP in insulin resistance and associated metabolic disturbances, and provide the first experimental evidence that GIP-R
antagonists might provide a novel and effective means of treating obesity-driven forms of type 2 diabetes.
Knock¨out of the GIP-R in normal mice has been shown to result in significant impairment of glucose tolerance and meal-induced insulin secretion without appreciable effects on food intake, body weight or basal glucose or insulin concentrations (Miyawaki, K. et al., 1999, Proc. Nat. Acad. Sci. USA 96:14843-14847). More recent studies with genetic GIP-R knockout mice have corroborated these findings and additionally shown that GIP has a significant involvement in the enteroinsular axis (Pederson, R.A. et al., 1998, Diabetes 47:1046-1052; Pamir, N. et aL, 2003, Am. J. Physiol. Endocrinol. Metab. 284:E931-939). However, double knockout of receptors for GLP-1 and GIP results in 9. surprisingly modest deterioration of glucose homeostasis (Hansotia, T., et al., 2004, Diabetes 53:1326-1335; Preitner, F., et aL, 2004, J. Clin. Invest. 113:635-645), indicating possible up-regulation of compensatory mechanisms during life-long deletion of GLP-1 and GIP
receptors.
The analogue (Pro3)GIP can be used as a specific and potent antagonist of the GIP-R that is highly stable and resistant to DPP IV-mediated degradation (Gault, V.A.
et al., 2002, Biochem. Biophys. Res. Commun. 290:1420-1426). Using (Pro3)GIP
acutely, the results disclosd herein highlight the involvement of GIP in the plasma insulin response to feeding and the enteroinsular axis of ob/ob mice (Gault, V.A. et al., 2003, Diabetologia 46:222-230). Comparison with the effects of the GLP-1-R
antagonist, exendin(9-39), indicates that GIP contributes substantially to the insulin releasing actions of the enteroinsular axis and represents the major physiological incretin (Gault, V.A. et al., 2003, Diabetologia 46:222-230). Once daily administration of (Pro3)GIP to normal mice for 11 days results in the reversible impairment of glucose tolerance associated with decreased insulin sensitivity (Irwin, N., 2004, Biol. Chem. 385:845-852). Basal and postprandial insulin secretion together with pancreatic insulin content and islet morphology were unchanged.
Thus longer-term chemical ablation of GIP-R function with daily (Pro3)GIP can mimic the phenotype induced by genetic GIP-R knockout in mice with the exception of revealing a potentially important additional effect of endogenous GIP on insulin action, which appears to be independent of enhanced insulin secretion.
Far from reproducing this predicted scenario and the metabolic deterioration observed following genetic or chemical knockout of the GIP-R in normal mice (Miyawaki, K. et al., 1999, Proc. Nat. Acad. ScL USA 96:14843-14847; Irwin, N., 2004, Biol. Chem. 385:845-852), ob/ob mice treated with daily (Pro3)GIP for 11 days exhibited a marked improvement in diabetic status. This included decreased fasting and basal hyperglycemia, lowered glycated hemoglobin, improved glucose tolerance and a significantly diminished glycemic excursion following feeding. Notably, basal and glucose-stimulated plasma insulin concentrations were decreased, suggesting that insulin sensitivity must have improved significantly following (Pro3)GIP in order to restrain the hyperglycemia. Indeed, insulin sensitivity tests conducted after 11 days of (Pro3)GIP administration revealed a 57% increase in the glucose-lowering action of exogenous insulin. Bearing in mind that the severity of the ob/ob syndrome represents a tough test for current antidiabetic drugs, including insulin, sulfonylureas, metformin and thiazolidenediones (Flatt, P.R. et al., "Defective insulin secretion in diabetes and insulinoma," in Nutrient regulation of insulin secretion, Flatt P.R., ed.
London, Portland Press, 1992, p. 341-386; Stevenson, R.W. et al., 1995, The Diabetes Annual 9:175-191; Wiernsperger, N.F., "Preclinical pharmacology of biguanides,"
Handbook of Experimental Pharmacology 119:305-358, 1996), induction of such rapid and reversible changes by GIP-R blockade using (Pro3)GIP is unprecedented.
It is important to note that the above effects were observed independently of any change in food intake or body weight in (Pro3)GIP treated ob/ob mice. This accords with the view that endogenous GIP lacks effects on feeding activity (Meier, J.J. et al., 2002, ReguL Pept. 107:1-13). However, the observation on body weight contrasts with findings in ob/ob mice cross-bred to genetically knockout GIP-R
function (Miyawaki, K. et al., 2002, Nat. Med. 8:738-742). Thus in these transgenic mice, life-long depletion of GIP-R function was associated with decreased body weight gain and significant amelioration of both adiposity and insulin resistance compared with control (Lee/Lee) mice (Miyawaki, K. et at., 2002, Nat. Med.
8:738-742). In this previous study, the improvement of insulin sensitivity may have been a simple consequence of reduced adipose tissue mass as this would significantly enhance peripheral glucose disposal (Bailey, C.J. et at., "Animal syndromes resembling type 2 diabetes," in Textbook of Diabetes, 3rd ed. Pickup J.C. &
Williams G., eds. Oxford, Blackwell Science Ltd., 2003, p. 25.1-25.30). However, the present results observed in rapid time and without effects on feeding or body weight clearly indicate the involvement of an alternative mechanism.
The most plausible explanation for the present data stem from appreciation of the key milestones in the age-dependent progression of the ob/ob syndrome on the Aston background as depicted in Fig. 49, which is an illustration of how the GIP-R
antagonist, (Pro3)GIP, counters beta cell hyperplasia, hyperinsulinemia and insulin resistance lead to improved glucose intolerance and diabetes control. Possible longer-term direct actions of GIP on adipocyte function and fat stores, suggested by studies in GIP-R knockout ob/ob mice have been omitted.
Due to double recessive ob mutation and resulting leptin deficiency, young ob/ob mice develop a profound early hyperphagia (Bailey, C.J., et at., 1982, Int. J.
Obes. 6:11-21). Substantial enteroendocrine stimulation results in K-cell hyperplasia and markedly elevated concentrations of intestinal and circulating GIP (Flatt, P.R. et al., 1983, Diabetes 32:433-435; Flatt, P.R. et at., 1984, J EndocrinoL 101:249-256;
Bailey, C.J. et at., 1986, Acta Endocrinol. (Copenh) 112:224-229). This in turn promotes islet hypertrophy and beta cell hyperplasia (Bailey, C.J., et at., 1982, Int. J.
Obes. 6:11-21) together with marked hyperinsulinemia and mounting insulin resistance (Flatt, P.R., et at., 1981, Horin Metab Res 13:556-560). This process manifests itself in ternis of rising basal hyperglycemia and glucose intolerance. A
vicious spiral is thus established wherein beta cell compensation results in marked hyperinsulinemia which attempts to moderate increasing insulin resistance (Bailey, C.J., et at., 1982, Int. J. Obes. 6:11-21; Flatt, P.R., et at., 1981, Horni Metab Res 13:556-560). Viewed in this context, it is clear that chemical ablation of GIP-R
function with daily (Pro3)GIP will decrease beta cell stimulation and hyperinsulinemia. However, instead of causing further impairment of glucose homeostasis, a preferentially marked improvement of insulin sensitivity results in a substantial improvement of the metabolic syndrome. Further support for this scenario, is the partial amelioration of islet hypertrophy and beta cell hyperplasia in (Pro3)GIP treated ob/ob mice (Fig. 48). Notably, average islet diameter was diminished with the largest islets (>15 mm) being replaced by a greater proportion with small or medium diameters (0.1-15mm). These effects were largely reversed by 9 day cessation of treatment, supporting the idea of active islet and beta cell growth in adult ob/ob mice (Bailey, C.J., et al., 1982, Int. J. Obes. 6:11-21). Recent observations indicate that GIP acts as a mitotic stimulus and anti-apoptotic agent to the beta cell (Pospisilik, J.A. et al., 2003, Diabetes 52:741-750; Trumper, A.
et al., 2001, MoL EndocrinoL 15:1559-1570; Ehses, J.A. et al., 2003, Endocrinology 144:4433-4445, Trumper, A. et al., 2002, J EndocrinoL 174:233-246). Thus, it is believed that negative effects of (Pro3)GIP on islet size reflects a combination of decreased proliferation and increased apoptosis of beta cells.
The results shown in Example 5 have demonstrated for the first time that daily administration of the GIP-R antagonist, (Pro3)GIP, improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure and function in ob/ob mice. Notably, these effects were reversed by discontinuation of (Pro3)GIP for 9 days. Freedom from any obvious side effects also accords with earlier observations in normal mice (Irwin, N., 2004, Biol. Chem. 385:845-852) and mice genetically engineered with life-long GIP-R deficiency (Miyawaki, K. et al., 2002, Nat.
Med.
8:738-742). The present observations point to a cardinal role of endogenous GIP in the pathogenesis of obese-insulin resistant-diabetes. More importantly, the data indicate that GIP-R antagonists, such as (Pro3)GIP, provide a novel, physiological and effective means to treat obese type 2 diabetes through the alleviation of insulin resistance.
In Example 6, fatty acid derivatisation was used to develop two novel long-acting, N-terminally modified GIP analogues (N-AcGIP(LysPAL16) and N-AcG1P(LysP AL37)).
Degradation studies were carried out with dipeptidylpeptidase IV (DPP IV).
Cyclic AMP production was assessed using GIP receptor transfected CHL
fibroblasts.
In vitro insulin release was assessed in BRIN-BD11 cells. Insulinotropic and glycaemic responses to acute and long-term peptide administration were evaluated in obese diabetic (ob/ob) mice.
Pancreatic insulin content was also decreased and the characteristic islet hypertrophy of the obese mutant was partially reversed. These latter observations indicate a decreased secretory demand for endogenous insulin following (Pro3)GIP as a result of improved =
insulin resistance.
Indeed, insulin sensitivity tests conducted in ob/ob mice 11 days into (Pro3)GIP treatment revealed a substantial improvement in tissue insulin insensitivity, which more than compensated for the functional ablation of the insulin-releasing GIP
component of the enteroinsular axis. The exact mechanism responsible for this effect on insulin sensitivity is unknown but ablation of direct action of circulating GIP on adipose tissue metabolism is a likely candidate. Also noteworthy was the fact that all these beneficial actions of (Pro3)GIP in obese diabetic ob/ob mice were reversed within 9 days cessation of treatment.
These results clearly indicate that (Pro3)GIP and other analogues based on G1u3-substituted or N-terminally truncated forms of the gastrointestinal hormone GIP
can offer an important therapeutic means of alleviating insulin resistance for the treatment of obesity, the so-called insulin resistant (metabolic) syndrome and type 2 diabetes in humans.
Some studies have attempted to enhance incretin action using DPP IV
inhibitors or stable analogs of GLP-1 and GIP for the treatment of type 2 diabetes (Green, B.D. et al., 2004, Curr. Pharm. Des. 10:In Press; Drucker, D.J. et al., Diabetes Care 10:2929-2940). Such an approach is reliant on the possibility that incretin action is defective in diabetes and that the underlying defects responsible for metabolic disarray might be over-ridden by exogenous GLP-1 or GIP
administration.
There is some evidence for a beneficial and possibly therapeutic role of both and GIP analogs in diabetes (Meier, J.J. et al., 2002, Regul. Pept. 107:1-13;
Gault, V.A. et al., 2003, Biochem Biophys Res Commun 308:207-213; Holst, J.J. et al., 2004, Am. J. PhysioL EndocrinoL Metab. 287:E199-E206; Green, B.D. et aL, 2004, Curr.
Pharm. Des. 10:In Press; Drucker, D.J. et al., Diabetes Care 10:2929-2940).
Nevertheless, understanding of the possible involvement of incretin hormones in the pathophysiology of diabetes is lacking, partly due to cross-reaction of classical GLP-1 and GIP radioimmunoassays with the predominant DPP IV-generated truncated peptide forms, GLP-1(9-36) and GIP(3-42), which circulate at particularly high concentrations (Meier, J.J. etal., 2002, ReguL Pept. 107:1-13). Some clinical studies seems to suggest existence of a defect in the secretion of GLP-1 and a defect in the action of GIP in type 2 diabetes (Hoist, J.J. et al., 2004, Am. J. PhysioL
EndocrinoL
Metab. 287:E199-E206). However, the basis for such a conclusion is not impressive given the many previous contradictory human studies (Morgan, L.M., "Insulin secretion and the enteroinsular axis," In: Nutrient regulation of insulin secretion, Flatt, P.R., ed.; London, Portland Press, 1992, p. 1-22), and the likelihood that the reported insensitivity of pancreatic beta cells to GIP (Vilsboll, T. etal., 2002, Diabetologia 45:1111-1119) may reflect a generalized secretory dysfunction rather than a specific cellular defect (Meier, J.J. etal., 2003, Metabolism 52:1579-1585).
Indeed, the insulin secretory response to all secretagogues, including GLP-1 is compromised in type 2 diabetes (Kjems, L.L. etal., 2003, Diabetes 52:380-386;
Flatt, P.R. etal., "Defective insulin secretion in diabetes and insulinoma," in Nutrient regulation of insulin secretion, Flatt P.R., ed. London, Portland Press, 1992, p. 341-386). Thus the proposed use of GLP-1 and GIP for diabetes therapy is reliant on peptide engineering to provide analogs of incretin hormones with improved potency due to DPP IV resistance, decreased renal clearance and/or enhanced GIP
receptor and post-receptor activity (Gault, V.A. etal., 2003, Biochem Biophys Res Commun 308:207-213).
Although no single animal model can match the complex etiology of type 2 diabetes in man, studies of the ob/ob syndrome in mice have highlighted notable abnormalities of GIP in relation to the interplay between hyperphagia, hyperinsulinemia and the metabolic demise associated with progressive obesity-diabetes (Flatt, P.R. etal., 1983, Diabetes 32:433-435; Flatt, P.R. etal., 1984, J.
Endocrinol. 101:249-256; Bailey, C.J. et al., 1986, Acta EndocrinoL (Copenh) 112:224-229). These animals constitute a model of non-insulin dependent diabetes associated with gross obesity and severe insulin resistance, driven by leptin deficiency (Bailey, C.J. etal., "Animal syndromes resembling type 2 diabetes," in Textbook of Diabetes, 3rd ed. Pickup J.C. & Williams G., eds. Oxford, Blackwell Science Ltd., 2003, p. 25.1-25.30). Furthermore, recent research suggests an interaction between leptin and the enteroinsular axis (Anini, Y. etal., 2003, Diabetes 52:252-259) and that over-stimulation of the GIP receptor ("GIP-R") on adipocytes appears to be an important contributor to fat deposition in ob/ob mice (Miyawaki, K. et al., 2002, Nat.
Med. 8:738-742).
As shown in Example 5, below, daily injections of the stable and specific GIP-S R antagonist, (Pro3)GIP can be used to chemically ablate the GIP-R and evaluate the role of endogenous circulating GIP in obesity-diabetes as manifested in ob/ob mice.
The results reveal a cardinal role for GIP in insulin resistance and associated metabolic disturbances, and provide the first experimental evidence that GIP-R
antagonists might provide a novel and effective means of treating obesity-driven forms of type 2 diabetes.
Knock¨out of the GIP-R in normal mice has been shown to result in significant impairment of glucose tolerance and meal-induced insulin secretion without appreciable effects on food intake, body weight or basal glucose or insulin concentrations (Miyawaki, K. et al., 1999, Proc. Nat. Acad. Sci. USA 96:14843-14847). More recent studies with genetic GIP-R knockout mice have corroborated these findings and additionally shown that GIP has a significant involvement in the enteroinsular axis (Pederson, R.A. et al., 1998, Diabetes 47:1046-1052; Pamir, N. et aL, 2003, Am. J. Physiol. Endocrinol. Metab. 284:E931-939). However, double knockout of receptors for GLP-1 and GIP results in 9. surprisingly modest deterioration of glucose homeostasis (Hansotia, T., et al., 2004, Diabetes 53:1326-1335; Preitner, F., et aL, 2004, J. Clin. Invest. 113:635-645), indicating possible up-regulation of compensatory mechanisms during life-long deletion of GLP-1 and GIP
receptors.
The analogue (Pro3)GIP can be used as a specific and potent antagonist of the GIP-R that is highly stable and resistant to DPP IV-mediated degradation (Gault, V.A.
et al., 2002, Biochem. Biophys. Res. Commun. 290:1420-1426). Using (Pro3)GIP
acutely, the results disclosd herein highlight the involvement of GIP in the plasma insulin response to feeding and the enteroinsular axis of ob/ob mice (Gault, V.A. et al., 2003, Diabetologia 46:222-230). Comparison with the effects of the GLP-1-R
antagonist, exendin(9-39), indicates that GIP contributes substantially to the insulin releasing actions of the enteroinsular axis and represents the major physiological incretin (Gault, V.A. et al., 2003, Diabetologia 46:222-230). Once daily administration of (Pro3)GIP to normal mice for 11 days results in the reversible impairment of glucose tolerance associated with decreased insulin sensitivity (Irwin, N., 2004, Biol. Chem. 385:845-852). Basal and postprandial insulin secretion together with pancreatic insulin content and islet morphology were unchanged.
Thus longer-term chemical ablation of GIP-R function with daily (Pro3)GIP can mimic the phenotype induced by genetic GIP-R knockout in mice with the exception of revealing a potentially important additional effect of endogenous GIP on insulin action, which appears to be independent of enhanced insulin secretion.
Far from reproducing this predicted scenario and the metabolic deterioration observed following genetic or chemical knockout of the GIP-R in normal mice (Miyawaki, K. et al., 1999, Proc. Nat. Acad. ScL USA 96:14843-14847; Irwin, N., 2004, Biol. Chem. 385:845-852), ob/ob mice treated with daily (Pro3)GIP for 11 days exhibited a marked improvement in diabetic status. This included decreased fasting and basal hyperglycemia, lowered glycated hemoglobin, improved glucose tolerance and a significantly diminished glycemic excursion following feeding. Notably, basal and glucose-stimulated plasma insulin concentrations were decreased, suggesting that insulin sensitivity must have improved significantly following (Pro3)GIP in order to restrain the hyperglycemia. Indeed, insulin sensitivity tests conducted after 11 days of (Pro3)GIP administration revealed a 57% increase in the glucose-lowering action of exogenous insulin. Bearing in mind that the severity of the ob/ob syndrome represents a tough test for current antidiabetic drugs, including insulin, sulfonylureas, metformin and thiazolidenediones (Flatt, P.R. et al., "Defective insulin secretion in diabetes and insulinoma," in Nutrient regulation of insulin secretion, Flatt P.R., ed.
London, Portland Press, 1992, p. 341-386; Stevenson, R.W. et al., 1995, The Diabetes Annual 9:175-191; Wiernsperger, N.F., "Preclinical pharmacology of biguanides,"
Handbook of Experimental Pharmacology 119:305-358, 1996), induction of such rapid and reversible changes by GIP-R blockade using (Pro3)GIP is unprecedented.
It is important to note that the above effects were observed independently of any change in food intake or body weight in (Pro3)GIP treated ob/ob mice. This accords with the view that endogenous GIP lacks effects on feeding activity (Meier, J.J. et al., 2002, ReguL Pept. 107:1-13). However, the observation on body weight contrasts with findings in ob/ob mice cross-bred to genetically knockout GIP-R
function (Miyawaki, K. et al., 2002, Nat. Med. 8:738-742). Thus in these transgenic mice, life-long depletion of GIP-R function was associated with decreased body weight gain and significant amelioration of both adiposity and insulin resistance compared with control (Lee/Lee) mice (Miyawaki, K. et at., 2002, Nat. Med.
8:738-742). In this previous study, the improvement of insulin sensitivity may have been a simple consequence of reduced adipose tissue mass as this would significantly enhance peripheral glucose disposal (Bailey, C.J. et at., "Animal syndromes resembling type 2 diabetes," in Textbook of Diabetes, 3rd ed. Pickup J.C. &
Williams G., eds. Oxford, Blackwell Science Ltd., 2003, p. 25.1-25.30). However, the present results observed in rapid time and without effects on feeding or body weight clearly indicate the involvement of an alternative mechanism.
The most plausible explanation for the present data stem from appreciation of the key milestones in the age-dependent progression of the ob/ob syndrome on the Aston background as depicted in Fig. 49, which is an illustration of how the GIP-R
antagonist, (Pro3)GIP, counters beta cell hyperplasia, hyperinsulinemia and insulin resistance lead to improved glucose intolerance and diabetes control. Possible longer-term direct actions of GIP on adipocyte function and fat stores, suggested by studies in GIP-R knockout ob/ob mice have been omitted.
Due to double recessive ob mutation and resulting leptin deficiency, young ob/ob mice develop a profound early hyperphagia (Bailey, C.J., et at., 1982, Int. J.
Obes. 6:11-21). Substantial enteroendocrine stimulation results in K-cell hyperplasia and markedly elevated concentrations of intestinal and circulating GIP (Flatt, P.R. et al., 1983, Diabetes 32:433-435; Flatt, P.R. et at., 1984, J EndocrinoL 101:249-256;
Bailey, C.J. et at., 1986, Acta Endocrinol. (Copenh) 112:224-229). This in turn promotes islet hypertrophy and beta cell hyperplasia (Bailey, C.J., et at., 1982, Int. J.
Obes. 6:11-21) together with marked hyperinsulinemia and mounting insulin resistance (Flatt, P.R., et at., 1981, Horin Metab Res 13:556-560). This process manifests itself in ternis of rising basal hyperglycemia and glucose intolerance. A
vicious spiral is thus established wherein beta cell compensation results in marked hyperinsulinemia which attempts to moderate increasing insulin resistance (Bailey, C.J., et at., 1982, Int. J. Obes. 6:11-21; Flatt, P.R., et at., 1981, Horni Metab Res 13:556-560). Viewed in this context, it is clear that chemical ablation of GIP-R
function with daily (Pro3)GIP will decrease beta cell stimulation and hyperinsulinemia. However, instead of causing further impairment of glucose homeostasis, a preferentially marked improvement of insulin sensitivity results in a substantial improvement of the metabolic syndrome. Further support for this scenario, is the partial amelioration of islet hypertrophy and beta cell hyperplasia in (Pro3)GIP treated ob/ob mice (Fig. 48). Notably, average islet diameter was diminished with the largest islets (>15 mm) being replaced by a greater proportion with small or medium diameters (0.1-15mm). These effects were largely reversed by 9 day cessation of treatment, supporting the idea of active islet and beta cell growth in adult ob/ob mice (Bailey, C.J., et al., 1982, Int. J. Obes. 6:11-21). Recent observations indicate that GIP acts as a mitotic stimulus and anti-apoptotic agent to the beta cell (Pospisilik, J.A. et al., 2003, Diabetes 52:741-750; Trumper, A.
et al., 2001, MoL EndocrinoL 15:1559-1570; Ehses, J.A. et al., 2003, Endocrinology 144:4433-4445, Trumper, A. et al., 2002, J EndocrinoL 174:233-246). Thus, it is believed that negative effects of (Pro3)GIP on islet size reflects a combination of decreased proliferation and increased apoptosis of beta cells.
The results shown in Example 5 have demonstrated for the first time that daily administration of the GIP-R antagonist, (Pro3)GIP, improves glucose tolerance and ameliorates insulin resistance and abnormalities of islet structure and function in ob/ob mice. Notably, these effects were reversed by discontinuation of (Pro3)GIP for 9 days. Freedom from any obvious side effects also accords with earlier observations in normal mice (Irwin, N., 2004, Biol. Chem. 385:845-852) and mice genetically engineered with life-long GIP-R deficiency (Miyawaki, K. et al., 2002, Nat.
Med.
8:738-742). The present observations point to a cardinal role of endogenous GIP in the pathogenesis of obese-insulin resistant-diabetes. More importantly, the data indicate that GIP-R antagonists, such as (Pro3)GIP, provide a novel, physiological and effective means to treat obese type 2 diabetes through the alleviation of insulin resistance.
In Example 6, fatty acid derivatisation was used to develop two novel long-acting, N-terminally modified GIP analogues (N-AcGIP(LysPAL16) and N-AcG1P(LysP AL37)).
Degradation studies were carried out with dipeptidylpeptidase IV (DPP IV).
Cyclic AMP production was assessed using GIP receptor transfected CHL
fibroblasts.
In vitro insulin release was assessed in BRIN-BD11 cells. Insulinotropic and glycaemic responses to acute and long-term peptide administration were evaluated in obese diabetic (ob/ob) mice.
In contrast to GIP both analogues displayed resistance to DPP IV degradation.
The analogues also stimulated cyclic AMP production and exhibited significantly improved in vitro insulin secretion compared to control. Administration of N-AcGIP(LysP AL16) or N-AcGIP(LysPAL37) together with glucose in ob/ob mice significantly reduced the glycaemic excursion and improved the insulinotropic response compared to GIP. Dose-response studies with N-AcGIP(LysPAL37) revealed highly significant decreases in the overall glycaemic excursion and increases in circulating insulin even with 6.25 nmoles/kg. Once daily injection of ob/ob mice with N-AcGIP(LysPAL37) over 14 days significantly decreased plasma glucose, glycated haemoglobin and improved glucose tolerance compared with saline or native GIP. Plasma and pancreatic insulin were significantly increased, together with a significant enhancement in the insulin response to glucose and a notable improvement of insulin sensitivity. No evidence was found for GIP-receptor desensitization and the metabolic effects of N-AcGIP(LysPAL37) were independent of any change in feeding or body weight.
These results show that novel fatty acid derivatised, N-terminally modified analogues of GIP such as N-AcGIP(LysPAL37), may have significant potential for the treatment of type 2 diabetes.
One approach to counter both renal clearance and enzyme degradation of GIP
concerns the utilisation of fatty acid derivatisation together with N-terminal modification. Fatty acid derivatisation has previously been shown to prolong the half-life of insulin (Kurtzhals, P. et aL, 1995, Biochem. J 312: 725-731) and the sister incretin glucagon-like peptide-1 (GLP-1) (Knudsen, L.B. et al., 2000, J Med.
Chem.
43: 1664-1669; Green, B.D. et aL, 2004, BioL Chem. 385: 169-177; Kim, J.G. et al., 2003, Diabetes 52: 751-759). A number of N-terminally modified GIP analogues have been developed which exhibit profound resistance to DPP IV (Hinke, S.A.
et al., 2002, Diabetes 51: 656-661; Gault, V.A. et al., 2002, Biochem. J 367: 913-920;
Gault, V.A. et aL, 2003, J. EndocrinoL 176: 133-141; O'Harte, F.P.M. et at, 1999, Diabetes 48: 758-765). Several of these, most notably those modified at Tyr1 of GIP
with an addition of an acetyl, glucitol, pyroglutamyl or Fmoc adduct, exhibit enhanced activity at the GIP receptor in vitro (Gault, V.A. et al., 2002, Biochem. J.
367: 913-920; O'Harte, F.P.M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291). As a result of degradation resistance and enhanced cellular activity, these analogues display enhanced and protracted antihyperglycaemic and insulin-releasing activity when administered acutely to animals with obesity-diabetes (Hinke, S.A. et al., 2002, Diabetes 51: 656-661;
Gault, >
V.A. et al., 2002, Biochem. 1 367: 913-920; Gault, V.A. et at., 2003,1 EndocrinoL
176: 133-141; O'Harte, F.P.M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291). Of these, N-AcGIP has emerged as the most effective DPP IV-resistant analogue, substantially augmenting the plasma insulin response and curtailing the glycaemic excursion following conjoint administration with glucose to ob/ob mice (O'Harte, F.P.M. et at., 2002, Diabetologia 45:
1291).
Example 6 was designed to evaluate the metabolic stability, biological activity and antidiabetic potential of novel second generation fatty acid derivatised, N-terminally modified N-AcGIP analogues, N-AcGIP(LysPAL16) and N-AcGIP(LysP AL37). Both GIP analogues contain a C-16 palmitate group linked to the epsilon-amino group of Lys at positions 16 or 37, in combination with an N-terminal (Tyr1) acetyl group (O'Harte, F.P.M. et at., 2002, Diabetologia 45: 1281-1291). The relative stability to DPP IV degradation, insulin secretion and cyclic AMP
properties were examined in vitro together with acute and dose-response studies in obese diabetic ob/ob mice. The most effective analogue, N-AcGIP(LysPAL37) was administered to ob/ob mice by once daily intraperitoneal injection for 14 days prior to evaluation of glucose homeostasis, pancreatic beta cell function and insulin sensitivity. Possible desensitization of GIP receptor action by prolonged exposure to elevated concentrations of N-AcGIP(LysPAL37) was also examined. The results indicate the particular promise of the novel second generation N-terminally acetylated GIP analogue, N-AcGIP(LysPAL37), as a potential therapeutic agent for the treatment of type 2 diabetes.
Despite their many attributes, DPP IV-resistant analogues of GIP and GLP-1, like their native counterparts, are still subject to renal filtration. To circumvent this problem, fatty acid derivatisation has been used to improve the duration of action of GLP-1 (Knudsen, L.B. et al., 2000, 1 Med. Chem. 43: 1664-1669; Green, B.D. et al., 2004, BioL Chem. 385: 169-177; Kim, J.G. et at., 2003, Diabetes 52: 751-759).
The most promising analogue, NN2211 (Liraglutide), appears effective in improving blood glucose control in type 2 diabetic subjects despite a tendency towards promotion of nausea possibly due to slowing of gastric emptying (Agerso, H. et al., 2002, Diabetologia 45: 195-202).
Example 6 describes the results of introducing two specific modifications to the native GIP hormone, namely N-terminal acetylation and C-terminal fatty acid derivatisation. N-terminal acetylation was employed, as previously described (O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291), to significantly enhance stability to DPP IV. In contrast, conjugation of a C-16 palmitate residue at the epsilon-amino group of Lys16 or Lys37 was introduced to extend the biological half-life through binding to circulating proteins (Kurtzhals, P. et al., 1995, Biochem. J.
312: 725-731). Unlike the native peptide, both GIP analogues appeared to be completely resistant to enzymatic breakdown by DPP IV, which corroborates previous observations with N-AcGIP (O'Harte, F.P.M. et al., 2002, Diabetologia 45:
1281-1291). Furthermore, both analogues displayed similar or slightly better insulin-releasing and cyclic AMP generating properties to native GIP and N-AcGIP when tested in the in vitro cellular systems (O'Harte, F.P.M. et al., 2002, Diabetologia 45:
1281-1291).
To assess the antihyperglycaemic and insulinotropic potential of the fatty acid derivatised GIP analogues in vivo, obese diabetic (ob/ob) mice were employed.
The ob/ob syndrome is an extensively studied model of spontaneous obesity and diabetes, exhibiting hyperphagia, marked obesity, moderate hyperglycaemia and severe hyperinsulinemia (Bailey, C.J. et al., 1982, Int. J. Obesity 6: 11-21). As described in previous studies (Gault, V.A. et al., 2002, Biochem. J 367: 913-920; Gault, V.A. et al., 2003, J Endocrinol. 176: 133-141), native GIP only modestly reduced the glycaemic excursion in ob/ob mice reflecting the severe insulin resistance of this mutant animal model (Bailey, C.J. et al., 1982, Int. J Obesity 6: 11-21). In sharp contrast, both N-acetylated GIP analogues additionally substituted with a palmitate molecule at Lys16 or Lys37 (N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)) significantly lowered plasma glucose levels compared to the native peptide.
This was accompanied by significantly enhanced insulin-releasing activity, especially in the case of N-AcGIP(LysPAL37). The significantly protracted insulinotropic response to both fatty acid derivatised GIP analogues at 60 minutes despite substantially lower plasma glucose is indicative of an extended plasma half-life. This may be due to binding of both palmitate derivatised GIP analogues to serum albumin, therefore significantly impairing their clearance via the kidneys (Meier, J.J. et al., 2004, Diabetes 53: 654-662). However, further studies including establishment of sensitive and specific immunoassays for the novel GIP analogues would be needed to confirm such actions.
N-AcGIP(LysPAL37) appeared to be the best fatty acid derivatised analogue displaying a more protracted, significantly enhanced insulin-releasing potency over N-AcGIP(LysP AL16) in vivo. Reasons for the increased potency of N-AcGIP(LysP AL37) remain unclear, but one explanation is an extended half-life.
Another possibility may be that a fatty acid chain linked to the Lys closer to the C-terminus of the peptide may have less of a detrimental effect upon the bioactive region of the molecule known to be located within the N-terminus (Gault, V.A.
et al., 2002, Biosci. Rep. 22: 523-528; Hinke, S.A. et al., 2001, Biochim. Biophys.
Acta 1547: 143-55; Manhart, S. et al., 2003, Biochemist/J.) 42: 3081-3088).
However, similarities between the in vitro biological activities of the two palmitate substituted analogues make this less likely.
Given that N-AcGIP(LysPAL37) was the more potent of the two analogues in vivo, it was further utilised in dose-response studies. Considering that native GIP
itself has only very modest effects in ob/ob mice, as sometimes observed with type 2 diabetic subjects (Nauck, M.A. et al., 1993, J. Clin. Invest. 91: 301-307;
Meier, J.J. et al., 2004, Diabetes 53: 220-224; Vilsboll, T. et al., 2002, Diabetologia 45:
1119), it is remarkable that N-AcGIP(LysPAL37), even at the lowest dose of 6.25 nmoles/kg, exhibited significant glucose-lowering and insulinotropic activity when administered with glucose. Considering N-AcGIP(LysPAL37) is subject to albumin binding, the fact that it is still highly biologically active even at lower concentrations indicates striking potency.
Daily administration of N-AcGIP(LysPAL37) to young adult ob/ob mice by intraperitoneal injection (12.5 nmoles/kg) resulted in a progressive lowering of plasma glucose concentrations and a significant decrease of glycated haemoglobin by 14 days. This was associated with a substantial improvement of glucose tolerance.
Importantly food intake and body weight were unchanged ruling out the possibility that improvement of glucose homeostasis was merely the consequence of body weight loss. These observations also indicate that N-AcGIP(LysPAL37) did not exert any untoward toxic actions affecting feeding over the study period. This is in harmony with recent studies showing that GIP does not inhibit gastric emptying (Meier, J.J. et al., 2003, Am. J PhysioL Endocrinol. Metab. 286: 621-625). Daily administration of native GIP to ob/ob mice for 14 days had no effect on any of the parameters measure, consistent with the very short half-life of the native GIP in vivo.
As expected, a key component of the beneficial action of N-AcGIP(LysPAL37) concerned effects on beta-cells. Thus although native GIP is a weak stimulus to insulin secretion in ob/ob mice at the age studied, plasma and pancreatic insulin concentrations were raised in ob/ob mice receiving the novel fatty acid derivatised analogue. This is consistent with the action of GIP as a promoter of proinsulin gene expression (Wang, Y. et at., 1996, Mot. Cell. EndocrinoL 116:81-87) and exemplifies the increased potency reported for N-terminally modified GIP analogues in this animal model of diabetes (Hinke, S.A. et at., 2002, Diabetes 51: 656-661;
Gault, V.A.
et al., 2002, Biochein. J. 367: 913-920; Gault, V.A. et al., 2003, J.
Endocrinol. 176:
133-141; O'Harte, F.P.M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.P.M.
et at., 2002, Diabetologia 45: 1281-1291). Furthermore, the insulin response to glucose was significantly enhanced in ob/ob mice receiving N-AcGIP(LysPAL37). This ability to augment or restore pancreatic beta cell glucose responsiveness has been similarly observed with GLP-1 (Holz, G.G. et at., 1993, Nature 28: 362-365; Flamez, D.
et at., 1998, Diabetes 47: 646-652). As with observations on glycaemic control, none of these attributes were reproduced by daily injections of native GIP.
Results of insulin sensitivity tests conducted after 14 days treatment indicate that the improvement of diabetic status achieved in ob/ob mice with N-AcGIP(LysP AL37) was not solely due to the potentiation of insulin secretion.
Thus, these animals also exhibited a significant improvement of insulin sensitivity compared to the GIP or saline treated groups. Given that hyperinsulinemia is generally believed to down-regulate insulin receptor function (Marshall, S. et at., 1981, Diabetes 30:
746-753), this suggests that N-AcGIP(LysPAL37) may exert other compensatory effects. Further study is necessary to evaluate this aspect but possibilities include inhibition of counter-regulatory hormones and effects on extrapancreatic sites such as muscle, adipose tissue and liver (Morgan, L.M. et at., 1996, Biochem. Soc.
Trans.
24:585-591; O'Harte, F.P.M. et at., 1998,J. EndocrinoL 156: 237-243; Yip, R.G.
et al., 1998, Endocrinology 139: 4004-4007).
The analogues also stimulated cyclic AMP production and exhibited significantly improved in vitro insulin secretion compared to control. Administration of N-AcGIP(LysP AL16) or N-AcGIP(LysPAL37) together with glucose in ob/ob mice significantly reduced the glycaemic excursion and improved the insulinotropic response compared to GIP. Dose-response studies with N-AcGIP(LysPAL37) revealed highly significant decreases in the overall glycaemic excursion and increases in circulating insulin even with 6.25 nmoles/kg. Once daily injection of ob/ob mice with N-AcGIP(LysPAL37) over 14 days significantly decreased plasma glucose, glycated haemoglobin and improved glucose tolerance compared with saline or native GIP. Plasma and pancreatic insulin were significantly increased, together with a significant enhancement in the insulin response to glucose and a notable improvement of insulin sensitivity. No evidence was found for GIP-receptor desensitization and the metabolic effects of N-AcGIP(LysPAL37) were independent of any change in feeding or body weight.
These results show that novel fatty acid derivatised, N-terminally modified analogues of GIP such as N-AcGIP(LysPAL37), may have significant potential for the treatment of type 2 diabetes.
One approach to counter both renal clearance and enzyme degradation of GIP
concerns the utilisation of fatty acid derivatisation together with N-terminal modification. Fatty acid derivatisation has previously been shown to prolong the half-life of insulin (Kurtzhals, P. et aL, 1995, Biochem. J 312: 725-731) and the sister incretin glucagon-like peptide-1 (GLP-1) (Knudsen, L.B. et al., 2000, J Med.
Chem.
43: 1664-1669; Green, B.D. et aL, 2004, BioL Chem. 385: 169-177; Kim, J.G. et al., 2003, Diabetes 52: 751-759). A number of N-terminally modified GIP analogues have been developed which exhibit profound resistance to DPP IV (Hinke, S.A.
et al., 2002, Diabetes 51: 656-661; Gault, V.A. et al., 2002, Biochem. J 367: 913-920;
Gault, V.A. et aL, 2003, J. EndocrinoL 176: 133-141; O'Harte, F.P.M. et at, 1999, Diabetes 48: 758-765). Several of these, most notably those modified at Tyr1 of GIP
with an addition of an acetyl, glucitol, pyroglutamyl or Fmoc adduct, exhibit enhanced activity at the GIP receptor in vitro (Gault, V.A. et al., 2002, Biochem. J.
367: 913-920; O'Harte, F.P.M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291). As a result of degradation resistance and enhanced cellular activity, these analogues display enhanced and protracted antihyperglycaemic and insulin-releasing activity when administered acutely to animals with obesity-diabetes (Hinke, S.A. et al., 2002, Diabetes 51: 656-661;
Gault, >
V.A. et al., 2002, Biochem. 1 367: 913-920; Gault, V.A. et at., 2003,1 EndocrinoL
176: 133-141; O'Harte, F.P.M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291). Of these, N-AcGIP has emerged as the most effective DPP IV-resistant analogue, substantially augmenting the plasma insulin response and curtailing the glycaemic excursion following conjoint administration with glucose to ob/ob mice (O'Harte, F.P.M. et at., 2002, Diabetologia 45:
1291).
Example 6 was designed to evaluate the metabolic stability, biological activity and antidiabetic potential of novel second generation fatty acid derivatised, N-terminally modified N-AcGIP analogues, N-AcGIP(LysPAL16) and N-AcGIP(LysP AL37). Both GIP analogues contain a C-16 palmitate group linked to the epsilon-amino group of Lys at positions 16 or 37, in combination with an N-terminal (Tyr1) acetyl group (O'Harte, F.P.M. et at., 2002, Diabetologia 45: 1281-1291). The relative stability to DPP IV degradation, insulin secretion and cyclic AMP
properties were examined in vitro together with acute and dose-response studies in obese diabetic ob/ob mice. The most effective analogue, N-AcGIP(LysPAL37) was administered to ob/ob mice by once daily intraperitoneal injection for 14 days prior to evaluation of glucose homeostasis, pancreatic beta cell function and insulin sensitivity. Possible desensitization of GIP receptor action by prolonged exposure to elevated concentrations of N-AcGIP(LysPAL37) was also examined. The results indicate the particular promise of the novel second generation N-terminally acetylated GIP analogue, N-AcGIP(LysPAL37), as a potential therapeutic agent for the treatment of type 2 diabetes.
Despite their many attributes, DPP IV-resistant analogues of GIP and GLP-1, like their native counterparts, are still subject to renal filtration. To circumvent this problem, fatty acid derivatisation has been used to improve the duration of action of GLP-1 (Knudsen, L.B. et al., 2000, 1 Med. Chem. 43: 1664-1669; Green, B.D. et al., 2004, BioL Chem. 385: 169-177; Kim, J.G. et at., 2003, Diabetes 52: 751-759).
The most promising analogue, NN2211 (Liraglutide), appears effective in improving blood glucose control in type 2 diabetic subjects despite a tendency towards promotion of nausea possibly due to slowing of gastric emptying (Agerso, H. et al., 2002, Diabetologia 45: 195-202).
Example 6 describes the results of introducing two specific modifications to the native GIP hormone, namely N-terminal acetylation and C-terminal fatty acid derivatisation. N-terminal acetylation was employed, as previously described (O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291), to significantly enhance stability to DPP IV. In contrast, conjugation of a C-16 palmitate residue at the epsilon-amino group of Lys16 or Lys37 was introduced to extend the biological half-life through binding to circulating proteins (Kurtzhals, P. et al., 1995, Biochem. J.
312: 725-731). Unlike the native peptide, both GIP analogues appeared to be completely resistant to enzymatic breakdown by DPP IV, which corroborates previous observations with N-AcGIP (O'Harte, F.P.M. et al., 2002, Diabetologia 45:
1281-1291). Furthermore, both analogues displayed similar or slightly better insulin-releasing and cyclic AMP generating properties to native GIP and N-AcGIP when tested in the in vitro cellular systems (O'Harte, F.P.M. et al., 2002, Diabetologia 45:
1281-1291).
To assess the antihyperglycaemic and insulinotropic potential of the fatty acid derivatised GIP analogues in vivo, obese diabetic (ob/ob) mice were employed.
The ob/ob syndrome is an extensively studied model of spontaneous obesity and diabetes, exhibiting hyperphagia, marked obesity, moderate hyperglycaemia and severe hyperinsulinemia (Bailey, C.J. et al., 1982, Int. J. Obesity 6: 11-21). As described in previous studies (Gault, V.A. et al., 2002, Biochem. J 367: 913-920; Gault, V.A. et al., 2003, J Endocrinol. 176: 133-141), native GIP only modestly reduced the glycaemic excursion in ob/ob mice reflecting the severe insulin resistance of this mutant animal model (Bailey, C.J. et al., 1982, Int. J Obesity 6: 11-21). In sharp contrast, both N-acetylated GIP analogues additionally substituted with a palmitate molecule at Lys16 or Lys37 (N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37)) significantly lowered plasma glucose levels compared to the native peptide.
This was accompanied by significantly enhanced insulin-releasing activity, especially in the case of N-AcGIP(LysPAL37). The significantly protracted insulinotropic response to both fatty acid derivatised GIP analogues at 60 minutes despite substantially lower plasma glucose is indicative of an extended plasma half-life. This may be due to binding of both palmitate derivatised GIP analogues to serum albumin, therefore significantly impairing their clearance via the kidneys (Meier, J.J. et al., 2004, Diabetes 53: 654-662). However, further studies including establishment of sensitive and specific immunoassays for the novel GIP analogues would be needed to confirm such actions.
N-AcGIP(LysPAL37) appeared to be the best fatty acid derivatised analogue displaying a more protracted, significantly enhanced insulin-releasing potency over N-AcGIP(LysP AL16) in vivo. Reasons for the increased potency of N-AcGIP(LysP AL37) remain unclear, but one explanation is an extended half-life.
Another possibility may be that a fatty acid chain linked to the Lys closer to the C-terminus of the peptide may have less of a detrimental effect upon the bioactive region of the molecule known to be located within the N-terminus (Gault, V.A.
et al., 2002, Biosci. Rep. 22: 523-528; Hinke, S.A. et al., 2001, Biochim. Biophys.
Acta 1547: 143-55; Manhart, S. et al., 2003, Biochemist/J.) 42: 3081-3088).
However, similarities between the in vitro biological activities of the two palmitate substituted analogues make this less likely.
Given that N-AcGIP(LysPAL37) was the more potent of the two analogues in vivo, it was further utilised in dose-response studies. Considering that native GIP
itself has only very modest effects in ob/ob mice, as sometimes observed with type 2 diabetic subjects (Nauck, M.A. et al., 1993, J. Clin. Invest. 91: 301-307;
Meier, J.J. et al., 2004, Diabetes 53: 220-224; Vilsboll, T. et al., 2002, Diabetologia 45:
1119), it is remarkable that N-AcGIP(LysPAL37), even at the lowest dose of 6.25 nmoles/kg, exhibited significant glucose-lowering and insulinotropic activity when administered with glucose. Considering N-AcGIP(LysPAL37) is subject to albumin binding, the fact that it is still highly biologically active even at lower concentrations indicates striking potency.
Daily administration of N-AcGIP(LysPAL37) to young adult ob/ob mice by intraperitoneal injection (12.5 nmoles/kg) resulted in a progressive lowering of plasma glucose concentrations and a significant decrease of glycated haemoglobin by 14 days. This was associated with a substantial improvement of glucose tolerance.
Importantly food intake and body weight were unchanged ruling out the possibility that improvement of glucose homeostasis was merely the consequence of body weight loss. These observations also indicate that N-AcGIP(LysPAL37) did not exert any untoward toxic actions affecting feeding over the study period. This is in harmony with recent studies showing that GIP does not inhibit gastric emptying (Meier, J.J. et al., 2003, Am. J PhysioL Endocrinol. Metab. 286: 621-625). Daily administration of native GIP to ob/ob mice for 14 days had no effect on any of the parameters measure, consistent with the very short half-life of the native GIP in vivo.
As expected, a key component of the beneficial action of N-AcGIP(LysPAL37) concerned effects on beta-cells. Thus although native GIP is a weak stimulus to insulin secretion in ob/ob mice at the age studied, plasma and pancreatic insulin concentrations were raised in ob/ob mice receiving the novel fatty acid derivatised analogue. This is consistent with the action of GIP as a promoter of proinsulin gene expression (Wang, Y. et at., 1996, Mot. Cell. EndocrinoL 116:81-87) and exemplifies the increased potency reported for N-terminally modified GIP analogues in this animal model of diabetes (Hinke, S.A. et at., 2002, Diabetes 51: 656-661;
Gault, V.A.
et al., 2002, Biochein. J. 367: 913-920; Gault, V.A. et al., 2003, J.
Endocrinol. 176:
133-141; O'Harte, F.P.M. et al., 1999, Diabetes 48: 758-765; O'Harte, F.P.M.
et at., 2002, Diabetologia 45: 1281-1291). Furthermore, the insulin response to glucose was significantly enhanced in ob/ob mice receiving N-AcGIP(LysPAL37). This ability to augment or restore pancreatic beta cell glucose responsiveness has been similarly observed with GLP-1 (Holz, G.G. et at., 1993, Nature 28: 362-365; Flamez, D.
et at., 1998, Diabetes 47: 646-652). As with observations on glycaemic control, none of these attributes were reproduced by daily injections of native GIP.
Results of insulin sensitivity tests conducted after 14 days treatment indicate that the improvement of diabetic status achieved in ob/ob mice with N-AcGIP(LysP AL37) was not solely due to the potentiation of insulin secretion.
Thus, these animals also exhibited a significant improvement of insulin sensitivity compared to the GIP or saline treated groups. Given that hyperinsulinemia is generally believed to down-regulate insulin receptor function (Marshall, S. et at., 1981, Diabetes 30:
746-753), this suggests that N-AcGIP(LysPAL37) may exert other compensatory effects. Further study is necessary to evaluate this aspect but possibilities include inhibition of counter-regulatory hormones and effects on extrapancreatic sites such as muscle, adipose tissue and liver (Morgan, L.M. et at., 1996, Biochem. Soc.
Trans.
24:585-591; O'Harte, F.P.M. et at., 1998,J. EndocrinoL 156: 237-243; Yip, R.G.
et al., 1998, Endocrinology 139: 4004-4007).
Irrespective of knowledge of the full range of actions contributing to the antihyperglycaemic effect of N-AcGIP(Ly5PAL37), a currently envisaged problem of long-term treatment with stable analogues of GIP or GLP-1 concerns desensitization of hormone receptor action (Delmeire, D. et al., 2004, Biochem. PharmacoL 68:
39). Although this has been observed during prolonged exposure of pancreatic beta cells to GIP in rats (Tseng, C.C. et al., 1996, Am. j. PhysioL 270: E661-E666), there was no evidence that treatment with N-AcGIP(LysPAL37) for 14 days compromised the glucose lowering or insulin releasing actions of N-AcGIP(LysPAL37). Thus the antidiabetic actions of N-AcGIP(LysPAL37) were clearly evident when the analogue was administered acutely together with glucose. Furthermore, the acute effects of N-AcGIP(LysP AL37) in such experiments were identical in groups of ob/ob mice receiving either N-AcGIP(LysPAL37), native GIP or saline injections for 14 days.
Such data clearly indicate that prolonged exposure to N-AcGIP(LysPAL37) does not induce and possibly overcomes inherent GIP receptor desensitization in ob/ob mice. Given the high circulating concentrations of GIP in these obese-diabetic rodents (Flat, P.R. et al., 1983, Diabetes 32: 433-435; Flatt, P.R. et al., 1984, J.
Endocrinol 101: 249-256), it is tempting to link beta cell refractoriness to GIP
evident in ob/ob mice to simple receptor desensitization at the hands of inappropriate secretion and metabolism of GIP.
The data shown herein demonstrate that N-terminally acetylated GIP carrying a palmitate group linked to Lys at position 37 displays resistance to DPP IV
and an impressive profile of bioactivity manifested by potent and long-acting glucose-lowering activity in a commonly employed animal model of obesity-diabetes.
This activity profile provides strong encouragement for the development of long-acting fatty acid derivatised N-terminally modified analogues of GIP for the once-daily treatment of type 2 diabetes.
The peptide analogues of the present invention have use in treating diseases and conditions caused by improper modulation of insulin levels, including diabetes, type 2 diabetes, insulin resistance, insulin resistant metabolic syndrome (Syndrome X), and obesity.
A peptide analogue produced by the methods of the present invention can be used in a pharmaceutical composition, wherein the analogue is combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to the analogue and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
Administration of the peptide analogue of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as by oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Administration can be internal or external; or local, topical or systemic.
The compositions containing a peptide analogue of this invention can be administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
When a therapeutically effective amount of the composition of the present invention is administered orally, the composition of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the composition of the present invention, and preferably from about 1 to 50% of the composition of the present invention.
When a therapeutically effective amount of the composition of the present invention is administered by intravenous, cutaneous or subcutaneous injection, the composition of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the composition of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
Use of timed release or sustained release delivery systems are also included.
A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).
The therapeutic compositions can include pharmaceutically acceptable salts of the components therein, e.g., which may be derived from inorganic or organic acids.
By "pharmaceutically acceptable salt" is meant those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, S. M. Berge, et al., describe pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences (1977) 66:1 et seq. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as,isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable organic acid.
Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptonoate, hexanoate, fumarate, hydrochloride, 'nydrobromide, hyciroiodide, 2-hydroxymethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quatemized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; diallcyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.
As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH
buffering agents and the like which enhance the effectiveness of the active ingredient.
The amount of peptide analogue of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, on the nature of prior treatments which the patient has undergone, and on a variety of other factors, including the type of injury, the age, weight, sex, medical condition of the individual. Ultimately, the attending physician will decide the amount of the analogue with which to treat each individual patient.
Initially, the attending physician will administer low doses of peptide analogue and observe the patient's response. Larger doses of peptide analogue may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
Additional guidance on methods of determining dosages can be found in standard references, for example, Spilker, Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, 1984, pp. 7-13 and 54-60;
Spilker, Guide to Clinical Trials, Raven Press, Ltd., New York, 1991, pp. 93-101; Craig et al., Modern Pharmacology, 2d ed., Little Brown and Co., Boston, 1986, pp. 127-133;
Speight, Aveiy's Drug Treatment: Principles and Practices of Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins, Baltimore, 1987, pp.
50-56; Tallarida et al., Principles in General Pharmacology, Springer-Verlag, New York, 1998, pp. 18-20; and Olson, Clinical Pharmacology Made Ridiculously Simple, MedMaster, Inc., Miami, 1993, pp. 1-5.
EXAMPLES
Example 1. Preparation of N-terminally modified GIP and analogues thereof.
The N-terminal modification of GIP is essentially a three step process.
Firstly, GIP is synthesized from its C-terminal (starting from a Fmoc-Gln (Trt)-Wang resin (Calbiochem Novabiochem, Beeston, Nottingham, UK) up to the penultimate N-terminal amino-acid (Ala2) on an automated peptide synthesizer (Applied Biosystems, California, USA). The synthesis follows standard Fmoc peptide chemistry protocols.
Secondly, the N-terminal amino acid of native GIP (Tyr) is added to a manual bubbler system as a Fmoc-protected Tyr(tBu)-Wang resin. This amino acid is deprotected at its N-terminus (piperidine in DMF (20% v/v)) and allowed to react with a high concentration of glucose (glycation, under reducing conditions with sodium cyanoborohydride), acetic anhydride (acetylation), pyroglutamic acid (pyroglutamyl) etc. for up to 24 hours as necessary to allow the reaction to go to completion. The completeness of reaction is monitored using the ninhydrin test which determines the presence of available free a-amino groups. Thirdly (once the reaction is complete), the now structurally modified Tyr is cleaved from the Wang resin (95% TFA, and 5%
of the appropriate scavengers. N.B. Tyr is considered to be a problematic amino acid and may need special consideration) and the required amount of N-terminally modified-Tyr consequently added directly to the automated peptide synthesiser, which will carry on the synthesis, thereby stitching the N-terminally modified-Tyr to the a-amino of GIP (Ala2), completing the synthesis of the GIP analogue.. This peptide is cleaved off the Wang resin (as above) and then worked up using the standard Buchner filtering, precipation, rotary evaporation and drying techniques.
Example 2. Preparation of Tyr'-Glucitol GIP and Its Properties in vivo.
The following example investigates preparation of Tyriglucitol GIP together with evaluation of its antihyperglycemie and insulin-releasing properties in vivo. The results clearly demonstrate that this novel GIP analogue exhibits a substantial resistance to aminopeptidase degradation and increased glucose lowering activity compared with the native GIP.
Research Design and Methods Materials. Human GIP was purchased from the American Peptide Company (Sunnyvale, California, USA). HPLC grade acetonitrile was obtained from Rathburn (Walkersbum, Scotland). Sequencing grade trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset, UK). All other chemicals purchased including dextran T-70, activated charcoal, sodium cyanoborohydride and bovine serum albumin fraction V were from Sigma (Poole, Dorset, UK). Diprotin A (DPA) was purchased from Calbiochem-Novabiochem (UK) Ltd. (Beeston, Nottingham, UK) and rat insulin standard for RIA was obtained from Novo Industria (Copenhagen, Denmark).
Reversed-phase Sep-Pak cartridges (C-18) were purchased from Millipore-Waters (Milford, MA, USA). All water used in these experiments was purified using a Milli-WO, Water Purification System (Millipore Corporation, Milford, Massachusetts, USA).
Preparation of Tyr' -glucitol GIP. Human GIP was incubated with glucose under reducing conditions in 10 mmoi/1 sodium phosphate buffer at pH 7.4 for 24 hours.
The reaction was stopped by addition of 0.5 mo1/1 acetic acid (30 pl) and the mixture applied to a Vydac (C18)(4.6 x 250mm) analytical HPLC column (The Separations Group, Hesperia, California, USA) and gradient elution conditions were established using aqueous/TFA and acetonitrile/TFA solvents. Fractions corresponding to the glycated peaks were pooled, taken to dryness under vacuum using an ABS 1000 Speed-Vac concentrator (Life Sciences International, Runcorn, UK) and purified to _ homogeneity on a Supelcosil (C-8) (4.6 x 150mm) column (Supelco Inc., Poole, Dorset, UK).
Degradation of GIP and Tyri -glucitol GIP by DPP 1V. HPLC-purified GIP or Tyr1-glucitol GIP were incubated at 37 C with DPP-IV (5mU) for various time periods in a reaction mixture made up to 5001.11 with 50 mmo1/1 triethanolamine-HC1, pH 7.8 (final peptide concentration 1 umo1/1). Enzymatic reactions were terminated after 0, 2, 4 and 12 hours by addition of 5 1 of 10% (v/v) TFA/water. Samples were made up to a final volume of 1.0 ml with 0.12% (v/v) TFA and stored at -20 C prior to HPLC
analysis.
Degradation of GIP and Tyr) -glucitol GIP by human plasma. Pooled human plasma (20 I) taken from six healthy fasted human subjects was incubated at 37 C
with GIP
or Tyrl-glucitol GIP (10 lig) for 0 and 4 hours in a reaction mixture made up to 500 p1, containing 50 mmol/ltriethanolamine/HCL buffer pH 7.8. Incubations for 4 hours were also performed in the presence of diprotin A (5 mU). The reactions were terminated by addition of 5 pi of TFA and the final volume adjusted to 1.0 ml using 0.1% v/v TFA/water. Samples were centrifuged (13,000g, 5 minutes) and the supernatant applied to a C-18 Sep-Pak cartridge (Millipore-Waters ) which was previously primed and washed with 0.1% (v/v) TFA/water. After washing with 20 ml 0.12% TFA/water, bound material was released by elution with 2 ml of 80% (v/v) acetonitrile/water and concentrated using a Speed-Vac concentrator (Runcorn, UK).
The volume was adjusted to 1.0m1 with 0.12% (v/v) TFA/water prior to HPLC
purification.
39). Although this has been observed during prolonged exposure of pancreatic beta cells to GIP in rats (Tseng, C.C. et al., 1996, Am. j. PhysioL 270: E661-E666), there was no evidence that treatment with N-AcGIP(LysPAL37) for 14 days compromised the glucose lowering or insulin releasing actions of N-AcGIP(LysPAL37). Thus the antidiabetic actions of N-AcGIP(LysPAL37) were clearly evident when the analogue was administered acutely together with glucose. Furthermore, the acute effects of N-AcGIP(LysP AL37) in such experiments were identical in groups of ob/ob mice receiving either N-AcGIP(LysPAL37), native GIP or saline injections for 14 days.
Such data clearly indicate that prolonged exposure to N-AcGIP(LysPAL37) does not induce and possibly overcomes inherent GIP receptor desensitization in ob/ob mice. Given the high circulating concentrations of GIP in these obese-diabetic rodents (Flat, P.R. et al., 1983, Diabetes 32: 433-435; Flatt, P.R. et al., 1984, J.
Endocrinol 101: 249-256), it is tempting to link beta cell refractoriness to GIP
evident in ob/ob mice to simple receptor desensitization at the hands of inappropriate secretion and metabolism of GIP.
The data shown herein demonstrate that N-terminally acetylated GIP carrying a palmitate group linked to Lys at position 37 displays resistance to DPP IV
and an impressive profile of bioactivity manifested by potent and long-acting glucose-lowering activity in a commonly employed animal model of obesity-diabetes.
This activity profile provides strong encouragement for the development of long-acting fatty acid derivatised N-terminally modified analogues of GIP for the once-daily treatment of type 2 diabetes.
The peptide analogues of the present invention have use in treating diseases and conditions caused by improper modulation of insulin levels, including diabetes, type 2 diabetes, insulin resistance, insulin resistant metabolic syndrome (Syndrome X), and obesity.
A peptide analogue produced by the methods of the present invention can be used in a pharmaceutical composition, wherein the analogue is combined with a pharmaceutically acceptable carrier. Such a composition may also contain (in addition to the analogue and a carrier) diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials well known in the art. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredient(s). The characteristics of the carrier will depend on the route of administration.
Administration of the peptide analogue of the present invention used in the pharmaceutical composition or to practice the method of the present invention can be carried out in a variety of conventional ways, such as by oral ingestion, inhalation, topical application or cutaneous, subcutaneous, intraperitoneal, parenteral or intravenous injection. Administration can be internal or external; or local, topical or systemic.
The compositions containing a peptide analogue of this invention can be administered intravenously, as by injection of a unit dose, for example. The term "unit dose" when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for the subject, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent, i.e., carrier or vehicle.
Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
When a therapeutically effective amount of the composition of the present invention is administered orally, the composition of the present invention will be in the form of a tablet, capsule, powder, solution or elixir. When administered in tablet form, the pharmaceutical composition of the invention may additionally contain a solid carrier such as a gelatin or an adjuvant. The tablet, capsule, and powder contain from about 5 to 95% protein of the present invention, and preferably from about 25 to 90% protein of the present invention. When administered in liquid form, a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, or sesame oil, or synthetic oils may be added. The liquid form of the pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol. When administered in liquid form, the pharmaceutical composition contains from about 0.5 to 90% by weight of the composition of the present invention, and preferably from about 1 to 50% of the composition of the present invention.
When a therapeutically effective amount of the composition of the present invention is administered by intravenous, cutaneous or subcutaneous injection, the composition of the present invention will be in the form of a pyrogen-free, parenterally acceptable aqueous solution. The preparation of such parenterally acceptable protein solutions, having due regard to pH, isotonicity, stability, and the like, is within the skill in the art. A preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the composition of the present invention, an isotonic vehicle such as Sodium Chloride Injection, Ringer's Injection, Dextrose Injection, Dextrose and Sodium Chloride Injection, Lactated Ringer's Injection, or other vehicle as known in the art.
The pharmaceutical composition of the present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
Use of timed release or sustained release delivery systems are also included.
A sustained-release matrix, as used herein, is a matrix made of materials, usually polymers, which are degradable by enzymatic or acid/base hydrolysis or by dissolution. Once inserted into the body, the matrix is acted upon by enzymes and body fluids. The sustained-release matrix desirably is chosen from biocompatible materials such as liposomes, polylactides (polylactic acid), polyglycolide (polymer of glycolic acid), polylactide co-glycolide (co-polymers of lactic acid and glycolic acid) polyanhydrides, poly(ortho)esters, polyproteins, hyaluronic acid, collagen, chondroitin sulfate, carboxylic acids, fatty acids, phospholipids, polysaccharides, nucleic acids, polyamino acids, amino acids such as phenylalanine, tyrosine, isoleucine, polynucleotides, polyvinyl propylene, polyvinylpyrrolidone and silicone.
A preferred biodegradable matrix is a matrix of one of either polylactide, polyglycolide, or polylactide co-glycolide (co-polymers of lactic acid and glycolic acid).
The therapeutic compositions can include pharmaceutically acceptable salts of the components therein, e.g., which may be derived from inorganic or organic acids.
By "pharmaceutically acceptable salt" is meant those salts which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well-known in the art. For example, S. M. Berge, et al., describe pharmaceutically acceptable salts in detail in J Pharmaceutical Sciences (1977) 66:1 et seq. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as,isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine and the like. The salts may be prepared in situ during the final isolation and purification of the compounds of the invention or separately by reacting a free base function with a suitable organic acid.
Representative acid addition salts include, but are not limited to acetate, adipate, alginate, citrate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, camphorate, camphorsufonate, digluconate, glycerophosphate, hemisulfate, heptonoate, hexanoate, fumarate, hydrochloride, 'nydrobromide, hyciroiodide, 2-hydroxymethanesulfonate (isethionate), lactate, maleate, methanesulfonate, nicotinate, 2-naphthalenesulfonate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartate, thiocyanate, phosphate, glutamate, bicarbonate, p-toluenesulfonate and undecanoate. Also, the basic nitrogen-containing groups can be quatemized with such agents as lower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides, bromides and iodides; diallcyl sulfates like dimethyl, diethyl, dibutyl, and diamyl sulfates; long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; arylalkyl halides like benzyl and phenethyl bromides and others. Water or oil-soluble or dispersible products are thereby obtained. Examples of acids which may be employed to form pharmaceutically acceptable acid addition salts include such inorganic acids as hydrochloric acid, hydrobromic acid, sulphuric acid and phosphoric acid and such organic acids as oxalic acid, maleic acid, succinic acid and citric acid.
As used herein, the terms "pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a mammal with a minimum of undesirable physiological effects such as nausea, dizziness, gastric upset and the like.
The preparation of a pharmacological composition that contains active ingredients dissolved or dispersed therein is well understood in the art and need not be limited based on formulation. Typically such compositions are prepared as injectables either as liquid solutions or suspensions, however, solid forms suitable for solution, or suspensions, in liquid prior to use can also be prepared. The preparation can also be emulsified.
The active ingredient can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient and in amounts suitable for use in the therapeutic methods described herein. Suitable excipients include, for example, water, saline, dextrose, glycerol, ethanol or the like and combinations thereof. In addition, if desired, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH
buffering agents and the like which enhance the effectiveness of the active ingredient.
The amount of peptide analogue of the present invention in the pharmaceutical composition of the present invention will depend upon the nature and severity of the condition being treated, on the nature of prior treatments which the patient has undergone, and on a variety of other factors, including the type of injury, the age, weight, sex, medical condition of the individual. Ultimately, the attending physician will decide the amount of the analogue with which to treat each individual patient.
Initially, the attending physician will administer low doses of peptide analogue and observe the patient's response. Larger doses of peptide analogue may be administered until the optimal therapeutic effect is obtained for the patient, and at that point the dosage is not increased further.
Additional guidance on methods of determining dosages can be found in standard references, for example, Spilker, Guide to Clinical Studies and Developing Protocols, Raven Press Books, Ltd., New York, 1984, pp. 7-13 and 54-60;
Spilker, Guide to Clinical Trials, Raven Press, Ltd., New York, 1991, pp. 93-101; Craig et al., Modern Pharmacology, 2d ed., Little Brown and Co., Boston, 1986, pp. 127-133;
Speight, Aveiy's Drug Treatment: Principles and Practices of Clinical Pharmacology and Therapeutics, 3d ed., Williams and Wilkins, Baltimore, 1987, pp.
50-56; Tallarida et al., Principles in General Pharmacology, Springer-Verlag, New York, 1998, pp. 18-20; and Olson, Clinical Pharmacology Made Ridiculously Simple, MedMaster, Inc., Miami, 1993, pp. 1-5.
EXAMPLES
Example 1. Preparation of N-terminally modified GIP and analogues thereof.
The N-terminal modification of GIP is essentially a three step process.
Firstly, GIP is synthesized from its C-terminal (starting from a Fmoc-Gln (Trt)-Wang resin (Calbiochem Novabiochem, Beeston, Nottingham, UK) up to the penultimate N-terminal amino-acid (Ala2) on an automated peptide synthesizer (Applied Biosystems, California, USA). The synthesis follows standard Fmoc peptide chemistry protocols.
Secondly, the N-terminal amino acid of native GIP (Tyr) is added to a manual bubbler system as a Fmoc-protected Tyr(tBu)-Wang resin. This amino acid is deprotected at its N-terminus (piperidine in DMF (20% v/v)) and allowed to react with a high concentration of glucose (glycation, under reducing conditions with sodium cyanoborohydride), acetic anhydride (acetylation), pyroglutamic acid (pyroglutamyl) etc. for up to 24 hours as necessary to allow the reaction to go to completion. The completeness of reaction is monitored using the ninhydrin test which determines the presence of available free a-amino groups. Thirdly (once the reaction is complete), the now structurally modified Tyr is cleaved from the Wang resin (95% TFA, and 5%
of the appropriate scavengers. N.B. Tyr is considered to be a problematic amino acid and may need special consideration) and the required amount of N-terminally modified-Tyr consequently added directly to the automated peptide synthesiser, which will carry on the synthesis, thereby stitching the N-terminally modified-Tyr to the a-amino of GIP (Ala2), completing the synthesis of the GIP analogue.. This peptide is cleaved off the Wang resin (as above) and then worked up using the standard Buchner filtering, precipation, rotary evaporation and drying techniques.
Example 2. Preparation of Tyr'-Glucitol GIP and Its Properties in vivo.
The following example investigates preparation of Tyriglucitol GIP together with evaluation of its antihyperglycemie and insulin-releasing properties in vivo. The results clearly demonstrate that this novel GIP analogue exhibits a substantial resistance to aminopeptidase degradation and increased glucose lowering activity compared with the native GIP.
Research Design and Methods Materials. Human GIP was purchased from the American Peptide Company (Sunnyvale, California, USA). HPLC grade acetonitrile was obtained from Rathburn (Walkersbum, Scotland). Sequencing grade trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset, UK). All other chemicals purchased including dextran T-70, activated charcoal, sodium cyanoborohydride and bovine serum albumin fraction V were from Sigma (Poole, Dorset, UK). Diprotin A (DPA) was purchased from Calbiochem-Novabiochem (UK) Ltd. (Beeston, Nottingham, UK) and rat insulin standard for RIA was obtained from Novo Industria (Copenhagen, Denmark).
Reversed-phase Sep-Pak cartridges (C-18) were purchased from Millipore-Waters (Milford, MA, USA). All water used in these experiments was purified using a Milli-WO, Water Purification System (Millipore Corporation, Milford, Massachusetts, USA).
Preparation of Tyr' -glucitol GIP. Human GIP was incubated with glucose under reducing conditions in 10 mmoi/1 sodium phosphate buffer at pH 7.4 for 24 hours.
The reaction was stopped by addition of 0.5 mo1/1 acetic acid (30 pl) and the mixture applied to a Vydac (C18)(4.6 x 250mm) analytical HPLC column (The Separations Group, Hesperia, California, USA) and gradient elution conditions were established using aqueous/TFA and acetonitrile/TFA solvents. Fractions corresponding to the glycated peaks were pooled, taken to dryness under vacuum using an ABS 1000 Speed-Vac concentrator (Life Sciences International, Runcorn, UK) and purified to _ homogeneity on a Supelcosil (C-8) (4.6 x 150mm) column (Supelco Inc., Poole, Dorset, UK).
Degradation of GIP and Tyri -glucitol GIP by DPP 1V. HPLC-purified GIP or Tyr1-glucitol GIP were incubated at 37 C with DPP-IV (5mU) for various time periods in a reaction mixture made up to 5001.11 with 50 mmo1/1 triethanolamine-HC1, pH 7.8 (final peptide concentration 1 umo1/1). Enzymatic reactions were terminated after 0, 2, 4 and 12 hours by addition of 5 1 of 10% (v/v) TFA/water. Samples were made up to a final volume of 1.0 ml with 0.12% (v/v) TFA and stored at -20 C prior to HPLC
analysis.
Degradation of GIP and Tyr) -glucitol GIP by human plasma. Pooled human plasma (20 I) taken from six healthy fasted human subjects was incubated at 37 C
with GIP
or Tyrl-glucitol GIP (10 lig) for 0 and 4 hours in a reaction mixture made up to 500 p1, containing 50 mmol/ltriethanolamine/HCL buffer pH 7.8. Incubations for 4 hours were also performed in the presence of diprotin A (5 mU). The reactions were terminated by addition of 5 pi of TFA and the final volume adjusted to 1.0 ml using 0.1% v/v TFA/water. Samples were centrifuged (13,000g, 5 minutes) and the supernatant applied to a C-18 Sep-Pak cartridge (Millipore-Waters ) which was previously primed and washed with 0.1% (v/v) TFA/water. After washing with 20 ml 0.12% TFA/water, bound material was released by elution with 2 ml of 80% (v/v) acetonitrile/water and concentrated using a Speed-Vac concentrator (Runcorn, UK).
The volume was adjusted to 1.0m1 with 0.12% (v/v) TFA/water prior to HPLC
purification.
HPLC analysis of degraded GIP and Tyr -glucitol GIP. Samples were applied to a Vydac C-18 widepore column equilibriated with 0.12% (v/v) TFA/H20 at a flow rate of 1.0 ml/minute. Using 0.1% (v/v) TFA in 70% acetonitrile/H20, the concentration of acetonitrile in the eluting solvent was raised from 0% to 31.5% over 15 min, to 38.5% over 30 minutes and from 38.5% to 70% over 5 minutes, using linear gradients. The absorbance was monitored at 206 nm and peak areas evaluated using a model 2221 LKB integrator. Samples recovered manually were concentrated using a Speed-Vac concentrator.
Electrospray ionization mass spectrometry (ESI-MS). Samples for ESI-MS
analysis containing intact and degradation fragments of GIP (from DPP IV and plasma incubations) as well as Tyr'-glucitol GIP, were further purified by HPLC.
Peptides were dissolved (approximately 400 pmol) in 100 ill of water and applied to the LCQ
benchtop mass spectrometer (Finnigan MAT, Hemel Hempstead, UK) equipped with a microhore C-18 HPLC column (150 x 2.0mm, Phenomenex , Ltd., Macclesfield, UK). Samples (300 direct loop injection) were injected at a flow rate of 0.2m1/min, under isocratic conditions 35% (v/v) acetonitile/water. Mass spectra were obtained from the quadripole ion trap mass analyzer and recorded. Spectra were collected using full ion scan mode over the mass-to-charge (rn/z) range 150-2000. The molecular masses of GIP and related structures were determined from ESI-MS
profiles using prominent multiple charged ions and the following equation MT = iMi -where Mr = molecular mass; Mi= m/z ratio; i = number of charges; Mh = mass of a proton.
In vivo biological activity of GIP and Tyr -glucitoi GIP. Effects of GIP and Tyri-glucitol GIP on plasma glucose and insulin concentrations were examined using 12 week old male Wistar rats. The animals were housed individually in an air conditioned room and 22 2 C with a 12 hour light/12 hour dark cycle. Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Belfast, Northern Ireland) were supplied ad libitum. Food was withdrawn for an 18 hour period prior to intraperitoneal injection of glucose alone (18mmol/kq body weight) or in combination with either GIP or Tyri-glucitol GIP (10 nmol/kg). Test solutions were administered in a final volume of 8 ml/kg body weight. Blood samples were collected at 0, 15, 30 and 60 minutes from the cut tip of the tail of conscious rats into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Samples were centrifuged using a Beckman microcentrifuge for about 30 seconds at 13,000 g.
Plasma samples were aliquoted and stored at -20 C prior to glucose and insulin determinations. All animal studies were done in accordance with the Animals (Scientific Procedures) Act 1986.
Analyses. Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman Glucose Analyzer II [33]. Plasma insulin was determined by dextran charcoal radioimmunoassay as described previously [34]. Incremental areas under plasma glucose and insulin area under the curve (AUC) were calculated using a computer program (CAREA) employing the trapezoidal rule [35] with baseline subtraction. Results are expressed as mean SEM and values were compared using the Student's unpaired t-test. Groups of data were considered to be significantly different if P<0.05.
Degradation of GIP and Tyr' -glueitol GIP by DPP IV. Fig. 1 illustrates the typical peak profiles obtained from the HPLC separation of the products obtained from the incubation of GIP (Fig la) or Tyrl-glucitol GIP (Fig lb) with DPP IV for 0, 2, 4 and 12 hours. The retention times of GIP and Tyr'-glucitol GIP at t=0 were 21.93 minutes and 21.75 minutes respectively. Degradation of GIP was evident after 4 hours incubation (54% intact), and by 12 hours the majority (60%) of intact GIP was converted to the single product with a retention time of 21.61 minutes. Tyrl-glucitol GIP remained almost completely intact throughout 2-12 hours incubation.
Separation was on a Vydac C-18 column using linear gradients of 0% to 31.5% acetonitrile over 15 minutes, to 38.5% over 30 minutes and from 38.5 to 70% acetonitrile over 5 minutes.
Degradation of GIP and Tyr' -glucitol GIP by human plasma. Fig. 2 shows a set of typical HPLC profiles of the products obtained from the incubation of GIP or Tyri-glucitol GIP with human plasma for 0 and 4 hours. GIP (Fig 2a) with a retention time of 22.06 minutes was readily metabolised by plasma within 4 hours incubation giving rise to the appearance of a major degradation peak with a retention time of 21.74 minutes. In contrast, the incubation of Tyr'-glucitol GIP under similar conditions (Fig 2b) did not result in the formation of any detectable degradation fragments during this time with only a single peak being observed with a retention time of 21.77 minutes.
Addition of diprotin A, a specific inhibitor of DPP IV, to GIP during the 4 hours incubation completely inhibited degradation of the peptide by plasma. Peaks corresponding with intact GIP, GIP (3-42) and Tyr'-glucitol GIP are indicated.
A
major peak corresponding to the specific DPP IV inhibitor tripeptide DPA
appears in the bottom peanels with retention time of 16-29 minutes.
Identification of GIP degradation fragments by ESI-MS. Fig. 3 shows the monoisotopic molecular masses obtained for GIP (Fig. 3A), Tyr'-glucitol GIP
(Fig.
3B) and the major plasma degradation fragment of GIP (Fig. 3C) using ESI-MS.
The peptides analyzed were purified from plasma incubations as shown in Fig. 2.
Peptides were dissolved (approximately 400 pmol) in 100 1 of water and applied to the LC/MS
equipped with a microbore C-18 HPLC column. Samples (300 direct loop injection) were applied at a flow rate of 0.2m1/min, under isocratic conditions 35%
acetonitrile/water. Mass spectra were recorded using a quadripole ion trap mass analyzer. Spectra were collected using full ion scan mode over the mass-to-charge (m/z) range 150-2000. The molecular masses (Mr) of GIP and related structures were determined from ESI-MS profiles using prominent multiple charged ions and the following equation Mr = iM, - iMh. The exact molecular mass (Mr) of the peptides were calculated using the equation Mr = iMi - iMh as defined above in Research Design and Methods. After spectral averaging was performed, prominent multiple charges species (M+3H )3+ and (M+4H )4+ were detected from GIP at m/z 1661.6 and 1246.8, corresponding to intact Mr 4981.8 and 4983.2 Da, respectively (Fig.
3A).
Similarly, for Tyrl-glucitol GIP ( (M+4H )4+ and (M+5H)5+) were detected at m/z 1287.7 and 1030.3, corresponding to intact molecular masses of Mr 5146.8 and 5146.5 Da, respectively (Fig. 3B). The difference between the observed molecular masses of the quadruply charged GIP and the N-terminally modified GIP species (163.6 Da) indicated that the latter peptide contained a single glucitol adduct corresponding to Tyri-glucitol GIP. Fig. 3C shows the prominent multiply charged species (M+3H )3+ and (M+4H)4+ detected from the major fragment of GIP at m/z 1583.8 and 1188.1, corresponding to intact Mr 4748.4 and 4748 Da, respectively (Fig.
3C). This corresponds with the theoretical mass of the N-terminally truncated form of the peptide GIP(3-42). This fragment was also the major degradation product of DPP
IV incubations (data not shown).
Effects of GIP and Tyr' -glucitol GIP on plasma glucose homeostasis. Figs. 4 and 5 show the effects of intraperitoneal (ip) glucose alone (18mmol/kg) (control group), and glucose in combination With GIP or Tyri-glucitol GIP (10nmol/kg) on plasma glucose and insulin concentrations.
Fig. 4A shows plasma glucose concentrations after i.p. glucose alone (18mmol/kg) (control group), or glucose in combination with either GIP of Tyrl-glucitol GIP (10nmol/kg). The time of injection is indicated by the arrow (0 minutes).
Fig. 4B shows plasma 'glucose AUC values for 0-60 minutes post injection.
Values are mean SEM for six rats. "P<0.01, ***P<0.001 compared with GIP and Tyr'"
glucitol GIP; TP<0.05, 11/3<0.01 compared with non-glucated GIP. Fig. 5A shows plasma insulin concentrates after i.p. glucose along (18 mmol/kg) (control group), or glucose in combination with either with GIP or glycated GIP (10nmol/kq). The time of injection is indicated by the arrow. Fig. 5B shows plasma insulin AUC
values were calculated for each of the 3 groups up to 90 minutes post injection. The time of injection is indicated by the arrow (0 minutes). Plasma insulin AUC values for minutes post injection. Values are mean SEM for six rats. *P<0.05, "P<0.001 compared with GIP and Tyri-glucitol GIP; fP<0.05, P<0.01 compared with non-glycated GIP.
Compared with the control group, plasma glucose concentrations and area under the curve (AUC) were significantly lower following administration of either GIP or Tyrl-glucitol GIP (Figs 4A, B). Furthermore, individual values at 15 and 30 minutes together with AUC were significantly lower following administration of Tyrl-glucitol GIP as compared to GIP. Consistent with the established insulin-releasing properties of GIP, plasma insulin concentrations of both peptide-treated groups were significantly raised at 15 and 30 minutes compared with the values after administration of glucose alone (Fig. 5A). The overall insulin responses, estimated as AUC were also significantly greater for the two peptide-treated groups (Fig.
5B).
Despite lower prevailing glucose concentrations than the GIP-treated group, plasma insulin response, calculated as AUC, following Tyrl-glucitol GIP was significantly greater than after GIP (Fig. 5B). The significant elevation of plasma insulin at 30 minutes is of particular note, suggesting that the insulin-releasing action of Tyrl-glucitol GIP is more protracted than GIP even in the face of a diminished glycemic stimulus (Figs. 4A, 5A).
Example 3. Additional N-Terminal Structural Modifications of GIP.
This example further looked at the ability of additional N-terminal structural modifications of GIP in preventing inactivation by DPP and in plasma and their associated increase in both the insulin-releasing potency and potential therapeutic value. Native human GIP, glycated GIP, acetylated GIP and a number of GIP
analogues with N-terminal amino acid substitutions were tested.
Materials and Methods. High-performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset, UK).
Dipeptidyl peptidase IV was purchased from Sigma (Poole, Dorset, UK), and Diprotin A was purchased from Calbiochem Novabiochem (Beeston, Nottingham, UK). RPM' 1640 tissue culture medium, foetal calf serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). All water used in these experiments was purified using a Water Purification System (Millipore, Milford, Massachusetts, USA). All other chemicals used were of the highest purity available.
Synthesis of GIP and N-terminally modified GIP analogues. GIP, GIP(Abu2), GIP(Sku2), GIP(Ser2), GIP(G1y2) and GIP(Pro3) were sequentially synthesized on an Applied Biosystems automated peptide synthesizer (model 432A) using standard solid-phase Fmoc procedure, starting with an Fmoc-Gln-Wang resin. Following cleavage from the resin by trifluoroacetic acid: water, thioanisole, ethanedithiol (90/2.5/5/2.5, a total volume of 20 ml/g resin), the resin was removed by filtration and the filtrate volume was decreased under reduced pressure. Dry diethyl ether was slowly added until a precipitate was observed. The precipitate was collected by low-speed centrifugation, resuspended in diethyl ether and centrifuged again, this =
procedure being carried out at least five times. The pellets were then dried in vacuo and judged pure by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5.). N-terminal glycated and acetylated GIP were prepared by minor modification of a published method.
Electrospray ionization-mass spectrometry (ESI-MS) was carried out as described in Example 2. Degradation of GIP and novel Gil' analogues by DPP IV
and human plasma was carried out as described in Example 2.
Culture of insulin secreting cells. BRIN-BD11 cells [30] were cultured in sterile tissue culture flasks Corning , Glass Works, UK) using RPMI-1640 tissue culture medium containing 10% (v/v) foetal calf serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. The cells were maintained at 37 C in an atmosphere of 5% CO2 and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).
Acute tests for insulin secretion, Before experimentation, the cells were harvested from the surface of the tissue culture flasks with the aid of trypsin/EDTA
(Gibco), seeded into 24-multiwell plates (Nunc, Roskilde, Denmark) at a density of 1.5 x 105 cells per well, and allowed to attach overnight at 37 C. Acute tests for insulin release were preceded by 40 minutes pre-incubation at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer (115 mM NaC1, 4.7 mM KC I, 1.28 mM CaC12, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM NaHCO3, 5 g/1 bovine serum albumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations were performed (n=12) at two glucose concentrations (5.6 mM and 16.7 mM) with a range of concentrations (1(-13 to M) of GIP or GIP analogues. After 20 minutes incubation, the buffer was removed from each well and aliquots (200 i) were used for measurement of insulin by radioimmunoassay [31].
Statistical analysis. Results are expressed as mean S.E.M. and values were compared using the Student's unpaired t-test. Groups of data were considered to be significantly different if P< 05.
Electrospray ionization mass spectrometry (ESI-MS). Samples for ESI-MS
analysis containing intact and degradation fragments of GIP (from DPP IV and plasma incubations) as well as Tyr'-glucitol GIP, were further purified by HPLC.
Peptides were dissolved (approximately 400 pmol) in 100 ill of water and applied to the LCQ
benchtop mass spectrometer (Finnigan MAT, Hemel Hempstead, UK) equipped with a microhore C-18 HPLC column (150 x 2.0mm, Phenomenex , Ltd., Macclesfield, UK). Samples (300 direct loop injection) were injected at a flow rate of 0.2m1/min, under isocratic conditions 35% (v/v) acetonitile/water. Mass spectra were obtained from the quadripole ion trap mass analyzer and recorded. Spectra were collected using full ion scan mode over the mass-to-charge (rn/z) range 150-2000. The molecular masses of GIP and related structures were determined from ESI-MS
profiles using prominent multiple charged ions and the following equation MT = iMi -where Mr = molecular mass; Mi= m/z ratio; i = number of charges; Mh = mass of a proton.
In vivo biological activity of GIP and Tyr -glucitoi GIP. Effects of GIP and Tyri-glucitol GIP on plasma glucose and insulin concentrations were examined using 12 week old male Wistar rats. The animals were housed individually in an air conditioned room and 22 2 C with a 12 hour light/12 hour dark cycle. Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Belfast, Northern Ireland) were supplied ad libitum. Food was withdrawn for an 18 hour period prior to intraperitoneal injection of glucose alone (18mmol/kq body weight) or in combination with either GIP or Tyri-glucitol GIP (10 nmol/kg). Test solutions were administered in a final volume of 8 ml/kg body weight. Blood samples were collected at 0, 15, 30 and 60 minutes from the cut tip of the tail of conscious rats into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Samples were centrifuged using a Beckman microcentrifuge for about 30 seconds at 13,000 g.
Plasma samples were aliquoted and stored at -20 C prior to glucose and insulin determinations. All animal studies were done in accordance with the Animals (Scientific Procedures) Act 1986.
Analyses. Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman Glucose Analyzer II [33]. Plasma insulin was determined by dextran charcoal radioimmunoassay as described previously [34]. Incremental areas under plasma glucose and insulin area under the curve (AUC) were calculated using a computer program (CAREA) employing the trapezoidal rule [35] with baseline subtraction. Results are expressed as mean SEM and values were compared using the Student's unpaired t-test. Groups of data were considered to be significantly different if P<0.05.
Degradation of GIP and Tyr' -glueitol GIP by DPP IV. Fig. 1 illustrates the typical peak profiles obtained from the HPLC separation of the products obtained from the incubation of GIP (Fig la) or Tyrl-glucitol GIP (Fig lb) with DPP IV for 0, 2, 4 and 12 hours. The retention times of GIP and Tyr'-glucitol GIP at t=0 were 21.93 minutes and 21.75 minutes respectively. Degradation of GIP was evident after 4 hours incubation (54% intact), and by 12 hours the majority (60%) of intact GIP was converted to the single product with a retention time of 21.61 minutes. Tyrl-glucitol GIP remained almost completely intact throughout 2-12 hours incubation.
Separation was on a Vydac C-18 column using linear gradients of 0% to 31.5% acetonitrile over 15 minutes, to 38.5% over 30 minutes and from 38.5 to 70% acetonitrile over 5 minutes.
Degradation of GIP and Tyr' -glucitol GIP by human plasma. Fig. 2 shows a set of typical HPLC profiles of the products obtained from the incubation of GIP or Tyri-glucitol GIP with human plasma for 0 and 4 hours. GIP (Fig 2a) with a retention time of 22.06 minutes was readily metabolised by plasma within 4 hours incubation giving rise to the appearance of a major degradation peak with a retention time of 21.74 minutes. In contrast, the incubation of Tyr'-glucitol GIP under similar conditions (Fig 2b) did not result in the formation of any detectable degradation fragments during this time with only a single peak being observed with a retention time of 21.77 minutes.
Addition of diprotin A, a specific inhibitor of DPP IV, to GIP during the 4 hours incubation completely inhibited degradation of the peptide by plasma. Peaks corresponding with intact GIP, GIP (3-42) and Tyr'-glucitol GIP are indicated.
A
major peak corresponding to the specific DPP IV inhibitor tripeptide DPA
appears in the bottom peanels with retention time of 16-29 minutes.
Identification of GIP degradation fragments by ESI-MS. Fig. 3 shows the monoisotopic molecular masses obtained for GIP (Fig. 3A), Tyr'-glucitol GIP
(Fig.
3B) and the major plasma degradation fragment of GIP (Fig. 3C) using ESI-MS.
The peptides analyzed were purified from plasma incubations as shown in Fig. 2.
Peptides were dissolved (approximately 400 pmol) in 100 1 of water and applied to the LC/MS
equipped with a microbore C-18 HPLC column. Samples (300 direct loop injection) were applied at a flow rate of 0.2m1/min, under isocratic conditions 35%
acetonitrile/water. Mass spectra were recorded using a quadripole ion trap mass analyzer. Spectra were collected using full ion scan mode over the mass-to-charge (m/z) range 150-2000. The molecular masses (Mr) of GIP and related structures were determined from ESI-MS profiles using prominent multiple charged ions and the following equation Mr = iM, - iMh. The exact molecular mass (Mr) of the peptides were calculated using the equation Mr = iMi - iMh as defined above in Research Design and Methods. After spectral averaging was performed, prominent multiple charges species (M+3H )3+ and (M+4H )4+ were detected from GIP at m/z 1661.6 and 1246.8, corresponding to intact Mr 4981.8 and 4983.2 Da, respectively (Fig.
3A).
Similarly, for Tyrl-glucitol GIP ( (M+4H )4+ and (M+5H)5+) were detected at m/z 1287.7 and 1030.3, corresponding to intact molecular masses of Mr 5146.8 and 5146.5 Da, respectively (Fig. 3B). The difference between the observed molecular masses of the quadruply charged GIP and the N-terminally modified GIP species (163.6 Da) indicated that the latter peptide contained a single glucitol adduct corresponding to Tyri-glucitol GIP. Fig. 3C shows the prominent multiply charged species (M+3H )3+ and (M+4H)4+ detected from the major fragment of GIP at m/z 1583.8 and 1188.1, corresponding to intact Mr 4748.4 and 4748 Da, respectively (Fig.
3C). This corresponds with the theoretical mass of the N-terminally truncated form of the peptide GIP(3-42). This fragment was also the major degradation product of DPP
IV incubations (data not shown).
Effects of GIP and Tyr' -glucitol GIP on plasma glucose homeostasis. Figs. 4 and 5 show the effects of intraperitoneal (ip) glucose alone (18mmol/kg) (control group), and glucose in combination With GIP or Tyri-glucitol GIP (10nmol/kg) on plasma glucose and insulin concentrations.
Fig. 4A shows plasma glucose concentrations after i.p. glucose alone (18mmol/kg) (control group), or glucose in combination with either GIP of Tyrl-glucitol GIP (10nmol/kg). The time of injection is indicated by the arrow (0 minutes).
Fig. 4B shows plasma 'glucose AUC values for 0-60 minutes post injection.
Values are mean SEM for six rats. "P<0.01, ***P<0.001 compared with GIP and Tyr'"
glucitol GIP; TP<0.05, 11/3<0.01 compared with non-glucated GIP. Fig. 5A shows plasma insulin concentrates after i.p. glucose along (18 mmol/kg) (control group), or glucose in combination with either with GIP or glycated GIP (10nmol/kq). The time of injection is indicated by the arrow. Fig. 5B shows plasma insulin AUC
values were calculated for each of the 3 groups up to 90 minutes post injection. The time of injection is indicated by the arrow (0 minutes). Plasma insulin AUC values for minutes post injection. Values are mean SEM for six rats. *P<0.05, "P<0.001 compared with GIP and Tyri-glucitol GIP; fP<0.05, P<0.01 compared with non-glycated GIP.
Compared with the control group, plasma glucose concentrations and area under the curve (AUC) were significantly lower following administration of either GIP or Tyrl-glucitol GIP (Figs 4A, B). Furthermore, individual values at 15 and 30 minutes together with AUC were significantly lower following administration of Tyrl-glucitol GIP as compared to GIP. Consistent with the established insulin-releasing properties of GIP, plasma insulin concentrations of both peptide-treated groups were significantly raised at 15 and 30 minutes compared with the values after administration of glucose alone (Fig. 5A). The overall insulin responses, estimated as AUC were also significantly greater for the two peptide-treated groups (Fig.
5B).
Despite lower prevailing glucose concentrations than the GIP-treated group, plasma insulin response, calculated as AUC, following Tyrl-glucitol GIP was significantly greater than after GIP (Fig. 5B). The significant elevation of plasma insulin at 30 minutes is of particular note, suggesting that the insulin-releasing action of Tyrl-glucitol GIP is more protracted than GIP even in the face of a diminished glycemic stimulus (Figs. 4A, 5A).
Example 3. Additional N-Terminal Structural Modifications of GIP.
This example further looked at the ability of additional N-terminal structural modifications of GIP in preventing inactivation by DPP and in plasma and their associated increase in both the insulin-releasing potency and potential therapeutic value. Native human GIP, glycated GIP, acetylated GIP and a number of GIP
analogues with N-terminal amino acid substitutions were tested.
Materials and Methods. High-performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkersburn, Scotland). Sequencing grade trifluoroacetic acid (TFA) was obtained from Aldrich (Poole, Dorset, UK).
Dipeptidyl peptidase IV was purchased from Sigma (Poole, Dorset, UK), and Diprotin A was purchased from Calbiochem Novabiochem (Beeston, Nottingham, UK). RPM' 1640 tissue culture medium, foetal calf serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). All water used in these experiments was purified using a Water Purification System (Millipore, Milford, Massachusetts, USA). All other chemicals used were of the highest purity available.
Synthesis of GIP and N-terminally modified GIP analogues. GIP, GIP(Abu2), GIP(Sku2), GIP(Ser2), GIP(G1y2) and GIP(Pro3) were sequentially synthesized on an Applied Biosystems automated peptide synthesizer (model 432A) using standard solid-phase Fmoc procedure, starting with an Fmoc-Gln-Wang resin. Following cleavage from the resin by trifluoroacetic acid: water, thioanisole, ethanedithiol (90/2.5/5/2.5, a total volume of 20 ml/g resin), the resin was removed by filtration and the filtrate volume was decreased under reduced pressure. Dry diethyl ether was slowly added until a precipitate was observed. The precipitate was collected by low-speed centrifugation, resuspended in diethyl ether and centrifuged again, this =
procedure being carried out at least five times. The pellets were then dried in vacuo and judged pure by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5.). N-terminal glycated and acetylated GIP were prepared by minor modification of a published method.
Electrospray ionization-mass spectrometry (ESI-MS) was carried out as described in Example 2. Degradation of GIP and novel Gil' analogues by DPP IV
and human plasma was carried out as described in Example 2.
Culture of insulin secreting cells. BRIN-BD11 cells [30] were cultured in sterile tissue culture flasks Corning , Glass Works, UK) using RPMI-1640 tissue culture medium containing 10% (v/v) foetal calf serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mM glucose. The cells were maintained at 37 C in an atmosphere of 5% CO2 and 95% air using a LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).
Acute tests for insulin secretion, Before experimentation, the cells were harvested from the surface of the tissue culture flasks with the aid of trypsin/EDTA
(Gibco), seeded into 24-multiwell plates (Nunc, Roskilde, Denmark) at a density of 1.5 x 105 cells per well, and allowed to attach overnight at 37 C. Acute tests for insulin release were preceded by 40 minutes pre-incubation at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer (115 mM NaC1, 4.7 mM KC I, 1.28 mM CaC12, 1.2 mM KH2PO4, 1.2 mM MgSO4, 10 mM NaHCO3, 5 g/1 bovine serum albumin, pH 7.4) supplemented with 1.1 mM glucose. Test incubations were performed (n=12) at two glucose concentrations (5.6 mM and 16.7 mM) with a range of concentrations (1(-13 to M) of GIP or GIP analogues. After 20 minutes incubation, the buffer was removed from each well and aliquots (200 i) were used for measurement of insulin by radioimmunoassay [31].
Statistical analysis. Results are expressed as mean S.E.M. and values were compared using the Student's unpaired t-test. Groups of data were considered to be significantly different if P< 05.
Structural identification of GIP and GIP analogues by ESI-MS. The monoisotopic molecular masses of the peptides were determined using ESI-MS. After spectral averaging was performed, prominent multiple charged species (M+3H)3+ and (M+4H)4+ were detected for each peptide. Calculated molecular masses confirmed the structural identity of synthetic GIP and each of the N-terminal analogues.
Degradation of GIP and novel GIP analogues by DPP-IV. Figs. 6-11 illustrate the typical peak profiles obtained from the HPLC separation of the reaction products obtained from the incubation of GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), glycated GIP
and acetylated GIP with DPP IV, for 0, 2, 4, 8 and 24 hours. The results summarized in Table 1 indicate that glycated GIP, acetylated GIP, GIP(Ser2) are GIP(Abu2) more resistant than native GIP to in vitro degradation with DPP IV. From these data GIP(Sar2) appears to be less resistant.
Table 1. Percent intact peptide remaining after incubation with DPPIV.
Peptide % Intact peptide remaining after time (h) GIP 1-42 100 52 1 23+1 0 0 Glycated GIP 100 100 100 100 100 GIP(Abu2) 100 38 1 28 2 0 0 GIP(Ser2) 100 77+2 60+1 32+4 0 GIP(Sar2) 100 28 + 2 8 0 0 N-Acetyl-GIP 100 100 100 100 Table represents the percentage of intact peptide (i.e., GIP 1-42) relative to the major degradation product GIP 3-42. Values were taken from HPLC traces performed in triplicate and the mean and S.E.M. values calculated. DPA is diprotin A, a specific inhibitor of DPPIV.
Degradation of GIP and GIP analogues by human plasma. Figs. 12-16 show a representative set of HPLC profiles obtained from the incubation of GIP and GIP
analogues with human plasma for 0, 2, 4, 8 and 24 hours. Observations were also made after incubation for 24 hours in the presence of DPA. These results are summarized in Table 2 are broadly comparable with DPP IV incubations, but conditions which more closely mirror in vivo conditions are less enzymatically severe.
GIP was rapidly degraded by plasma. In comparison, all analogues tested exhibited resistance to plasma degradation, including GIP(Sar2) which from DPP IV data appeared least resistant of the peptides tested. DPA substantially inhibited degradation of GIP and all analogues tested with complete abolition of degradation in the cases of GIP(Abu2), GIP(Ser2) and glycated GIP. This indicates that DPP IV
is a key factor in the in vivo degradation of GIP.
Table 2. Percent intact peptide remaining after incubation with human plasma.
Peptide % Intact peptide remaining after incubations with human plasma Glycated GIP 100 100 100 100 100 100 GIP (Abu2) 100 38 1 28 2 0 0 100 GIP(Ser2) 100 77 2 60 1 32 .1 4 0 63 3 GIP(Sar2) 100 28 1 2 8 0 0 100 Table represents the percentage of intact peptide (i.e., GIP 1-42) relative to the major degradation product GIP 3-42. Values were taken from HPLC traces performed in triplicate and the mean and S.E.M. values calculated. DPA is diprotin A, a specific inhibitor of DPPIV.
Dose-dependent effects of GIP and novel GIP analogues on insulin secretion.
Figs.
17-30 show the effects of a range of concentrations of GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), acetylated GIP, glycated GIP, GIP(G1y2) and GIP(Pro3) on insulin secretion from BRIN-BD11 cells at 5.6 and 16.7 mM glucose. Native GIP provoked a prominent and dose-related stimulation of insulin secretion. Consistent with previous studies [28], the glycated GIP analogue exhibited a considerably greater insulinotropic response compared with native peptide. N-terminal acetylated GIP
exhibited a similar pattern and the GIP(Ser2) analogue also evoked a strong response.
From these tests, GIP(G1y2) and GIP(Pro3) appeared to be the least potent analogues in terms of insulin release. Other stable analogues tested, namely GIP(Abu2) and GIP(Sar2), exhibited a complex pattern of responsiveness dependent on glucose concentration and dose employed. Thus very low concentrations were extremely potent under hyperglycemic conditions (16.7 mM glucose). This suggests that even these analogues may prove therapeutically useful in the treatment of type 2 diabetes where insulinotropic capacity combined with in vivo degradation dictates peptide potency.
Example 4. Glu3 substituted GIP improves obesity-related insulin resistance and associated glucose intolerance.
This example examines GIP receptor antagonism and obesity-related insulin resistance and associated glucose intolerance using a G1u3-substituted form of GIP, namely, (Pro3)GIP.
Cell lines and animals. In vitro insulin secretion was evaluated using the clonal pancreatic beta-cell line, BRIN-BD11 (McClenaghan, N.H. et al., 1996, Diabetes 45:1132-1140). In vitro cyclic AMP generation was measured using Chinese hamster lung (CHL) fibroblast cells stably transfected with the human GIP receptor (Gremlich, S. et al., 1995, Diabetes 44:1202-1208). In vivo studies were conducted in 8-week-old obese diabetic ob/ob mice (Bailey C.J. et al., 1982, Int. J Obesity 6:11-21) and normal control mice.
Peptide synthesis and characterisation. Glu3-substituted analogues were sequentially synthesised on an Applied Biosystems automated peptide synthesiser (Model 432A) using standard solid-phase Fmoc peptide chemistry (Fields, G.B. et aL,1990, Int. J.
Pept Protein Res. 35:161-214), from a pre-loaded Fmoc-Gln-Wang resin. The synthetic peptides were judged pure by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5). The molecular masses of the purified peptide analogues were determined using Matrix Assisted Laser Desorption lonisation-Time of Flight (MALDI-TOF) mass spectrometry. Samples were dissolved in 10 [11 H20 (approximately 40 pmo1/1), placed on a stainless steel sample plate and allowed to dry at room temperature. Samples were then mixed with a matrix solution (10 mg/ml solution of a-cyano-4-hydroxycinnamic acid) in acetonitrile/ethanol (1/1) and allowed to dry at room temperature. The molecular masses were then recorded as mass-to-charge (m/z) ratio versus relative peak intensity =
and compared using theoretical values on a Voyager-DE BioSpectrometry Workstation (PerSeptive Biosystems 0, Framingham, MA, USA).
Tissue culture. Chinese hamster lung (CHL) fibroblast cells stably fransfected with 5 the human GIP receptor were cultured in DMEM tissue culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 Uhnl penicillin, 0.1 mg/ml streptomycin). BRIN-BD11 cells were cultured using RPMI-1640 tissue culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Cells were maintained in sterile tissue culture 10 flasks (Corning Glass Works, Sunderland, UK) at 37 C in an atmosphere of 5% CO2 and 95% air using an LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).
Acute studies of insulin release. Insulin release from BRIN-BD11 cells was 15 determined using cell monolayers (McClenaghan, N.H. et al., 1996, Diabetes 45:1132-1140). Cells were harvested with the aid of trypsin/EDTA (Gibco), seeded into 24-multiwell plates (Nunc, Roskilde, Denmark) at a density of 1.0 x 105cells per well, and allowed to attach overnight at 37 C. Prior to acute test, cells were preincubated for 40 minutes at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer (115 20 mM NaCl, 4.7 mM KCI, 1.28 mM CaC12, 1.2 niM KH2PO4, 1.2 mM MgSO4, 10 mM
NaHCO3, 0.5% (w/v) bovine serum albumin, pH 7.4) supplemented with 1.1 mM
glucose. Acute tests for insulin release were performed for 20 minutes at 37 C
at 5.6 mM glucose using various concentrations of Glu3-substituted analogues and GIP(3-42) in the presence of native GIP (1 0-7 M) as indicated in the Figures. After 25 incubation, aliquots of buffer were removed and stored at -20 C for insulin radioimmunoassay (Flatt, P.R. et al., 1981, Diabetologia 20:573-577).
Acute studies of cyclic AMP generation. GIP receptor transfected CHL cells were seeded into 12-well plates (Nunc, Roskilde, Denmark) at a density of 1.0 x 105 cells 30 per well. The cells were then allowed to grow for 48 hours before being loaded with tritiated adenine (2 p.Ci; TRK311, Amersham, Buckinghamshire, UK) and incubated at 37 C for 6 hours in 1 ml DMEM, supplemented with 0.5% (w/v) foetal bovine serum. The cells were then washed twice with FIBS buffer (130 mM NaCl, 20 mM
HEPES, 0.9 mM NaHPO4, 0.8 mM MgSO4, 5.4 mM KC1, 1.8 mM CaC12, 25 mM
glucose, 25 1.1.M phenol red, pH 7.4). The cells were then exposed for 10 minutes at 37 C to forskolin (FSK, 10 M) or varying concentrations of (Pro3)GIP in the absence (control) or presence of native GIP (10-7 M). After removal of the medium, cells were lysed with 1 ml of 5% trichloroacetic acid (TCA) containing 0.1 mM
unlabelled cAMP and 0.1 mM unlabelled ATP. The intracellular tritiated cAMP
was then separated on Dowex and alumina exchange resins as previously described (Widmann, C. et al., 1993, Mol. Pharmacol. 45:1029-1035).
Acute in vivo effects of (Pro3)GIP administration in obese diabetic ob/ob mice.
Plasma glucose and insulin responses were evaluated using 8- to 12-week old obese diabetic ob/ob mice following intraperitoneal (i.p.) injection of native GIP, (Pro3)GIP
(25 nmol/kg body weight) or saline (0.9% (w/v) NaCl; control) immediately following the combined injection of GIP (25 nmol/kg body weight) with glucose (18 mmol/kg body weight). All test solutions were administered in a final volume of 8m1/kg body weight. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30 and 60 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -20 prior to glucose and insulin determinations.
Degradation of GIP and novel GIP analogues by DPP-IV. Figs. 6-11 illustrate the typical peak profiles obtained from the HPLC separation of the reaction products obtained from the incubation of GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), glycated GIP
and acetylated GIP with DPP IV, for 0, 2, 4, 8 and 24 hours. The results summarized in Table 1 indicate that glycated GIP, acetylated GIP, GIP(Ser2) are GIP(Abu2) more resistant than native GIP to in vitro degradation with DPP IV. From these data GIP(Sar2) appears to be less resistant.
Table 1. Percent intact peptide remaining after incubation with DPPIV.
Peptide % Intact peptide remaining after time (h) GIP 1-42 100 52 1 23+1 0 0 Glycated GIP 100 100 100 100 100 GIP(Abu2) 100 38 1 28 2 0 0 GIP(Ser2) 100 77+2 60+1 32+4 0 GIP(Sar2) 100 28 + 2 8 0 0 N-Acetyl-GIP 100 100 100 100 Table represents the percentage of intact peptide (i.e., GIP 1-42) relative to the major degradation product GIP 3-42. Values were taken from HPLC traces performed in triplicate and the mean and S.E.M. values calculated. DPA is diprotin A, a specific inhibitor of DPPIV.
Degradation of GIP and GIP analogues by human plasma. Figs. 12-16 show a representative set of HPLC profiles obtained from the incubation of GIP and GIP
analogues with human plasma for 0, 2, 4, 8 and 24 hours. Observations were also made after incubation for 24 hours in the presence of DPA. These results are summarized in Table 2 are broadly comparable with DPP IV incubations, but conditions which more closely mirror in vivo conditions are less enzymatically severe.
GIP was rapidly degraded by plasma. In comparison, all analogues tested exhibited resistance to plasma degradation, including GIP(Sar2) which from DPP IV data appeared least resistant of the peptides tested. DPA substantially inhibited degradation of GIP and all analogues tested with complete abolition of degradation in the cases of GIP(Abu2), GIP(Ser2) and glycated GIP. This indicates that DPP IV
is a key factor in the in vivo degradation of GIP.
Table 2. Percent intact peptide remaining after incubation with human plasma.
Peptide % Intact peptide remaining after incubations with human plasma Glycated GIP 100 100 100 100 100 100 GIP (Abu2) 100 38 1 28 2 0 0 100 GIP(Ser2) 100 77 2 60 1 32 .1 4 0 63 3 GIP(Sar2) 100 28 1 2 8 0 0 100 Table represents the percentage of intact peptide (i.e., GIP 1-42) relative to the major degradation product GIP 3-42. Values were taken from HPLC traces performed in triplicate and the mean and S.E.M. values calculated. DPA is diprotin A, a specific inhibitor of DPPIV.
Dose-dependent effects of GIP and novel GIP analogues on insulin secretion.
Figs.
17-30 show the effects of a range of concentrations of GIP, GIP(Abu2), GIP(Sar2), GIP(Ser2), acetylated GIP, glycated GIP, GIP(G1y2) and GIP(Pro3) on insulin secretion from BRIN-BD11 cells at 5.6 and 16.7 mM glucose. Native GIP provoked a prominent and dose-related stimulation of insulin secretion. Consistent with previous studies [28], the glycated GIP analogue exhibited a considerably greater insulinotropic response compared with native peptide. N-terminal acetylated GIP
exhibited a similar pattern and the GIP(Ser2) analogue also evoked a strong response.
From these tests, GIP(G1y2) and GIP(Pro3) appeared to be the least potent analogues in terms of insulin release. Other stable analogues tested, namely GIP(Abu2) and GIP(Sar2), exhibited a complex pattern of responsiveness dependent on glucose concentration and dose employed. Thus very low concentrations were extremely potent under hyperglycemic conditions (16.7 mM glucose). This suggests that even these analogues may prove therapeutically useful in the treatment of type 2 diabetes where insulinotropic capacity combined with in vivo degradation dictates peptide potency.
Example 4. Glu3 substituted GIP improves obesity-related insulin resistance and associated glucose intolerance.
This example examines GIP receptor antagonism and obesity-related insulin resistance and associated glucose intolerance using a G1u3-substituted form of GIP, namely, (Pro3)GIP.
Cell lines and animals. In vitro insulin secretion was evaluated using the clonal pancreatic beta-cell line, BRIN-BD11 (McClenaghan, N.H. et al., 1996, Diabetes 45:1132-1140). In vitro cyclic AMP generation was measured using Chinese hamster lung (CHL) fibroblast cells stably transfected with the human GIP receptor (Gremlich, S. et al., 1995, Diabetes 44:1202-1208). In vivo studies were conducted in 8-week-old obese diabetic ob/ob mice (Bailey C.J. et al., 1982, Int. J Obesity 6:11-21) and normal control mice.
Peptide synthesis and characterisation. Glu3-substituted analogues were sequentially synthesised on an Applied Biosystems automated peptide synthesiser (Model 432A) using standard solid-phase Fmoc peptide chemistry (Fields, G.B. et aL,1990, Int. J.
Pept Protein Res. 35:161-214), from a pre-loaded Fmoc-Gln-Wang resin. The synthetic peptides were judged pure by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5). The molecular masses of the purified peptide analogues were determined using Matrix Assisted Laser Desorption lonisation-Time of Flight (MALDI-TOF) mass spectrometry. Samples were dissolved in 10 [11 H20 (approximately 40 pmo1/1), placed on a stainless steel sample plate and allowed to dry at room temperature. Samples were then mixed with a matrix solution (10 mg/ml solution of a-cyano-4-hydroxycinnamic acid) in acetonitrile/ethanol (1/1) and allowed to dry at room temperature. The molecular masses were then recorded as mass-to-charge (m/z) ratio versus relative peak intensity =
and compared using theoretical values on a Voyager-DE BioSpectrometry Workstation (PerSeptive Biosystems 0, Framingham, MA, USA).
Tissue culture. Chinese hamster lung (CHL) fibroblast cells stably fransfected with 5 the human GIP receptor were cultured in DMEM tissue culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 Uhnl penicillin, 0.1 mg/ml streptomycin). BRIN-BD11 cells were cultured using RPMI-1640 tissue culture medium containing 10% (v/v) foetal bovine serum, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Cells were maintained in sterile tissue culture 10 flasks (Corning Glass Works, Sunderland, UK) at 37 C in an atmosphere of 5% CO2 and 95% air using an LEEC incubator (Laboratory Technical Engineering, Nottingham, UK).
Acute studies of insulin release. Insulin release from BRIN-BD11 cells was 15 determined using cell monolayers (McClenaghan, N.H. et al., 1996, Diabetes 45:1132-1140). Cells were harvested with the aid of trypsin/EDTA (Gibco), seeded into 24-multiwell plates (Nunc, Roskilde, Denmark) at a density of 1.0 x 105cells per well, and allowed to attach overnight at 37 C. Prior to acute test, cells were preincubated for 40 minutes at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer (115 20 mM NaCl, 4.7 mM KCI, 1.28 mM CaC12, 1.2 niM KH2PO4, 1.2 mM MgSO4, 10 mM
NaHCO3, 0.5% (w/v) bovine serum albumin, pH 7.4) supplemented with 1.1 mM
glucose. Acute tests for insulin release were performed for 20 minutes at 37 C
at 5.6 mM glucose using various concentrations of Glu3-substituted analogues and GIP(3-42) in the presence of native GIP (1 0-7 M) as indicated in the Figures. After 25 incubation, aliquots of buffer were removed and stored at -20 C for insulin radioimmunoassay (Flatt, P.R. et al., 1981, Diabetologia 20:573-577).
Acute studies of cyclic AMP generation. GIP receptor transfected CHL cells were seeded into 12-well plates (Nunc, Roskilde, Denmark) at a density of 1.0 x 105 cells 30 per well. The cells were then allowed to grow for 48 hours before being loaded with tritiated adenine (2 p.Ci; TRK311, Amersham, Buckinghamshire, UK) and incubated at 37 C for 6 hours in 1 ml DMEM, supplemented with 0.5% (w/v) foetal bovine serum. The cells were then washed twice with FIBS buffer (130 mM NaCl, 20 mM
HEPES, 0.9 mM NaHPO4, 0.8 mM MgSO4, 5.4 mM KC1, 1.8 mM CaC12, 25 mM
glucose, 25 1.1.M phenol red, pH 7.4). The cells were then exposed for 10 minutes at 37 C to forskolin (FSK, 10 M) or varying concentrations of (Pro3)GIP in the absence (control) or presence of native GIP (10-7 M). After removal of the medium, cells were lysed with 1 ml of 5% trichloroacetic acid (TCA) containing 0.1 mM
unlabelled cAMP and 0.1 mM unlabelled ATP. The intracellular tritiated cAMP
was then separated on Dowex and alumina exchange resins as previously described (Widmann, C. et al., 1993, Mol. Pharmacol. 45:1029-1035).
Acute in vivo effects of (Pro3)GIP administration in obese diabetic ob/ob mice.
Plasma glucose and insulin responses were evaluated using 8- to 12-week old obese diabetic ob/ob mice following intraperitoneal (i.p.) injection of native GIP, (Pro3)GIP
(25 nmol/kg body weight) or saline (0.9% (w/v) NaCl; control) immediately following the combined injection of GIP (25 nmol/kg body weight) with glucose (18 mmol/kg body weight). All test solutions were administered in a final volume of 8m1/kg body weight. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30 and 60 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -20 prior to glucose and insulin determinations.
Acute in vivo effects of (Pro3)GIP on plasma glucose and insulin responses to feeding in obese diabetic ob/ob mice. Plasma glucose and insulin responses were evaluated using 8- to 12-week old ob/ob mice where food was withdrawn for an 18-hour period prior to intraperitoneal injection of saline (0.9% (w/v) NaCl; control) or (Pro3)GIP (25 nmol/kg body weight). Following injection, the mice were allowed to re-feed for 15 minutes. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30, 60 and 120 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -20 prior to glucose and insulin determinations.
Effects of chronic (Pro3 )GIP administration on plasma glucose, insulin and glycated HMI, in obese diabetic ob/ob mice and normal mice. Obese diabetic ob/ob mice and normal control mice aged 8-12 weeks were randomly divided into groups which received once daily subcutaneous injections (17:00h) of either saline (0.9%
w/v NaC1) or (Pro3)GIP (25 nmol/kg body weight in saline). After 11 days, treatment was ceased. Food intake and body weight were recorded daily. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin coated glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g prior to glucose, insulin and HbAie determinations.
Effects of chronic treatment with (Pro3)GIP on glucose tolerance in ob/ob mice and normal mice. Plasma glucose and insulin concentrations were measured following intraperitoneal administration of glucose (18 mmol/kg body weight) in ob/ob and normal mice treated with either saline (0.9% w/v NaC1) or (Pro3)GIP (25 nmol/kg body weight/day) for 11 days. This test was repeated 9 days after cessation of chronic (Pro3)GIP treatment. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30 and 60 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -200 prior to glucose and insulin determinations.
Effects of chronic treatment with (Pro3)GIP on the glucose lowering effects of exogenous insulin in ob/ob mice. The glucose lowering effects of insulin were evaluated by measuring plasma glucose response in 11-day saline (0.9% w/v NaC1) and (Pro3)GIP (25 nmol/kg body weight/day) treated ob/ob mice following acute intraperitoneal administration of insulin (50 U/kg bodyweight). Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 30 and 60 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -20 prior to glucose determination.
Effects of chronic treatment with (Pro3)GIP on pancreatic insulin content and associated islet hypertrophy in ob/ob mice. Pancreatic tissue was excised from non-fasted ob/ob mice after 11 days treatment with either saline (0.9% WIT NaC1) or (Pro3)GIP (25 nmol/kg body weight/day). Pancreatic samples were individually wrapped in aluminium foil and snap frozen in liquid nitrogen. Individual excised pancreatic samples were then either embedded, sectioned and immunohistochemically stained for insulin or permeabilised for determination of pancreatic insulin content.
Determination of HbA lc, plasma glucose and insulin concentrations. HbAic was measured in whole blood by ion-exchange high-performance liquid chromatography using the Menari HA-8140 kit (BIOMEN, Berkshire, UK). Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman Glucose Analyzer II (Stevens, J.F., 1971, Clinica Chemica Acta 32:199-201) and plasma insulin was determined by RIA (Flatt, P.R. et al., 1981, Diabetologia 20:573-577).
Incremental areas under plasma glucose and insulin curves (AUC) were calculated using a computer generated program (CAREA) employing the trapezoidal rule (Burington, R.S., 1973, Handbook of Mathematical Tables and Formulae, New York, McGraw Hill) with baseline subtraction.
Effects of chronic (Pro3 )GIP administration on plasma glucose, insulin and glycated HMI, in obese diabetic ob/ob mice and normal mice. Obese diabetic ob/ob mice and normal control mice aged 8-12 weeks were randomly divided into groups which received once daily subcutaneous injections (17:00h) of either saline (0.9%
w/v NaC1) or (Pro3)GIP (25 nmol/kg body weight in saline). After 11 days, treatment was ceased. Food intake and body weight were recorded daily. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin coated glucose microcentrifuge tubes (Sarstedt, Numbrecht, Germany). Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g prior to glucose, insulin and HbAie determinations.
Effects of chronic treatment with (Pro3)GIP on glucose tolerance in ob/ob mice and normal mice. Plasma glucose and insulin concentrations were measured following intraperitoneal administration of glucose (18 mmol/kg body weight) in ob/ob and normal mice treated with either saline (0.9% w/v NaC1) or (Pro3)GIP (25 nmol/kg body weight/day) for 11 days. This test was repeated 9 days after cessation of chronic (Pro3)GIP treatment. Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 15, 30 and 60 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -200 prior to glucose and insulin determinations.
Effects of chronic treatment with (Pro3)GIP on the glucose lowering effects of exogenous insulin in ob/ob mice. The glucose lowering effects of insulin were evaluated by measuring plasma glucose response in 11-day saline (0.9% w/v NaC1) and (Pro3)GIP (25 nmol/kg body weight/day) treated ob/ob mice following acute intraperitoneal administration of insulin (50 U/kg bodyweight). Blood samples were collected from the cut tip of the tail of conscious mice into chilled fluoride/heparin microcentrifuge tubes (Sarstedt, Numbrecht, Germany) immediately prior to injection and at 30 and 60 minutes post injection. Blood samples were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13000g and stored at -20 prior to glucose determination.
Effects of chronic treatment with (Pro3)GIP on pancreatic insulin content and associated islet hypertrophy in ob/ob mice. Pancreatic tissue was excised from non-fasted ob/ob mice after 11 days treatment with either saline (0.9% WIT NaC1) or (Pro3)GIP (25 nmol/kg body weight/day). Pancreatic samples were individually wrapped in aluminium foil and snap frozen in liquid nitrogen. Individual excised pancreatic samples were then either embedded, sectioned and immunohistochemically stained for insulin or permeabilised for determination of pancreatic insulin content.
Determination of HbA lc, plasma glucose and insulin concentrations. HbAic was measured in whole blood by ion-exchange high-performance liquid chromatography using the Menari HA-8140 kit (BIOMEN, Berkshire, UK). Plasma glucose was assayed by an automated glucose oxidase procedure using a Beckman Glucose Analyzer II (Stevens, J.F., 1971, Clinica Chemica Acta 32:199-201) and plasma insulin was determined by RIA (Flatt, P.R. et al., 1981, Diabetologia 20:573-577).
Incremental areas under plasma glucose and insulin curves (AUC) were calculated using a computer generated program (CAREA) employing the trapezoidal rule (Burington, R.S., 1973, Handbook of Mathematical Tables and Formulae, New York, McGraw Hill) with baseline subtraction.
Statistical analysis. Results are expressed as means SEM. Values were compared using Student's unpaired t-test and groups of data were considered to be significantly different if P<0.05.
Results GIP-stimulated cyclic AMP production and insulin secretion were inhibited in dose-dependent fashion by (Pro3)GIP, showing that (Pro3)GIP is a potent functional GIP receptor antagonist.
GIP receptor transfected Chinese hamster lung (CHL) fibroblasts were incubated with 1012 to 10-6 M (Pro3)GIP in the presence of native GIP (10-7 M). The results are shown in Figs. 32A and 32B. Fig. 32A is a line graph showing 3H-cAMP
production as a percent of maximal response (y-axis) with increasing peptide concentration (M) (x-axis). Fig. 32B is a bar graph showing insulin secretion (y-axis) with increasing peptide concentration (M) (x-axis) for 5.6 inM glucose (control) (white bar), GIP (gray bars), (Pro3)GIP (lined bars) and (Pro3)GIP+GIP(10-7M) (black bars). *P<0.05, **P<0.01, ***P<0.001 compared to glucose control. A6P<0.01, AAAP<0.001 compared with native GIP at the same concentration. Values are means SEM for 3-8 observations.
(Pro3)GIP inhibited GIP-induced cAMP formation with an 1050 value of 2.6 M. Insulin-releasing activity of BRIN-BD11 cells exposed to native GIP and (Pro3)GIP (in the absence and presence of 10-7M GIP).
GIP-stimulated insulin secretion was inhibited in a dose-dependent fashion by GIP(3-42), (Hyp3)GIP, (Lys3)GIP, (Tyr3)GIP, (Trp3)GIP, and (Phe3)GIP, as shown in Figs. 33A through 33F, which are bar charts. Fig. 33A shows 3H-cAMP production as a percent of 10-7M GIP (y-axis) versus logio of GIP (10-7M) (white bar, control) and GIP (10-7M)+GIP(3-42) (black bars). Figs. 33B through 33F show insulin secretion (in ng/106 cells/20 minutes) (y-axis) as a function of peptide concentration (M) (x-axis) for GIP (10-7M) (white bar, control) and a G1u3-substituted form of GIP
(black bars), including (Hyp3)GIP (Fig. 33B), (Lys3)GIP (Fig. 33C), (Tyr3)GIP
(Fig.
33D), (Trp3)GIP (Fig. 33E), and (Phe3)GIP (Fig. 33F). *P<0.05, **P<0.01 compared to GIP (10-7 M) control. Values are means SEM for 3-8 observations.
Figs. 34A through 34D are a set of two line graphs (Figs. 34A, 34C) and two bar graphs (Figs. 34B, 34D) showing that acute administration of (Pro3)GIP
Results GIP-stimulated cyclic AMP production and insulin secretion were inhibited in dose-dependent fashion by (Pro3)GIP, showing that (Pro3)GIP is a potent functional GIP receptor antagonist.
GIP receptor transfected Chinese hamster lung (CHL) fibroblasts were incubated with 1012 to 10-6 M (Pro3)GIP in the presence of native GIP (10-7 M). The results are shown in Figs. 32A and 32B. Fig. 32A is a line graph showing 3H-cAMP
production as a percent of maximal response (y-axis) with increasing peptide concentration (M) (x-axis). Fig. 32B is a bar graph showing insulin secretion (y-axis) with increasing peptide concentration (M) (x-axis) for 5.6 inM glucose (control) (white bar), GIP (gray bars), (Pro3)GIP (lined bars) and (Pro3)GIP+GIP(10-7M) (black bars). *P<0.05, **P<0.01, ***P<0.001 compared to glucose control. A6P<0.01, AAAP<0.001 compared with native GIP at the same concentration. Values are means SEM for 3-8 observations.
(Pro3)GIP inhibited GIP-induced cAMP formation with an 1050 value of 2.6 M. Insulin-releasing activity of BRIN-BD11 cells exposed to native GIP and (Pro3)GIP (in the absence and presence of 10-7M GIP).
GIP-stimulated insulin secretion was inhibited in a dose-dependent fashion by GIP(3-42), (Hyp3)GIP, (Lys3)GIP, (Tyr3)GIP, (Trp3)GIP, and (Phe3)GIP, as shown in Figs. 33A through 33F, which are bar charts. Fig. 33A shows 3H-cAMP production as a percent of 10-7M GIP (y-axis) versus logio of GIP (10-7M) (white bar, control) and GIP (10-7M)+GIP(3-42) (black bars). Figs. 33B through 33F show insulin secretion (in ng/106 cells/20 minutes) (y-axis) as a function of peptide concentration (M) (x-axis) for GIP (10-7M) (white bar, control) and a G1u3-substituted form of GIP
(black bars), including (Hyp3)GIP (Fig. 33B), (Lys3)GIP (Fig. 33C), (Tyr3)GIP
(Fig.
33D), (Trp3)GIP (Fig. 33E), and (Phe3)GIP (Fig. 33F). *P<0.05, **P<0.01 compared to GIP (10-7 M) control. Values are means SEM for 3-8 observations.
Figs. 34A through 34D are a set of two line graphs (Figs. 34A, 34C) and two bar graphs (Figs. 34B, 34D) showing that acute administration of (Pro3)GIP
completely antagonises the actions of GIP on glucose tolerance (Figs. 34A, 34B) and plasma insulin (Figs. 34C, 34D) responses in obese diabetic ob/ob mice. Figs.
and 34C are line graphs show plasma glucose levels (Fig. 34A, y-axis) and plasma insulin levels (Fig. 34C, y-axis) over time (x-axis) for glucose (control; =), glucose +
GIP (4) and glucose + (GIP+Pro3GIP)) (A). Figs. 34B and 34D are bar graphs showing plasmia glucose AUC for glucose alone (white bars), GIP (grey bars) and glucose + (GIP+Pro3GIP)) (black bars).
Plasma glucose and insulin concentrations after i.p. administration of glucose alone (18 mmol/kg body weight) or in combination with either native GIP or native GIP plus (Pro3)GIP (25 nmol/kg body weight). The time of injection is indicated by the arrow (0 minutes). Plasma glucose and insulin AUC values are given for 0-minutes post-injection. Values are means SEM for 8 mice. *P<0.05, "P<0.01, ***P<0.001 compared with glucose alone. 6P<0.01, mP<0.001 compared with native GIP.
Acute administration of (Pro3)GIP completely antagonised the insulin-releasing action of GIP and the associated improvement of glucose tolerance in ob/ob mice. Indeed, the glycemic excursion following (Pro3)GIP (A) was worse than when glucose was administered alone (V).
Figs. 35A through 35D show the effects of (Pro3)GIP on physiological meal-stimulated insulin release and glycemic excursion in obese diabetic ob/ob mice.
Plasma glucose and insulin concentrations were measured in mice allowed to re-feed for 15 minutes prior to i.p. administration of saline (0.9% (w/v) NaC1) as control or (Pro3)GIP (25 nmol/kg body weight). The time of injection is indicated by the arrow (15 minutes).
The results are shown in Figs. 35A through 35D, which are a set of two line graphs (Figs. 35A, 35C) and two bar graphs (Figs. 35B, 35D). The figures show plasma insulin (Figs. 35A) and plasma glucose (Fig. 35C) over time for saline control (V) and (Pro3)GIP (0), and plasma insulin AUC (Fig. 35B) and plasma glucose AUC
(Fig. 35D) for saline control (white bars) and (Pro3)GIP (black bars), respectively.
Values are means SEM for 8 mice. *P<0.05, "P<0.01, ***P<0.001 compared with saline alone.
Acute administration of (Pro3)GIP decreased the insulin response to feeding and worsened the associated glycemic excursion in ob/ob mice. These effects of functional ablation of endogenous GIP by the (Pro3)GIP antagonist are fully consistent with the accepted role of GIP in the regulation of insulin secretion and glycemic excursion following feeding.
The effects of chronic administration of (Pro3)GIP for 11 days on plasma glucose and insulin concentrations of obese diabetic ob/ob mice were also studied.
According to classical thinking and the experiments described above and the results shown in Figs. 32-35, functional ablation of endogenous GIP by daily administration of (Pro3)GIP over 11 days would be expected to inhibit insulin secretion and cause a marked deterioration in glucose tolerance.
However, the exact opposite occurred during chronic treatment with (Pro3)GIP
in ob/ob mice. This is shown in Fig. 36, which is a set of two bar graphs showing plasma glucose (Fig. 36A) and insulin (Fig. 36B) concentrations after daily subcutaneous administration of saline alone (0.9% (w/v) NaCl; as control;
white bars) or (Pro3)GIP (25 nmol/kg body weight; black bars) for 11 days. Values are means SEM for 6 mice and *P0.05 compared with saline alone. Chronic administration of (Pro3)GIP (black bars) for 11 days decreases plasma glucose and plasma insulin concentrations of obese diabetic ob/ob mice, relative to controls.
The effects of chronic administration of (Pro3)GIP for 11 days on HbAlc, (Fig.
37A), pancreatic insulin content (Fig. 37B) and associated islet hypertrophy (Fig.
37C) were examined in obese diabetic ob/ob mice treated with saline (control, white bars) and (Pro3)GIP were examined. HbAic, pancreatic insulin content and average islet diameter were measured after 11 daily subcutaneous injections of either saline alone (white bars) or (Pro3)GIP (25 nmol/kg body weight; black bars) to obese diabetic ob/ob mice. Values are means SEM for 6 mice and *P<0.05, ***P<0.001 compared with saline-treated group.
Beneficial effects of chronic (Pro3)GIP administration in ob/ob mice were associated with significant decreases in HbAlc and pancreatic insulin stores, with partial correction of the marked islet hypertrophy of the ob/ob mutant. There was also an approximate 7% decrease in body weight in (Pro3)GIP-treated ob/ob mice without any change in food intake. This effect did not achieve significance over the short study period, but this observation clearly suggests that GIP antagonism may also have a longer-term anti-obesity action.
and 34C are line graphs show plasma glucose levels (Fig. 34A, y-axis) and plasma insulin levels (Fig. 34C, y-axis) over time (x-axis) for glucose (control; =), glucose +
GIP (4) and glucose + (GIP+Pro3GIP)) (A). Figs. 34B and 34D are bar graphs showing plasmia glucose AUC for glucose alone (white bars), GIP (grey bars) and glucose + (GIP+Pro3GIP)) (black bars).
Plasma glucose and insulin concentrations after i.p. administration of glucose alone (18 mmol/kg body weight) or in combination with either native GIP or native GIP plus (Pro3)GIP (25 nmol/kg body weight). The time of injection is indicated by the arrow (0 minutes). Plasma glucose and insulin AUC values are given for 0-minutes post-injection. Values are means SEM for 8 mice. *P<0.05, "P<0.01, ***P<0.001 compared with glucose alone. 6P<0.01, mP<0.001 compared with native GIP.
Acute administration of (Pro3)GIP completely antagonised the insulin-releasing action of GIP and the associated improvement of glucose tolerance in ob/ob mice. Indeed, the glycemic excursion following (Pro3)GIP (A) was worse than when glucose was administered alone (V).
Figs. 35A through 35D show the effects of (Pro3)GIP on physiological meal-stimulated insulin release and glycemic excursion in obese diabetic ob/ob mice.
Plasma glucose and insulin concentrations were measured in mice allowed to re-feed for 15 minutes prior to i.p. administration of saline (0.9% (w/v) NaC1) as control or (Pro3)GIP (25 nmol/kg body weight). The time of injection is indicated by the arrow (15 minutes).
The results are shown in Figs. 35A through 35D, which are a set of two line graphs (Figs. 35A, 35C) and two bar graphs (Figs. 35B, 35D). The figures show plasma insulin (Figs. 35A) and plasma glucose (Fig. 35C) over time for saline control (V) and (Pro3)GIP (0), and plasma insulin AUC (Fig. 35B) and plasma glucose AUC
(Fig. 35D) for saline control (white bars) and (Pro3)GIP (black bars), respectively.
Values are means SEM for 8 mice. *P<0.05, "P<0.01, ***P<0.001 compared with saline alone.
Acute administration of (Pro3)GIP decreased the insulin response to feeding and worsened the associated glycemic excursion in ob/ob mice. These effects of functional ablation of endogenous GIP by the (Pro3)GIP antagonist are fully consistent with the accepted role of GIP in the regulation of insulin secretion and glycemic excursion following feeding.
The effects of chronic administration of (Pro3)GIP for 11 days on plasma glucose and insulin concentrations of obese diabetic ob/ob mice were also studied.
According to classical thinking and the experiments described above and the results shown in Figs. 32-35, functional ablation of endogenous GIP by daily administration of (Pro3)GIP over 11 days would be expected to inhibit insulin secretion and cause a marked deterioration in glucose tolerance.
However, the exact opposite occurred during chronic treatment with (Pro3)GIP
in ob/ob mice. This is shown in Fig. 36, which is a set of two bar graphs showing plasma glucose (Fig. 36A) and insulin (Fig. 36B) concentrations after daily subcutaneous administration of saline alone (0.9% (w/v) NaCl; as control;
white bars) or (Pro3)GIP (25 nmol/kg body weight; black bars) for 11 days. Values are means SEM for 6 mice and *P0.05 compared with saline alone. Chronic administration of (Pro3)GIP (black bars) for 11 days decreases plasma glucose and plasma insulin concentrations of obese diabetic ob/ob mice, relative to controls.
The effects of chronic administration of (Pro3)GIP for 11 days on HbAlc, (Fig.
37A), pancreatic insulin content (Fig. 37B) and associated islet hypertrophy (Fig.
37C) were examined in obese diabetic ob/ob mice treated with saline (control, white bars) and (Pro3)GIP were examined. HbAic, pancreatic insulin content and average islet diameter were measured after 11 daily subcutaneous injections of either saline alone (white bars) or (Pro3)GIP (25 nmol/kg body weight; black bars) to obese diabetic ob/ob mice. Values are means SEM for 6 mice and *P<0.05, ***P<0.001 compared with saline-treated group.
Beneficial effects of chronic (Pro3)GIP administration in ob/ob mice were associated with significant decreases in HbAlc and pancreatic insulin stores, with partial correction of the marked islet hypertrophy of the ob/ob mutant. There was also an approximate 7% decrease in body weight in (Pro3)GIP-treated ob/ob mice without any change in food intake. This effect did not achieve significance over the short study period, but this observation clearly suggests that GIP antagonism may also have a longer-term anti-obesity action.
The effects of chronic administration of (Pro3)GIP for 11 days on glucose tolerance and plasma insulin in obese diabetic ob/ob mice is shown in Figs.
38A-38D, which are a set of line graphs (Figs. 38A, 38C) and bar graphs (Figs. 38B, 38C) showing plasma glucose levels (Figs. 38A, 38B) and plasma insulin levels (Figs. 38C, 38D) in obese diabetic ob/ob mice treated with saline (control, white) or (Pro3)GIP
(black). Plasma glucose and insulin concentrations were measured prior to and at intervals after intraperitoneal administration of glucose (18 mmol/kg body weight).
Arrow indicates time of injection (t=0). Values are means SEM for 6 mice and *P<0.05, **P<0.01, ***P<0.001 compared with saline-treated group.
After 11 days treatment with (Pro3)GIP, glucose tolerance of ob/ob mice was substantially improved without change of circulating insulin (Fig. 38).
Fig. 39 shows the effects of chronic administration of (Pro3)GIP for 11 days on insulin sensitivity in obese diabetic ob/ob mice. Plasma glucose concentrations of saline and (Pro3)GIP treated ob/ob mice were measured prior to and at intervals after intraperitoneal administration of exogenous insulin (50 U/kg body weight;
t=0).
Values are means SEM for 6 mice and *P<0.05 compared with saline-treated group.
As shown in Fig. 39, chronic administration of (Pro3)GIP caused a significant improvement of insulin sensitivity.
Interestingly, the beneficial effects of chronic administration of (Pro3)GIP
for 11 days in obese diabetic ob/ob mice was reversed 9 days after cessation of treatment.
This is consistent with a physiological effect, and is shown in Fig. 40.
Plasma glucose concentrations were measured prior to and after intraperitoneal administration of glucose (18 mmol/kg body weight) for mice that had been treated with saline (control, ID) or (Pro3)GIP (A). Arrow indicates time of injection (t=0). Values are means SEM for 6.
Figs. 41A and 41B are a pair of line graphs showing the effects of chronic administration of (Pro3)GIP for 11 days on glucose tolerance in normal mice.
Plasma glucose concentrations were measured prior to and after intraperitoneal administration of glucose (18 mmol/kg body weight). Arrow indicates time of injection (t=0).
Values are means SEM for 6 and 'I./)<0.05, **P<0.01 compared to saline-treated group.
In total contrast to beneficial actions in ob/ob mice, chronic daily treatment of normal mice with (Pro3)GIP (A) for 11 days resulted in a marked deterioration of glucose tolerance (Fig. 41A) relative to controls (m), which was reversed 9 days after cessation of treatment (Fig. 41B).
Example 5. Chemical Ablation of Gastric Inhibitory Polypeptide Receptor Action By Daily (Pro3)GIP Administration Improves Glucose Tolerance and Ameliorates Insulin Resistance and Abnormalities of Islet Structure in Obesity-Diabetes.
Gastric inhibitory polypeptide (GIP) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. This study investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult oh/oh mice received once daily i.p. injections of saline vehicle or (Pro3)GIP over an 11-day period. Non-fasting plasma glucose levels and the overall glycemic excursion (AUC) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared to controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding.
These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP
reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and beta cell hyperplasia of ob/ob mice.
These comprehensive beneficial effects of (Pro3)GIP were reversed following 9 days cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.
Research Design And Methods Animals. Obese diabetic (oh/oh) mice derived from the colony maintained at Aston University, UK (Bailey, C.J., et al., 1982, Int. *I Obes. 6:11-21) were used at 12-16 weeks of age. Animals were age-matched, divided into groups and housed individually in an air-conditioned room at 222 C with a 12 hour light:12 hour dark cycle. Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse effects were observed following administration of (Pro3)GIP.
Synthesis, purification and characterization of (Pro3)GIP. (Pro3)GIP was sequentially synthesized on an Applied Biosystems automated peptide synthesizer (Model 432 A). (Pro3)GIP was purified by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5) and subsequently characterized using electrospray ionization mass spectrometry (ESI-MS).
Experimental protocols for ob/ob mouse studies. Initially, extended biological activity of (Pro3)GIP was examined in 18-hour fasted ob/ob mice 4 hours after administration. Thereafter, over an 11-day period, mice received once daily i.p.
injections (17:00 hours) of either saline vehicle (0.9% (w/v), NaCl) or (Pro3)GIP (25 nmol/kg body wt). During a subsequent 9-day period, observations were continued following discontinuation of (Pro3)GIP administration. Food intake and body weight were recorded daily whilst plasma glucose and insulin concentrations were monitored at intervals of 2-6 days. Whole blood for the measurement of glycated hemoglobin was taken on days 11 and 20. Intraperitoneal glucose tolerance (18 mmol/kg body wt), metabolic response to native GIP (25 nmol/kg body wt) and insulin sensitivity (50 U/kg body wt) tests were performed on days 11 and 20. Mice fasted for 18 hours were used to examine the metabolic response to 15 minutes feeding. In a separate series, pancreatic tissues were excised at the end of the 11-day treatment period or 9 days following discontinuation of (Pro3)GIP and processed for immunohistochemistry or measurement of insulin following extraction with 5 ml/g of ice-cold acid ethanol (750 ml ethanol, 235 ml water, 15 ml concentrated HC1). Blood samples taken from the cut tip of the tail vein of conscious mice at the times indicated in the Figures were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13,000 g. The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at -20 C prior to glucose and insulin determinations.
Biochemical analysis. Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, J.F., 1971, Clin. Chem. Acta 32:199-201) using a Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Plasma and pancreatic insulin were assayed by a modified dextran-coated charcoal radioimmunoassay (Flatt, P.R. et al., 1981, Diabetologia 20:573-577). Glycated hemoglobin was determined using cation-exchange columns (Sigma, Poole, Dorset, UK) with measurement of absorbance (415 nm) in wash and eluting buffer using a VersaMaxo Microplate Spectrophotometer (Molecular Devices, Wolcingham, Berkshire, UK).
Immunocytochemistry. Tissue fixed in 4% paraformaldehyde/PBS and embedded in paraffin was sectioned at 8 um. After de-waxing, sections were incubated with blocking serum (Vector Laboratories, CA, USA) prior to exposure to insulin antibody.
Tissue samples were then incubated consecutively with secondary biotinylated universal, pan-specific antibody (Vector Laboratories, CA, USA) and streptavidin/peroxidase preformed complex (Vector Laboratories, CA, USA).
Following washing, the stained pancreatic tissue was counterstained with hematoxylin (BDH Chemicals, Dorset, UK) and then plunged into acid methanol (500 ml methanol, 500 ml H20 and 2.5 ml concentrated HCI) prior to dehydration and mounting in Depex (BDH Chemicals, Dorset, UK). The stained slides were viewed under a microscope (Nikon Eclipse E2000 , Diagnostic Instruments Incorporated, Michigan, USA) attached to a NC camera Model KY-F55B (JVC, London, UK) and analyzed using Kromoscan imaging software (Kinetic Imaging Limited, Faversham, Kent, UK). The average number and diameter of every islet in each section was estimated in a blinded manner using an eyepiece graticule calibrated with a stage micrometer (Graticules Limited, Tonbridge, Kent, UK). The longest and shortest diameters of each islet were determined with the graticule. Half of the sum of these two values was then considered to be the average islet diameter. Approximately 70 random sections were examined from the pancreas of each mouse.
Statistics. Results are expressed as mean SEM. Data were compared using ANOVA, followed by a Student-Newman-Keuls post hoc test. Area under the curve (AUC) analyzes were calculated using the trapezoidal rule with baseline subtraction (Burington, R.S., Handbook of Mathematical Tables and Formulae, New York, McGraw-Hill, 1973). P < 0.05 was considered to be statistically significant.
38A-38D, which are a set of line graphs (Figs. 38A, 38C) and bar graphs (Figs. 38B, 38C) showing plasma glucose levels (Figs. 38A, 38B) and plasma insulin levels (Figs. 38C, 38D) in obese diabetic ob/ob mice treated with saline (control, white) or (Pro3)GIP
(black). Plasma glucose and insulin concentrations were measured prior to and at intervals after intraperitoneal administration of glucose (18 mmol/kg body weight).
Arrow indicates time of injection (t=0). Values are means SEM for 6 mice and *P<0.05, **P<0.01, ***P<0.001 compared with saline-treated group.
After 11 days treatment with (Pro3)GIP, glucose tolerance of ob/ob mice was substantially improved without change of circulating insulin (Fig. 38).
Fig. 39 shows the effects of chronic administration of (Pro3)GIP for 11 days on insulin sensitivity in obese diabetic ob/ob mice. Plasma glucose concentrations of saline and (Pro3)GIP treated ob/ob mice were measured prior to and at intervals after intraperitoneal administration of exogenous insulin (50 U/kg body weight;
t=0).
Values are means SEM for 6 mice and *P<0.05 compared with saline-treated group.
As shown in Fig. 39, chronic administration of (Pro3)GIP caused a significant improvement of insulin sensitivity.
Interestingly, the beneficial effects of chronic administration of (Pro3)GIP
for 11 days in obese diabetic ob/ob mice was reversed 9 days after cessation of treatment.
This is consistent with a physiological effect, and is shown in Fig. 40.
Plasma glucose concentrations were measured prior to and after intraperitoneal administration of glucose (18 mmol/kg body weight) for mice that had been treated with saline (control, ID) or (Pro3)GIP (A). Arrow indicates time of injection (t=0). Values are means SEM for 6.
Figs. 41A and 41B are a pair of line graphs showing the effects of chronic administration of (Pro3)GIP for 11 days on glucose tolerance in normal mice.
Plasma glucose concentrations were measured prior to and after intraperitoneal administration of glucose (18 mmol/kg body weight). Arrow indicates time of injection (t=0).
Values are means SEM for 6 and 'I./)<0.05, **P<0.01 compared to saline-treated group.
In total contrast to beneficial actions in ob/ob mice, chronic daily treatment of normal mice with (Pro3)GIP (A) for 11 days resulted in a marked deterioration of glucose tolerance (Fig. 41A) relative to controls (m), which was reversed 9 days after cessation of treatment (Fig. 41B).
Example 5. Chemical Ablation of Gastric Inhibitory Polypeptide Receptor Action By Daily (Pro3)GIP Administration Improves Glucose Tolerance and Ameliorates Insulin Resistance and Abnormalities of Islet Structure in Obesity-Diabetes.
Gastric inhibitory polypeptide (GIP) is an important incretin hormone secreted by endocrine K-cells in response to nutrient ingestion. This study investigated the effects of chemical ablation of GIP receptor (GIP-R) action on aspects of obesity-diabetes using a stable and specific GIP-R antagonist, (Pro3)GIP. Young adult oh/oh mice received once daily i.p. injections of saline vehicle or (Pro3)GIP over an 11-day period. Non-fasting plasma glucose levels and the overall glycemic excursion (AUC) to a glucose load were significantly reduced (1.6-fold; P <0.05) in (Pro3)GIP-treated mice compared to controls. GIP-R ablation also significantly lowered overall plasma glucose (1.4-fold; P <0.05) and insulin (1.5-fold; P <0.05) responses to feeding.
These changes were associated with significantly enhanced (1.6-fold; P <0.05) insulin sensitivity in the (Pro3)GIP-treated group. Daily injection of (Pro3)GIP
reduced pancreatic insulin content (1.3-fold; P <0.05) and partially corrected the obesity-related islet hypertrophy and beta cell hyperplasia of ob/ob mice.
These comprehensive beneficial effects of (Pro3)GIP were reversed following 9 days cessation of treatment and were independent of food intake and body weight, which were unchanged. These studies highlight a role for GIP in obesity-related glucose intolerance and emphasize the potential of specific GIP-R antagonists as a new class of drugs for the alleviation of insulin resistance and treatment of type 2 diabetes.
Research Design And Methods Animals. Obese diabetic (oh/oh) mice derived from the colony maintained at Aston University, UK (Bailey, C.J., et al., 1982, Int. *I Obes. 6:11-21) were used at 12-16 weeks of age. Animals were age-matched, divided into groups and housed individually in an air-conditioned room at 222 C with a 12 hour light:12 hour dark cycle. Drinking water and a standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse effects were observed following administration of (Pro3)GIP.
Synthesis, purification and characterization of (Pro3)GIP. (Pro3)GIP was sequentially synthesized on an Applied Biosystems automated peptide synthesizer (Model 432 A). (Pro3)GIP was purified by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5) and subsequently characterized using electrospray ionization mass spectrometry (ESI-MS).
Experimental protocols for ob/ob mouse studies. Initially, extended biological activity of (Pro3)GIP was examined in 18-hour fasted ob/ob mice 4 hours after administration. Thereafter, over an 11-day period, mice received once daily i.p.
injections (17:00 hours) of either saline vehicle (0.9% (w/v), NaCl) or (Pro3)GIP (25 nmol/kg body wt). During a subsequent 9-day period, observations were continued following discontinuation of (Pro3)GIP administration. Food intake and body weight were recorded daily whilst plasma glucose and insulin concentrations were monitored at intervals of 2-6 days. Whole blood for the measurement of glycated hemoglobin was taken on days 11 and 20. Intraperitoneal glucose tolerance (18 mmol/kg body wt), metabolic response to native GIP (25 nmol/kg body wt) and insulin sensitivity (50 U/kg body wt) tests were performed on days 11 and 20. Mice fasted for 18 hours were used to examine the metabolic response to 15 minutes feeding. In a separate series, pancreatic tissues were excised at the end of the 11-day treatment period or 9 days following discontinuation of (Pro3)GIP and processed for immunohistochemistry or measurement of insulin following extraction with 5 ml/g of ice-cold acid ethanol (750 ml ethanol, 235 ml water, 15 ml concentrated HC1). Blood samples taken from the cut tip of the tail vein of conscious mice at the times indicated in the Figures were immediately centrifuged using a Beckman microcentrifuge (Beckman Instruments, UK) for 30 seconds at 13,000 g. The resulting plasma was then aliquoted into fresh Eppendorf tubes and stored at -20 C prior to glucose and insulin determinations.
Biochemical analysis. Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, J.F., 1971, Clin. Chem. Acta 32:199-201) using a Beckman Glucose Analyzer II (Beckman Instruments, Galway, Ireland). Plasma and pancreatic insulin were assayed by a modified dextran-coated charcoal radioimmunoassay (Flatt, P.R. et al., 1981, Diabetologia 20:573-577). Glycated hemoglobin was determined using cation-exchange columns (Sigma, Poole, Dorset, UK) with measurement of absorbance (415 nm) in wash and eluting buffer using a VersaMaxo Microplate Spectrophotometer (Molecular Devices, Wolcingham, Berkshire, UK).
Immunocytochemistry. Tissue fixed in 4% paraformaldehyde/PBS and embedded in paraffin was sectioned at 8 um. After de-waxing, sections were incubated with blocking serum (Vector Laboratories, CA, USA) prior to exposure to insulin antibody.
Tissue samples were then incubated consecutively with secondary biotinylated universal, pan-specific antibody (Vector Laboratories, CA, USA) and streptavidin/peroxidase preformed complex (Vector Laboratories, CA, USA).
Following washing, the stained pancreatic tissue was counterstained with hematoxylin (BDH Chemicals, Dorset, UK) and then plunged into acid methanol (500 ml methanol, 500 ml H20 and 2.5 ml concentrated HCI) prior to dehydration and mounting in Depex (BDH Chemicals, Dorset, UK). The stained slides were viewed under a microscope (Nikon Eclipse E2000 , Diagnostic Instruments Incorporated, Michigan, USA) attached to a NC camera Model KY-F55B (JVC, London, UK) and analyzed using Kromoscan imaging software (Kinetic Imaging Limited, Faversham, Kent, UK). The average number and diameter of every islet in each section was estimated in a blinded manner using an eyepiece graticule calibrated with a stage micrometer (Graticules Limited, Tonbridge, Kent, UK). The longest and shortest diameters of each islet were determined with the graticule. Half of the sum of these two values was then considered to be the average islet diameter. Approximately 70 random sections were examined from the pancreas of each mouse.
Statistics. Results are expressed as mean SEM. Data were compared using ANOVA, followed by a Student-Newman-Keuls post hoc test. Area under the curve (AUC) analyzes were calculated using the trapezoidal rule with baseline subtraction (Burington, R.S., Handbook of Mathematical Tables and Formulae, New York, McGraw-Hill, 1973). P < 0.05 was considered to be statistically significant.
Results Effects of (Pro3)GIP on plasma glucose and insulin concentrations 4 hours after administration were examined. The results are shown in Figs. 42A through 42D, which are a set of two line graphs (Figs. 42A, 42C) and two bar graphs (Figs.
42B, 42D) showing the effects of (Pro3)GIP on plasma glucose and insulin response to native GIP 4 hours after administration. Tests were conducted 4 hours after administration of (Pro3)GIP (25 nmoles/kg body weight) or saline (0.9% NaC1) in 18 hour-fasted oh/oh mice. Plasma glucose and insulin concentrations were measured prior to and after i.p. administration of glucose (18 mmoles/kg body weight) in combination with native GIP (25 nmoles/kg body weight). The incremental area under the glucose or insulin curves (AUC) between 0 and 60 min are shown in the right panels. Values represent means SEM for 8 mice. *P < 0.05 and **P <
0.01 compared with saline alone group.
As shown in Figs. 42A through 42D, administration of (Pro3)GIP for 4 hours previously impaired the plasma glucose and insulin responses to native GIP, given together with glucose. AUC glucose and insulin values were increased by 151%
(P <
0.05) and decreased by 25% (P < 0.05); respectively, compared with saline-treated controls. This supports a protracted biological half-life and forms the basis of the once-daily injection.
The effects of (Pro3)GIP on food intake, body weight and non-fasting plasma glucose and insulin concentrations were studied. The results are shown in Figs. 43A
through 43D, which are a set of two line graphs and two bar graphs showing the effects of daily (Pro3)GIP administration on food intake (Fig. 43A), body weight (Fig.
43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D) concentrations in oh/oh mice.
Parameters were measured for 5 days prior to, 11 days during (indicated by black bar) and 9 days after treatment with saline or (Pro3)GIP (25 nmol/kg bw/day).
Values are mean SEM for eight mice. *P < 0.05 compared with saline group.
Administration of (Pro3)GIP had no effect on food intake and body weight (Fig. 43A and 43B). On day 11, plasma glucose had declined to significantly reduced (P <0.05) concentrations in oh/oh mice receiving (Pro3)GIP (Fig. 43C).
Cessation of treatment returned plasma glucose concentrations towards control levels.
Consistent with this pattern, glycated hemoglobin was significantly lower (P < 0.05) after 11 days treatment with (Pro3)GIP than either before or 9 days following cessation of daily injection (8.0 0.3%, 6.9 0.2%, 7.7 0.4%, respectively). No significant changes in plasma insulin levels were noted during or after the treatment period.
However, there was a general trend for plasma insulin concentrations to decrease progressively during (Pro3)GIP treatment (Fig. 43D).
The effects of (Pro3)GIP on glucose tolerance are shown in Figs. 44A through 44D, which are a set of four line graphs with inset bar graphs showing the effects of daily (Pro3)GIP administration on glucose tolerance and plasma insulin response to glucose in ob/ob mice. Tests were conducted after daily treatment with (Pro3)GIP (25 nmoles/kg body weight/day; = ; black bars) or saline (control; o; white bars) for 11 days (Fig. 44A, 44C) or 9 days after cessation of treatment (Fig. 44B, 44B).
Glucose (18 mmoles/kg body weight) was administered at the time indicated by the arrow.
Plasma glucose (Fig. 44A, 44B) and insulin (Fig. 44C, 44D) AUC values for 0-60 minutes post injection, with identical baseline subtractions in each case to demonstrate the complete effect of (Pro3)GIP treatment, are shown in insets.
Values are mean + SEM for eight mice. *P < 0.05, **P <0.01 and ***P <0.001 compared with saline group.
Daily administration of (Pro3)GIP for 11 days resulted in significantly reduced (P <0.001) plasma glucose concentrations at 15, 30 and 60 minutes following intraperitoneal glucose (Fig. 44A). This was corroborated by a significantly decreased 0-60 minutes AUC value (Fig. 44A) which was 2.1-fold reduced (P <
0.01) compared to controls. Plasma insulin concentrations were also significantly (P
<
0.05) reduced 15, 30 and 60 minutes following intraperitoneal glucose injection in the (Pro3)GIP treated group (Fig. 44A). AUC, 0-60 minutes values were also significantly decreased (P < 0.001). Interestingly, an almost identical pattern was observed when 11 day treated ob/ob mice were administered glucose together with native GIP (25 nmoles/kg body weight) (data not shown). This supports the view that GIP action was effectively antagonized in the (Pro3)GIP treated group.
Discontinuation of (Pro3)GIP treatment for 9 days (day 20 of study) resulted in almost identical plasma glucose and insulin responses to intraperitoneal glucose (Fig. 44), with lower glucose-mediated plasma insulin concentrations noted at one time point (15 minutes; P <0.05).
The effects of (Pro3)GIP on metabolic response to feeding and insulin sensitivity are shown in Figs. 45 and 46. Figs. 45A through 45D are a set of two line graphs (Figs. 45A, 45C) and two bar graphs (Figs. 45B, 45D) showing the effects of daily (Pro3)GIP administration (A; black bars) or saline (o; white bars) on glucose (Figs. 45A, 45B) and insulin (Figs. 45C, 45D) responses to feeding in ob/ob mice fasted for 18 hours. Tests were conducted after daily treatment with (Pro3)GIP
(25 nmol/kg body weight/day) or saline for 11 days. The arrow indicates the time of feeding (15 minutes). AUC values for 0 105 minutes post-feeding are also shown.
Values are mean SEM for eight mice. *P < 0.05 compared with saline group.
Figs. 46A through 46D are a set of two line graphs (Figs. 46A, 46C) and two bar graphs (Figs. 46B, 46D) showing the effects of daily (Pro3)GIP
administration on insulin sensitivity in ob/ob mice. Tests were conducted after daily treatment with (Pro3)GIP (25 nmol/kg body weight/day; = ; black bars) or saline (o; white bars) for 11 days (Fig. 46A, 46B) or 9 days after cessation of treatment (Fig. 46C, 46D).
Insulin (50 U/kg body weight) was administered by intraperitoneal injection at the time indicated by the arrow. AUC values for 0 60 minutes post-injection are also shown. Values are mean SEM for eight mice. *13 <0.05 compared with saline group.
Plasma glucose and insulin responses to 15 minutes feeding were significantly lowered (P < 0.05) at 30 and 60 minutes in ob/ob mice treated with (Pro3)GIP
for 11 days (Fig. 45). Similarly, AUC glucose and insulin were significantly (P <
0.05) decreased in (Pro3)GIP treated ob/ob mice, despite similar food intakes of 0.3 ¨0.5 g/mouse/15 minutes. As shown in Fig. 46A and 45B, the hypoglycemic action of insulin was significantly (P < 0.05) augmented in terms of AUC measures and post injection values in ob/ob mice treated with (Pro3)GIP for 11 days. The responses following 9 days discontinuation of (Pro3)GIP treatment were similar to saline treated controls (Fig. 45C, 45D).
The effects of (Pro3)GIP on pancreatic insulin and islet morphology are shown in Figs. 47A through 47D, and 48A through 48F. Figs. 47A through 47D are a set of four bar graphs showing the effects of daily (Pro3)GIP administration on pancreatic weight (Fig. 47A), insulin content (Fig. 47B), islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice. Parameters were measured after daily treatment with (Pro3)GIP (25 nmol/kg body weight/day; black bars) or saline (white bars) for 11 days and 9 days after cessation of treatment (day 20). Values are meanSEM for eight mice. *P <0.05 and ***P < 0.001 compared with saline group. Figs. 48A
through 48F are a set of two bar graphs (Figs. 48A, 48D) and four photomicrographs (Figs. 48B, 48C, 48E, 48F), showing the effects of daily (Pro3)GIP
administration on islet size and morphology in ob/ob)mice.
(Pro3)GIP treatment had no effect on pancreatic weight (Fig. 47A). However, pancreatic insulin content was significantly (P <0.05) decreased in ob/ob mice receiving (Pro3)GIP for 11 days compared to controls (Fig. 47B). No significant differences were observed in islet number per pancreatic section (Fig. 47C), but average islet diameter was markedly and significantly reduced (P <0.001) in (Pro3)GIP treated ob/ob mice (Fig. 47D). These effects were effectively reversed by discontinuation of (Pro3)GIP on day 20, however average islet diameter was still significantly reduced (P <0.05). As shown in Fig. 48A, more detailed analysis revealed that the reduction is islet diameter on day 11 was due to a significant decrease (P < 0.001) in the percentage of larger diameter (>0.15 mm) islets with increases in the proportion of islets with small (<0.10 mm) and medium (0.1 -0.15 mm) diameters. Figure 48D presents similar analysis following cessation of treatment, with a significant (P < 0.05) increase in the percentage of small islets still apparent. Representative images (x40 magnification) of pancreata immunohistologically stained for insulin from 11-day (Pro3)GIP treated ob/ob mice (Fig. 48B) and saline treated controls (Fig. 48C) illustrate the dramatic changes in pancreatic islet morphology induced by (Pro3)GIP treatment. Pancreata immunohistologically stained for insulin on day 20 are also shown (Fig. 48E, 48F).
Parameters were measured after daily treatment with (Pro3)GIP (25 nmol/kg body weight/day) or saline for 11 days (Fig. 48A) and 9 days after cessation of treatment (Fig. 48D). Proportion of islets classified as large (> 0.15 mm) diameter, medium (0.1 ¨0.15 mm) diameter and small (<0.1 mm) diameter are shown. Values are meanSEM for eight mice Figs. 48B, 48C, 48E and 48F are representative images (x 40 magnification) of pancreata stained for insulin following 11 days treatment with (Pro3)GIP (Fig. 48B) or saline (Fig. 48C). Corresponding images days after cessation of treatment with (Pro3)GIP (Fig. 48E) or saline (Fig.
48F) are also shown. The arrows indicate islets.
Example 6. N-Terminally Acetylated and Ly16 and Lys37-substituted GIP
This example examines the metabolic stability, biological activity and antidiabetic potential of fatty acid derivatized N-terminally modified GIP
analogues.
42B, 42D) showing the effects of (Pro3)GIP on plasma glucose and insulin response to native GIP 4 hours after administration. Tests were conducted 4 hours after administration of (Pro3)GIP (25 nmoles/kg body weight) or saline (0.9% NaC1) in 18 hour-fasted oh/oh mice. Plasma glucose and insulin concentrations were measured prior to and after i.p. administration of glucose (18 mmoles/kg body weight) in combination with native GIP (25 nmoles/kg body weight). The incremental area under the glucose or insulin curves (AUC) between 0 and 60 min are shown in the right panels. Values represent means SEM for 8 mice. *P < 0.05 and **P <
0.01 compared with saline alone group.
As shown in Figs. 42A through 42D, administration of (Pro3)GIP for 4 hours previously impaired the plasma glucose and insulin responses to native GIP, given together with glucose. AUC glucose and insulin values were increased by 151%
(P <
0.05) and decreased by 25% (P < 0.05); respectively, compared with saline-treated controls. This supports a protracted biological half-life and forms the basis of the once-daily injection.
The effects of (Pro3)GIP on food intake, body weight and non-fasting plasma glucose and insulin concentrations were studied. The results are shown in Figs. 43A
through 43D, which are a set of two line graphs and two bar graphs showing the effects of daily (Pro3)GIP administration on food intake (Fig. 43A), body weight (Fig.
43B), plasma glucose (Fig. 43C) and insulin (Fig. 43D) concentrations in oh/oh mice.
Parameters were measured for 5 days prior to, 11 days during (indicated by black bar) and 9 days after treatment with saline or (Pro3)GIP (25 nmol/kg bw/day).
Values are mean SEM for eight mice. *P < 0.05 compared with saline group.
Administration of (Pro3)GIP had no effect on food intake and body weight (Fig. 43A and 43B). On day 11, plasma glucose had declined to significantly reduced (P <0.05) concentrations in oh/oh mice receiving (Pro3)GIP (Fig. 43C).
Cessation of treatment returned plasma glucose concentrations towards control levels.
Consistent with this pattern, glycated hemoglobin was significantly lower (P < 0.05) after 11 days treatment with (Pro3)GIP than either before or 9 days following cessation of daily injection (8.0 0.3%, 6.9 0.2%, 7.7 0.4%, respectively). No significant changes in plasma insulin levels were noted during or after the treatment period.
However, there was a general trend for plasma insulin concentrations to decrease progressively during (Pro3)GIP treatment (Fig. 43D).
The effects of (Pro3)GIP on glucose tolerance are shown in Figs. 44A through 44D, which are a set of four line graphs with inset bar graphs showing the effects of daily (Pro3)GIP administration on glucose tolerance and plasma insulin response to glucose in ob/ob mice. Tests were conducted after daily treatment with (Pro3)GIP (25 nmoles/kg body weight/day; = ; black bars) or saline (control; o; white bars) for 11 days (Fig. 44A, 44C) or 9 days after cessation of treatment (Fig. 44B, 44B).
Glucose (18 mmoles/kg body weight) was administered at the time indicated by the arrow.
Plasma glucose (Fig. 44A, 44B) and insulin (Fig. 44C, 44D) AUC values for 0-60 minutes post injection, with identical baseline subtractions in each case to demonstrate the complete effect of (Pro3)GIP treatment, are shown in insets.
Values are mean + SEM for eight mice. *P < 0.05, **P <0.01 and ***P <0.001 compared with saline group.
Daily administration of (Pro3)GIP for 11 days resulted in significantly reduced (P <0.001) plasma glucose concentrations at 15, 30 and 60 minutes following intraperitoneal glucose (Fig. 44A). This was corroborated by a significantly decreased 0-60 minutes AUC value (Fig. 44A) which was 2.1-fold reduced (P <
0.01) compared to controls. Plasma insulin concentrations were also significantly (P
<
0.05) reduced 15, 30 and 60 minutes following intraperitoneal glucose injection in the (Pro3)GIP treated group (Fig. 44A). AUC, 0-60 minutes values were also significantly decreased (P < 0.001). Interestingly, an almost identical pattern was observed when 11 day treated ob/ob mice were administered glucose together with native GIP (25 nmoles/kg body weight) (data not shown). This supports the view that GIP action was effectively antagonized in the (Pro3)GIP treated group.
Discontinuation of (Pro3)GIP treatment for 9 days (day 20 of study) resulted in almost identical plasma glucose and insulin responses to intraperitoneal glucose (Fig. 44), with lower glucose-mediated plasma insulin concentrations noted at one time point (15 minutes; P <0.05).
The effects of (Pro3)GIP on metabolic response to feeding and insulin sensitivity are shown in Figs. 45 and 46. Figs. 45A through 45D are a set of two line graphs (Figs. 45A, 45C) and two bar graphs (Figs. 45B, 45D) showing the effects of daily (Pro3)GIP administration (A; black bars) or saline (o; white bars) on glucose (Figs. 45A, 45B) and insulin (Figs. 45C, 45D) responses to feeding in ob/ob mice fasted for 18 hours. Tests were conducted after daily treatment with (Pro3)GIP
(25 nmol/kg body weight/day) or saline for 11 days. The arrow indicates the time of feeding (15 minutes). AUC values for 0 105 minutes post-feeding are also shown.
Values are mean SEM for eight mice. *P < 0.05 compared with saline group.
Figs. 46A through 46D are a set of two line graphs (Figs. 46A, 46C) and two bar graphs (Figs. 46B, 46D) showing the effects of daily (Pro3)GIP
administration on insulin sensitivity in ob/ob mice. Tests were conducted after daily treatment with (Pro3)GIP (25 nmol/kg body weight/day; = ; black bars) or saline (o; white bars) for 11 days (Fig. 46A, 46B) or 9 days after cessation of treatment (Fig. 46C, 46D).
Insulin (50 U/kg body weight) was administered by intraperitoneal injection at the time indicated by the arrow. AUC values for 0 60 minutes post-injection are also shown. Values are mean SEM for eight mice. *13 <0.05 compared with saline group.
Plasma glucose and insulin responses to 15 minutes feeding were significantly lowered (P < 0.05) at 30 and 60 minutes in ob/ob mice treated with (Pro3)GIP
for 11 days (Fig. 45). Similarly, AUC glucose and insulin were significantly (P <
0.05) decreased in (Pro3)GIP treated ob/ob mice, despite similar food intakes of 0.3 ¨0.5 g/mouse/15 minutes. As shown in Fig. 46A and 45B, the hypoglycemic action of insulin was significantly (P < 0.05) augmented in terms of AUC measures and post injection values in ob/ob mice treated with (Pro3)GIP for 11 days. The responses following 9 days discontinuation of (Pro3)GIP treatment were similar to saline treated controls (Fig. 45C, 45D).
The effects of (Pro3)GIP on pancreatic insulin and islet morphology are shown in Figs. 47A through 47D, and 48A through 48F. Figs. 47A through 47D are a set of four bar graphs showing the effects of daily (Pro3)GIP administration on pancreatic weight (Fig. 47A), insulin content (Fig. 47B), islet number (Fig. 47C) and islet diameter (Fig. 47D) in ob/ob mice. Parameters were measured after daily treatment with (Pro3)GIP (25 nmol/kg body weight/day; black bars) or saline (white bars) for 11 days and 9 days after cessation of treatment (day 20). Values are meanSEM for eight mice. *P <0.05 and ***P < 0.001 compared with saline group. Figs. 48A
through 48F are a set of two bar graphs (Figs. 48A, 48D) and four photomicrographs (Figs. 48B, 48C, 48E, 48F), showing the effects of daily (Pro3)GIP
administration on islet size and morphology in ob/ob)mice.
(Pro3)GIP treatment had no effect on pancreatic weight (Fig. 47A). However, pancreatic insulin content was significantly (P <0.05) decreased in ob/ob mice receiving (Pro3)GIP for 11 days compared to controls (Fig. 47B). No significant differences were observed in islet number per pancreatic section (Fig. 47C), but average islet diameter was markedly and significantly reduced (P <0.001) in (Pro3)GIP treated ob/ob mice (Fig. 47D). These effects were effectively reversed by discontinuation of (Pro3)GIP on day 20, however average islet diameter was still significantly reduced (P <0.05). As shown in Fig. 48A, more detailed analysis revealed that the reduction is islet diameter on day 11 was due to a significant decrease (P < 0.001) in the percentage of larger diameter (>0.15 mm) islets with increases in the proportion of islets with small (<0.10 mm) and medium (0.1 -0.15 mm) diameters. Figure 48D presents similar analysis following cessation of treatment, with a significant (P < 0.05) increase in the percentage of small islets still apparent. Representative images (x40 magnification) of pancreata immunohistologically stained for insulin from 11-day (Pro3)GIP treated ob/ob mice (Fig. 48B) and saline treated controls (Fig. 48C) illustrate the dramatic changes in pancreatic islet morphology induced by (Pro3)GIP treatment. Pancreata immunohistologically stained for insulin on day 20 are also shown (Fig. 48E, 48F).
Parameters were measured after daily treatment with (Pro3)GIP (25 nmol/kg body weight/day) or saline for 11 days (Fig. 48A) and 9 days after cessation of treatment (Fig. 48D). Proportion of islets classified as large (> 0.15 mm) diameter, medium (0.1 ¨0.15 mm) diameter and small (<0.1 mm) diameter are shown. Values are meanSEM for eight mice Figs. 48B, 48C, 48E and 48F are representative images (x 40 magnification) of pancreata stained for insulin following 11 days treatment with (Pro3)GIP (Fig. 48B) or saline (Fig. 48C). Corresponding images days after cessation of treatment with (Pro3)GIP (Fig. 48E) or saline (Fig.
48F) are also shown. The arrows indicate islets.
Example 6. N-Terminally Acetylated and Ly16 and Lys37-substituted GIP
This example examines the metabolic stability, biological activity and antidiabetic potential of fatty acid derivatized N-terminally modified GIP
analogues.
These are N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37), which have an N-terminal Tyr' acetyl group, and a C-16 palmitate group linked to the epsilon-amino group of the lysine at either position 16 or position 37 of the GIP protein.
Materials and methods Animals. Obese diabetic (ob/ob) mice derived from the colony maintained at Aston University, UK were used at 12-17 weeks of age. The genetic background and characteristics of the colony used have been outlined in detail elsewhere (Bailey, C.J.
etal., 1982, Int. J. Obesity 6:11-21; Gault, V.A. et al., 2003, J. Endocrinol.
176: 133-141). Animals were housed in an air-conditioned room at 22+_2 C with a 12 hours light:12 hours dark cycle. Drinking water and standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available. All test solutions were administered by i.p. injection in a final volume of 5 ml/kg bw. Blood was collected from the cut tip of the tail vein of conscious mice into chilled fluoride/heparin microcentrifuge tubes immediately prior to injection and at the times indicated in the Figures. Plasma was separated using a Beckman microcentrifuge (Beckman Instruments, UK) at 13,000 g for 30 second and stored at ¨20 C prior to glucose and insulin determinations. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse effects were observed following acute or long-term administration of any of the peptides.
Materials. High performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkersburn, UK). Trifluoroacefic acid (TFA) and trichloroacetic acid (TCA) were obtained from Aldrich (Poole, Dorset, UK). DPP
IV, isobutylmethylxanthine (IBMX), alpha-cyano-4-hydroxycinnamic acid, cyclic AMP
and ATP were all purchased from Sigma (Poole, Dorset, UK). Fmoc-protected amino acids were from Calbiochem Novabiochem (Nottingham, UK). RPMI-1640 and DMEM tissue culture medium, foetal bovine serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). The chromatography columns used for cyclic AMP assay, Dowex AG50 WX and neutral alumina AG7 were obtained from Bio-Rad Life Science Research, Alpha Analytical, Larne, UK).
All water used in these experiments was purified using a Water Purification System (Millipore, Milford, MA, USA). All other chemicals used were of the highest purity available.
Synthesis, purification and characterisation of GIP and related analogues.
Native GIP was sequentially synthesised using standard solid-phase Fmoc peptide chemistry (ABI 432A Peptide Synthesiser) as described previously (O'Harte, F.P.M. et at., 2002, Diabetologia 45: 1281-1291). N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) were synthesised in the same way as native GIP but with the exception that the epsilon-amino groups of Lys at positions 16 or 37 were conjugated with a C-16 palmitate fatty acid. In addition, an acetyl adduct was incorporated at the N-terminal Tyr'. The synthetic peptides were judged pure by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5) and subsequently characterised using matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-ToF MS) as described previously (Gault, V.A. et at., 2002, Cell. Biol. Int. 27: 41-46).
DPP IV degradation studies. GIP and fatty acid derivatised GIP analogues were incubated at 37 C with purified porcine dipeptidylpeptidase IV (5 mU in 50 mmo1/1 triethanolamine-HC1; pH 7.8) for 0, 2, 4, 8 and 24 hours (final peptide concentration 2 mmo1/1). The reactions were subsequently terminated by addition of 10% (v/v) TFA/water and the reaction products separated using HPLC. Reaction products were applied to a Vydac C-4 column (4.6 x 250 mm; The Separations Group, Hesparia, CA) and the major degradation product GIP(3-42) separated from intact GIP. The column was equilibrated with 0.12% (v/v) TFA/water at a flow rate of 1.0 ml/minute using 0.1% (v/v) TFA in 70% acetonitrile/water with the concentration of acetonitrile in the eluting solvent being raised from 0% to 40% over 10 minutes, and then from 40% to 75% over 35 minutes. The absorbance was monitored at 206 nm using a SpectraSystem UV 2000 Detector (Thermoquest Limited, Manchester, UK) and the peaks collected manually prior to MALDI-ToF MS analysis. HPLC peak area data were used to calculate % intact peptide remaining throughout the incubation.
Cells and cell culture. Chinese hamster lung (CHL) fibroblasts stably transfected with the human GIP receptor (Gremlich, S. et al., 1995, Diabetes 44: 1202-1208) were cultured in DMEM tissue culture medium containing 10% (v/v) PBS, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Clonal pancreatic BRIN-BD11 cells (McClenaghan, N.H. et al., 1996, Diabetes 45: 1132-1140) were cultured using RPMI-1640 culture medium containing 10% (v/v) PBS, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mmo1/1 glucose. Cells were maintained at 37 C in an atmosphere of 5% CO2 and 95% air using an LEEC
incubator (Laboratory Technical Engineering, Nottingham, UK).
In vitro biological activity. Intracellular cyclic AMP production was measured using GIP-receptor transfected CHL fibroblasts (O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291). In brief, CHL cells were seeded into 12-well plates (Niinc, Roskilde, Denmark) at a density of 105 cells per well and allowed to grow for 48 hours before being loaded with tritiated adenine (2 uCi; TRK311; Amersham, Buckinghamshire, UK). The cells were then incubated at 37 C for 6 hours in 1 ml DMEM
supplemented with 0.5% (w/v) BSA and subsequently washed twice with HBS buffer (pH 7.4). Cells were then exposed to GIP/GIP analogues (10-13 to 10-6 mo1/1) in HBS
buffer in the presence of 1 mmo1/1 IBMX for 15 minutes at 37 C. The medium was subsequently removed and the cells lysed with 1 ml of 5% TCA containing 0.1 mmo1/1 unlabelled cyclic AMP and 0.1 mmo1/1 unlabelled ATP. The intracellular cyclic AMP was then separated on Dowex and alumina exchange resins as described previously (O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291).
Insulin-release studies were carried out using clonal pancreatic BRIN-BD11 cells as described previously (O'Harte, F.P.M. et al., 2002, Diabetologia 45:
1291). Briefly, BRIN-BD11 cells were seeded into 24-well plates at a density of 105 cells per well, and allowed to attach overnight at 37 C. Acute tests for insulin release were preceded by 40 minutes pre-incubation at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer supplemented with 1.1 mmo1/1 glucose. Test incubations were performed in the presence of 5.6 mmo1/1 glucose with a range of concentrations (1043 to 10-6 mo1/1) of GIP and GIP analogues. After 20 minutes incubation, the buffer was removed from each well and aliquots (200 1.t1) used for measurement of insulin.
Effects ofN-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) in ob/ob mice. Metabolic and dose-response effects of GIP and N-AcGIP(LysPAL) analogues (at 6.25 ¨25 nmoles/kg bw) following glucose administration (18 mmoles/kg bw) were examined in mice fasted for 18 hours. To evaluate long-term effects, groups of ob/ob mice received once daily intraperitoneal injections (17:00 h) for 14 days of either saline vehicle (0.9%, w/v, NaC1), native GIP or N-AcGIP(LysPAL37) (both at 12.5 nmoles/kg body weight/day). Food intake and body weight were recorded daily.
Plasma glucose and insulin concentrations were monitored at 2-6 day intervals.
At 14 days, groups of animals were used to evaluate intraperitoneal glucose tolerance (18 mmoles/kg) and insulin sensitivity (50 U/kg). In a separate series, two experimental protocols were employed to examine the possibility of GIP receptor desensitization after 14 days treatment. Acute metabolic effects of the usual injection of either saline, GIP or N-AcGIP(LysPAL37) were monitored when administered together with glucose (18 mmoles/kg). In the second, acute effects of N-AcGIP(LysPAL37) given together with glucose were examined in all 3 groups of mice. At the end of the day treatment period, pancreatic tissues were excised for measurement of insulin following extraction with 5 mug ice-cold acid ethanol (75% ethanol, 2.35% H20, 1.5% HC1). Whole blood was taken for determination of glycated hemoglobin.
Biochemical analyses. Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, J.F., 1971, Clin. Chem. Acta 32:199-201) using a Beckman Glucose Analyser II (Beckman, Galway, Ireland). Plasma insulin was determined by dextran-charcoal MA as described previously (Flaft, P.R. et al., 1981, Diabetologia 20: 573-577). Glycated hemoglobin was determined using cation-exchange columns (Sigma, Poole, Dorset, UK) with measurement of absorbance (415 nm) in wash and eluting buffers using a VersaMax microplate spectrophotometer (Molecular Devices, Wokingham, Berkshire, UK).
Statistics. Results are expressed as mean SEM. Data were compared using the unpaired Student's t-test. Where appropriate, data were compared using repeated measures ANOVA or one-way ANOVA, followed by the Student-Newman-Keuls post hoc test. Incremental areas under plasma glucose and insulin curves (AUC) were calculated using a computer-generated program employing the trapezoidal rule (Burington, R.S., 1973, Handbook of Mathematical Tables and Formulae, McGraw-Hill, New York) with baseline subtraction. Groups of data were considered to be significantly different ifp<0.05.
Results Structural characterisation by MALDI-ToF MS. Following synthesis and HPLC
purification, the molecular masses were obtained for GIP, N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) using MALDI-ToF MS (Table 3, below). The mass-to-charge (m/z) ratio for native GIP was calculated to be 4983.7 Da, corresponding very closely to the theoretical mass of 4982.4 Da. Similarly, the m/z ratios for N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) were 5268.9 Da and 5267.7 Da, respectively. These values correlate very closely to the theoretical mass (5266.1 Da), therefore, confirming the correct structures for each of the synthetic peptides.
Table 3. Structural characterisation of GIP and GIP analogues by MALDI-ToF MS.
Peptide Experimental Theoretical Difference Mr (Da) Mr (Da) (Da) GIP 4983.7 4982.4 1.3 N-AcGIP(Ly5PAL16) 5268.9 5266.1 2.8 N-AcGIP(LysPAL37) 5267.7 5266.1 1.6 Peptide samples were mixed with matrix (alpha-cyano-4-hydroxycinnamic acid) and m/z ratio vs. relative peak intensity recorded using a Voyager-DE
BioSpectrometry Workstation.
Degradation by DPP IV. Table 4, below, illustrates the % intact peptide remaining after incubation with DPP IV. Degradation of native GIP was evident after just hours with only 523% of the peptide remaining intact. After 8 hours incubation the native peptide was entirely degraded to GIP(3-42). In contrast, both N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) remained completely intact (no degradation fragment evident) even after 24 hours incubation with DPP IV.
Table 4. Percentage intact peptide remaining after incubation with DPP IV.
Peptide % Intact peptide remaining after time (hours) Native GIP 100 52 3 0 0 N-AcGIP(LysPAL116) 100 100 100 100 N-AcGIP(LysPAL37) 100 100 100 100 Values represent the % intact peptide remaining relative to the major degradation product GIP(3-42) following incubation with DPP IV as determined from HPLC
peak area data. The reactions were performed in triplicate and the means+SEM values calculated.
Changes in Cyclic AMP production. Fig. 50A shows intracellular cyclic AMP
production by GIP (A) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL16) (o) and N-AcGIP(LysPAL37) (*), as determined by column chromatography, in CHL
cells stably expressing the human GIP receptor. Each experiment was performed in triplicate (n=3) and the results expressed (means SEM) as a percentage of the maximum GIP response.
A concentration-dependent (1043 to 10-6 mo1/1) increase in cyclic AMP
production was observed with native GIP (EC50 value 18.2 nmo1/1) using CHL
cells transfected with the human GIP receptor (Fig. 50A). Likewise, both N-AcGIP(Ly5PAL16) and N-AcGIP(LysPAL37) followed a similar pattern of stimulation to that of native GIP with calculated EC50 values of 12.1 and 13.0 nmo1/1, respectively. The lower EC50 values for both analogues suggest an enhanced cyclic AMP-stimulating potency.
In vitro insulin-releasing activity. Fig. 50B shows insulin-releasing activity of glucose (5.6 mmo/1 glucose; white bars), GIP (lined bars) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL16) (grey bars) and N-AcGIP(LysPAL37) (black bars) in the clonal pancreatic beta cell line, BRIN-BD11. After a pre-incubation (40 minutes), the effects of various concentrations of peptide were tested on insulin-release during a 20 minutes incubation. Values are means SEM for 8 separate observations. *p<0.05, **p<0.01, ***p<0.001 compared to control (5.6 mmo1/1 glucose alone).
Consistent with its role as a potent insulinotropic hormone, native GIP dose-dependently stimulated insulin secretion (p<0.01 to p<0.001) compared to control (5.6 mmo1/1 glucose alone) (Fig. 50B). Likewise, both N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) significantly stimulated glucose-induced insulin secretion (p<0.05 to p<0.001). On the basis of cyclic AMP and insulin secretory data, both GIP
analogues appear to be at least equipotent to the native peptide.
Metaboliceffects in ob/ob mice. Figs. 51A through 51D are a set of two line graphs (Figs. 51A, 51C) and two bar graphs (Figs. 51B, 51D) showing glucose lowering effects (Figs. 51A, 51B) and insulin-releasing activity (Figs. 51C, 51D) of GIP and fatty acid derivatised GIP analogues in 18 hour-fasted ob/ob mice. Plasma glucose and insulin concentrations were measured prior to and after i.p.
administration of glucose alone (18 mmoles/lcg bw; o; white bars) as a control, or in combination with GIP (A; lined bars) or GIP analogues N-AcGIP(LysPAL16) (a; grey bars) and N-AcGIP(LysPAL37) (0; black bars) (25 nmoles/kg bw). The incremental area under the glucose or insulin curves (AUC) between 0 and 60 minutes are shown in the right panels. Values represent means SEM for 8 mice. *p<0.05, **p<0.01, ***p<0.001 compared to glucose alone, Ap<0.05, AAp<0.01 and Amp<0.001 compared to native GIP, rnp<0.001 compared with N-AcGIP(LysPAL16).
Basal blood glucose levels of the experimental groups were not significantly different at the start of the study (p>0.05). After injection of glucose alone, plasma glucose levels increased rapidly, attaining values of 40.3+1.5 mmo1/1 at 60 min.
Native GIP reduced plasma glucose at each of the time points monitored, however, this failed to reach significance in terms of overall glucose excursion as revealed by the AUC values (Fig. 52B). Administration of N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) produced a significant reduction in plasma glucose at each time point (p<0.01 to p<0.001) and significantly lowered glucose AUC (p<0.001 to p<0.001) when compared to glucose alone. Additionally, N-AcGIP(LysPAL16) and N-AcGIP(Ly5PAL37) decreased the overall glucose excursion (p<0.05 to p<0.001) when compared to native GIP.
The corresponding plasma insulin responses are illustrated in Figs. 51C and 51D. After administration of glucose alone (control) the maximal rise in plasma insulin was observed at 15 minutes, which then fell towards basal levels over the remaining 45 minutes. Administration of native GIP significantly elevated the overall insulinotropic response (p<0.05) compared with glucose alone. When N-AcGIP(LysPALI6) or N-AcGIP(LysPAL37) where administered together with glucose, a maximum plasma insulin concentration was observed at 15 minutes. Protracted biological activity for both analogues was clearly evident from 30 to 60 minutes.
Glucose-mediated plasma insulin concentrations were significantly higher compared in both control (p<0.01 to p<0.001) and GIP-treated animals (p<0.05 to p<0.001).
The corresponding AUC values for N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) revealed significant enhancements in overall glucose-mediated insulin release compared to native GIP (1.5-fold and 2.3-fold, respectively; p<0.01 to p<0.001). N-AcGIP(LysP AL37) was significantly more potent (1.5-fold: p<0.001) than N-AcGIP(LysPAL16) at stimulating insulin secretion.
Dose-dependent metabolic effects in ob/ob mice. Figs. 52A and 52B illustrate the dose-dependent antihyperglycaemic and insulinotropic effects of GIP and the more potent analogue N-AcGIP(LysPAL37) when administered with glucose to ob/ob mice.
They are are a pair of bar graphs showing dose-dependent effects of GIP and N-AcGIP(LysP AL37) in ob/ob mice fasted for 18 hours. The incremental area under the curve (AUC) for glucose (Fig. 52A) and insulin (Fig. 52B) between 0 and 60 minutes after i.p. administration of glucose alone (18 mmoles/kg bw; white bars) or in combination with GIP (lined bars) or N-AcGIP(LysPAL37) (each at 6.25, 12.5 and nmoles/kg bw; black bars). Values represent means SEM for 8 mice. "p<0.01 and ***p<0.001 compared to glucose alone. Ap<0.01 and p<0.001 compared to native GIP at the same dose.
Data are presented as overall AUC responses for convenience. Expressed in this manner, native GIP did not significantly affect AUC glucose and insulin at any of the doses tested. N-AcGIP(LysPAL37) was substantially more potent than native GIP
(p<0.01 to p<0.001) and exhibited prominent dose-dependent antihyperglycaemic and insulinotropic actions at all doses administered (Figs. 52A, 52B). Remarkably, even the lowest concentration of N-AcGIP(LysPAL37) tested (6.25 nmoles/kg) had highly significant antihyperglycaemic properties compared to glucose alone (p<0.001).
Consistent with this observation, 6.25 nmoles/kg N-AcGIP(LysPAL37) elicited a prominent insulin response (2.0-fold; p<0.01) compared to glucose alone.
Materials and methods Animals. Obese diabetic (ob/ob) mice derived from the colony maintained at Aston University, UK were used at 12-17 weeks of age. The genetic background and characteristics of the colony used have been outlined in detail elsewhere (Bailey, C.J.
etal., 1982, Int. J. Obesity 6:11-21; Gault, V.A. et al., 2003, J. Endocrinol.
176: 133-141). Animals were housed in an air-conditioned room at 22+_2 C with a 12 hours light:12 hours dark cycle. Drinking water and standard rodent maintenance diet (Trouw Nutrition, Cheshire, UK) were freely available. All test solutions were administered by i.p. injection in a final volume of 5 ml/kg bw. Blood was collected from the cut tip of the tail vein of conscious mice into chilled fluoride/heparin microcentrifuge tubes immediately prior to injection and at the times indicated in the Figures. Plasma was separated using a Beckman microcentrifuge (Beckman Instruments, UK) at 13,000 g for 30 second and stored at ¨20 C prior to glucose and insulin determinations. All animal experiments were carried out in accordance with the UK Animals (Scientific Procedures) Act 1986. No adverse effects were observed following acute or long-term administration of any of the peptides.
Materials. High performance liquid chromatography (HPLC) grade acetonitrile was obtained from Rathburn (Walkersburn, UK). Trifluoroacefic acid (TFA) and trichloroacetic acid (TCA) were obtained from Aldrich (Poole, Dorset, UK). DPP
IV, isobutylmethylxanthine (IBMX), alpha-cyano-4-hydroxycinnamic acid, cyclic AMP
and ATP were all purchased from Sigma (Poole, Dorset, UK). Fmoc-protected amino acids were from Calbiochem Novabiochem (Nottingham, UK). RPMI-1640 and DMEM tissue culture medium, foetal bovine serum, penicillin and streptomycin were all purchased from Gibco (Paisley, Strathclyde, UK). The chromatography columns used for cyclic AMP assay, Dowex AG50 WX and neutral alumina AG7 were obtained from Bio-Rad Life Science Research, Alpha Analytical, Larne, UK).
All water used in these experiments was purified using a Water Purification System (Millipore, Milford, MA, USA). All other chemicals used were of the highest purity available.
Synthesis, purification and characterisation of GIP and related analogues.
Native GIP was sequentially synthesised using standard solid-phase Fmoc peptide chemistry (ABI 432A Peptide Synthesiser) as described previously (O'Harte, F.P.M. et at., 2002, Diabetologia 45: 1281-1291). N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) were synthesised in the same way as native GIP but with the exception that the epsilon-amino groups of Lys at positions 16 or 37 were conjugated with a C-16 palmitate fatty acid. In addition, an acetyl adduct was incorporated at the N-terminal Tyr'. The synthetic peptides were judged pure by reversed-phase HPLC on a Waters Millenium 2010 chromatography system (Software version 2.1.5) and subsequently characterised using matrix assisted laser desorption ionisation-time of flight mass spectrometry (MALDI-ToF MS) as described previously (Gault, V.A. et at., 2002, Cell. Biol. Int. 27: 41-46).
DPP IV degradation studies. GIP and fatty acid derivatised GIP analogues were incubated at 37 C with purified porcine dipeptidylpeptidase IV (5 mU in 50 mmo1/1 triethanolamine-HC1; pH 7.8) for 0, 2, 4, 8 and 24 hours (final peptide concentration 2 mmo1/1). The reactions were subsequently terminated by addition of 10% (v/v) TFA/water and the reaction products separated using HPLC. Reaction products were applied to a Vydac C-4 column (4.6 x 250 mm; The Separations Group, Hesparia, CA) and the major degradation product GIP(3-42) separated from intact GIP. The column was equilibrated with 0.12% (v/v) TFA/water at a flow rate of 1.0 ml/minute using 0.1% (v/v) TFA in 70% acetonitrile/water with the concentration of acetonitrile in the eluting solvent being raised from 0% to 40% over 10 minutes, and then from 40% to 75% over 35 minutes. The absorbance was monitored at 206 nm using a SpectraSystem UV 2000 Detector (Thermoquest Limited, Manchester, UK) and the peaks collected manually prior to MALDI-ToF MS analysis. HPLC peak area data were used to calculate % intact peptide remaining throughout the incubation.
Cells and cell culture. Chinese hamster lung (CHL) fibroblasts stably transfected with the human GIP receptor (Gremlich, S. et al., 1995, Diabetes 44: 1202-1208) were cultured in DMEM tissue culture medium containing 10% (v/v) PBS, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin). Clonal pancreatic BRIN-BD11 cells (McClenaghan, N.H. et al., 1996, Diabetes 45: 1132-1140) were cultured using RPMI-1640 culture medium containing 10% (v/v) PBS, 1% (v/v) antibiotics (100 U/ml penicillin, 0.1 mg/ml streptomycin) and 11.1 mmo1/1 glucose. Cells were maintained at 37 C in an atmosphere of 5% CO2 and 95% air using an LEEC
incubator (Laboratory Technical Engineering, Nottingham, UK).
In vitro biological activity. Intracellular cyclic AMP production was measured using GIP-receptor transfected CHL fibroblasts (O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291). In brief, CHL cells were seeded into 12-well plates (Niinc, Roskilde, Denmark) at a density of 105 cells per well and allowed to grow for 48 hours before being loaded with tritiated adenine (2 uCi; TRK311; Amersham, Buckinghamshire, UK). The cells were then incubated at 37 C for 6 hours in 1 ml DMEM
supplemented with 0.5% (w/v) BSA and subsequently washed twice with HBS buffer (pH 7.4). Cells were then exposed to GIP/GIP analogues (10-13 to 10-6 mo1/1) in HBS
buffer in the presence of 1 mmo1/1 IBMX for 15 minutes at 37 C. The medium was subsequently removed and the cells lysed with 1 ml of 5% TCA containing 0.1 mmo1/1 unlabelled cyclic AMP and 0.1 mmo1/1 unlabelled ATP. The intracellular cyclic AMP was then separated on Dowex and alumina exchange resins as described previously (O'Harte, F.P.M. et al., 2002, Diabetologia 45: 1281-1291).
Insulin-release studies were carried out using clonal pancreatic BRIN-BD11 cells as described previously (O'Harte, F.P.M. et al., 2002, Diabetologia 45:
1291). Briefly, BRIN-BD11 cells were seeded into 24-well plates at a density of 105 cells per well, and allowed to attach overnight at 37 C. Acute tests for insulin release were preceded by 40 minutes pre-incubation at 37 C in 1.0 ml Krebs Ringer bicarbonate buffer supplemented with 1.1 mmo1/1 glucose. Test incubations were performed in the presence of 5.6 mmo1/1 glucose with a range of concentrations (1043 to 10-6 mo1/1) of GIP and GIP analogues. After 20 minutes incubation, the buffer was removed from each well and aliquots (200 1.t1) used for measurement of insulin.
Effects ofN-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) in ob/ob mice. Metabolic and dose-response effects of GIP and N-AcGIP(LysPAL) analogues (at 6.25 ¨25 nmoles/kg bw) following glucose administration (18 mmoles/kg bw) were examined in mice fasted for 18 hours. To evaluate long-term effects, groups of ob/ob mice received once daily intraperitoneal injections (17:00 h) for 14 days of either saline vehicle (0.9%, w/v, NaC1), native GIP or N-AcGIP(LysPAL37) (both at 12.5 nmoles/kg body weight/day). Food intake and body weight were recorded daily.
Plasma glucose and insulin concentrations were monitored at 2-6 day intervals.
At 14 days, groups of animals were used to evaluate intraperitoneal glucose tolerance (18 mmoles/kg) and insulin sensitivity (50 U/kg). In a separate series, two experimental protocols were employed to examine the possibility of GIP receptor desensitization after 14 days treatment. Acute metabolic effects of the usual injection of either saline, GIP or N-AcGIP(LysPAL37) were monitored when administered together with glucose (18 mmoles/kg). In the second, acute effects of N-AcGIP(LysPAL37) given together with glucose were examined in all 3 groups of mice. At the end of the day treatment period, pancreatic tissues were excised for measurement of insulin following extraction with 5 mug ice-cold acid ethanol (75% ethanol, 2.35% H20, 1.5% HC1). Whole blood was taken for determination of glycated hemoglobin.
Biochemical analyses. Plasma glucose was assayed by an automated glucose oxidase procedure (Stevens, J.F., 1971, Clin. Chem. Acta 32:199-201) using a Beckman Glucose Analyser II (Beckman, Galway, Ireland). Plasma insulin was determined by dextran-charcoal MA as described previously (Flaft, P.R. et al., 1981, Diabetologia 20: 573-577). Glycated hemoglobin was determined using cation-exchange columns (Sigma, Poole, Dorset, UK) with measurement of absorbance (415 nm) in wash and eluting buffers using a VersaMax microplate spectrophotometer (Molecular Devices, Wokingham, Berkshire, UK).
Statistics. Results are expressed as mean SEM. Data were compared using the unpaired Student's t-test. Where appropriate, data were compared using repeated measures ANOVA or one-way ANOVA, followed by the Student-Newman-Keuls post hoc test. Incremental areas under plasma glucose and insulin curves (AUC) were calculated using a computer-generated program employing the trapezoidal rule (Burington, R.S., 1973, Handbook of Mathematical Tables and Formulae, McGraw-Hill, New York) with baseline subtraction. Groups of data were considered to be significantly different ifp<0.05.
Results Structural characterisation by MALDI-ToF MS. Following synthesis and HPLC
purification, the molecular masses were obtained for GIP, N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) using MALDI-ToF MS (Table 3, below). The mass-to-charge (m/z) ratio for native GIP was calculated to be 4983.7 Da, corresponding very closely to the theoretical mass of 4982.4 Da. Similarly, the m/z ratios for N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) were 5268.9 Da and 5267.7 Da, respectively. These values correlate very closely to the theoretical mass (5266.1 Da), therefore, confirming the correct structures for each of the synthetic peptides.
Table 3. Structural characterisation of GIP and GIP analogues by MALDI-ToF MS.
Peptide Experimental Theoretical Difference Mr (Da) Mr (Da) (Da) GIP 4983.7 4982.4 1.3 N-AcGIP(Ly5PAL16) 5268.9 5266.1 2.8 N-AcGIP(LysPAL37) 5267.7 5266.1 1.6 Peptide samples were mixed with matrix (alpha-cyano-4-hydroxycinnamic acid) and m/z ratio vs. relative peak intensity recorded using a Voyager-DE
BioSpectrometry Workstation.
Degradation by DPP IV. Table 4, below, illustrates the % intact peptide remaining after incubation with DPP IV. Degradation of native GIP was evident after just hours with only 523% of the peptide remaining intact. After 8 hours incubation the native peptide was entirely degraded to GIP(3-42). In contrast, both N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) remained completely intact (no degradation fragment evident) even after 24 hours incubation with DPP IV.
Table 4. Percentage intact peptide remaining after incubation with DPP IV.
Peptide % Intact peptide remaining after time (hours) Native GIP 100 52 3 0 0 N-AcGIP(LysPAL116) 100 100 100 100 N-AcGIP(LysPAL37) 100 100 100 100 Values represent the % intact peptide remaining relative to the major degradation product GIP(3-42) following incubation with DPP IV as determined from HPLC
peak area data. The reactions were performed in triplicate and the means+SEM values calculated.
Changes in Cyclic AMP production. Fig. 50A shows intracellular cyclic AMP
production by GIP (A) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL16) (o) and N-AcGIP(LysPAL37) (*), as determined by column chromatography, in CHL
cells stably expressing the human GIP receptor. Each experiment was performed in triplicate (n=3) and the results expressed (means SEM) as a percentage of the maximum GIP response.
A concentration-dependent (1043 to 10-6 mo1/1) increase in cyclic AMP
production was observed with native GIP (EC50 value 18.2 nmo1/1) using CHL
cells transfected with the human GIP receptor (Fig. 50A). Likewise, both N-AcGIP(Ly5PAL16) and N-AcGIP(LysPAL37) followed a similar pattern of stimulation to that of native GIP with calculated EC50 values of 12.1 and 13.0 nmo1/1, respectively. The lower EC50 values for both analogues suggest an enhanced cyclic AMP-stimulating potency.
In vitro insulin-releasing activity. Fig. 50B shows insulin-releasing activity of glucose (5.6 mmo/1 glucose; white bars), GIP (lined bars) and fatty acid derivatised GIP analogues N-AcGIP(LysPAL16) (grey bars) and N-AcGIP(LysPAL37) (black bars) in the clonal pancreatic beta cell line, BRIN-BD11. After a pre-incubation (40 minutes), the effects of various concentrations of peptide were tested on insulin-release during a 20 minutes incubation. Values are means SEM for 8 separate observations. *p<0.05, **p<0.01, ***p<0.001 compared to control (5.6 mmo1/1 glucose alone).
Consistent with its role as a potent insulinotropic hormone, native GIP dose-dependently stimulated insulin secretion (p<0.01 to p<0.001) compared to control (5.6 mmo1/1 glucose alone) (Fig. 50B). Likewise, both N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) significantly stimulated glucose-induced insulin secretion (p<0.05 to p<0.001). On the basis of cyclic AMP and insulin secretory data, both GIP
analogues appear to be at least equipotent to the native peptide.
Metaboliceffects in ob/ob mice. Figs. 51A through 51D are a set of two line graphs (Figs. 51A, 51C) and two bar graphs (Figs. 51B, 51D) showing glucose lowering effects (Figs. 51A, 51B) and insulin-releasing activity (Figs. 51C, 51D) of GIP and fatty acid derivatised GIP analogues in 18 hour-fasted ob/ob mice. Plasma glucose and insulin concentrations were measured prior to and after i.p.
administration of glucose alone (18 mmoles/lcg bw; o; white bars) as a control, or in combination with GIP (A; lined bars) or GIP analogues N-AcGIP(LysPAL16) (a; grey bars) and N-AcGIP(LysPAL37) (0; black bars) (25 nmoles/kg bw). The incremental area under the glucose or insulin curves (AUC) between 0 and 60 minutes are shown in the right panels. Values represent means SEM for 8 mice. *p<0.05, **p<0.01, ***p<0.001 compared to glucose alone, Ap<0.05, AAp<0.01 and Amp<0.001 compared to native GIP, rnp<0.001 compared with N-AcGIP(LysPAL16).
Basal blood glucose levels of the experimental groups were not significantly different at the start of the study (p>0.05). After injection of glucose alone, plasma glucose levels increased rapidly, attaining values of 40.3+1.5 mmo1/1 at 60 min.
Native GIP reduced plasma glucose at each of the time points monitored, however, this failed to reach significance in terms of overall glucose excursion as revealed by the AUC values (Fig. 52B). Administration of N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) produced a significant reduction in plasma glucose at each time point (p<0.01 to p<0.001) and significantly lowered glucose AUC (p<0.001 to p<0.001) when compared to glucose alone. Additionally, N-AcGIP(LysPAL16) and N-AcGIP(Ly5PAL37) decreased the overall glucose excursion (p<0.05 to p<0.001) when compared to native GIP.
The corresponding plasma insulin responses are illustrated in Figs. 51C and 51D. After administration of glucose alone (control) the maximal rise in plasma insulin was observed at 15 minutes, which then fell towards basal levels over the remaining 45 minutes. Administration of native GIP significantly elevated the overall insulinotropic response (p<0.05) compared with glucose alone. When N-AcGIP(LysPALI6) or N-AcGIP(LysPAL37) where administered together with glucose, a maximum plasma insulin concentration was observed at 15 minutes. Protracted biological activity for both analogues was clearly evident from 30 to 60 minutes.
Glucose-mediated plasma insulin concentrations were significantly higher compared in both control (p<0.01 to p<0.001) and GIP-treated animals (p<0.05 to p<0.001).
The corresponding AUC values for N-AcGIP(LysPAL16) and N-AcGIP(LysPAL37) revealed significant enhancements in overall glucose-mediated insulin release compared to native GIP (1.5-fold and 2.3-fold, respectively; p<0.01 to p<0.001). N-AcGIP(LysP AL37) was significantly more potent (1.5-fold: p<0.001) than N-AcGIP(LysPAL16) at stimulating insulin secretion.
Dose-dependent metabolic effects in ob/ob mice. Figs. 52A and 52B illustrate the dose-dependent antihyperglycaemic and insulinotropic effects of GIP and the more potent analogue N-AcGIP(LysPAL37) when administered with glucose to ob/ob mice.
They are are a pair of bar graphs showing dose-dependent effects of GIP and N-AcGIP(LysP AL37) in ob/ob mice fasted for 18 hours. The incremental area under the curve (AUC) for glucose (Fig. 52A) and insulin (Fig. 52B) between 0 and 60 minutes after i.p. administration of glucose alone (18 mmoles/kg bw; white bars) or in combination with GIP (lined bars) or N-AcGIP(LysPAL37) (each at 6.25, 12.5 and nmoles/kg bw; black bars). Values represent means SEM for 8 mice. "p<0.01 and ***p<0.001 compared to glucose alone. Ap<0.01 and p<0.001 compared to native GIP at the same dose.
Data are presented as overall AUC responses for convenience. Expressed in this manner, native GIP did not significantly affect AUC glucose and insulin at any of the doses tested. N-AcGIP(LysPAL37) was substantially more potent than native GIP
(p<0.01 to p<0.001) and exhibited prominent dose-dependent antihyperglycaemic and insulinotropic actions at all doses administered (Figs. 52A, 52B). Remarkably, even the lowest concentration of N-AcGIP(LysPAL37) tested (6.25 nmoles/kg) had highly significant antihyperglycaemic properties compared to glucose alone (p<0.001).
Consistent with this observation, 6.25 nmoles/kg N-AcGIP(LysPAL37) elicited a prominent insulin response (2.0-fold; p<0.01) compared to glucose alone.
Long-acting effects in ob/ob mice. The effects of daily injection of N-AcGIP(LysPAL37) for 14 days on food intake, body weight, glycated hemoglobin and non-fasting plasma glucose and insulin concentrations of ob/ob mice are shown in Figs. 53A through 53E, which are a set of graphs showing the effects of daily N-AcGIP(LysPAL37) (.;black bars) administration on food intake (Fig. 53A), body weight (Fig. 53B), plasma glucose (Fig. 53C), insulin (Fig. 53D) and glycated hemoglobin N-AcGIP(LysPAL37) (12.5 nmoles/kg/day) (Fig. 53E). Native GIP (12.5 nmoles/kg/day; = ; lined bars) or saline vehicle (control; o; white bars) were administered for the 14-day period indicated by the horizontal black bar.
Values are means SEM for 8 mice. *p<0.05, "p<0.01 compared to control. mp<0.01 compared to native GIP.
GIP or N-AcGIP(LysPAL37) had no effect on body weight or food intake (Figs. 53A, 53B). Plasma glucose and insulin concentrations were also unchanged by treatment with native GIP for 14 days (Figs. 53C, 53D). In contrast, daily injection of N-AcGIP(LysPAL37) resulted in a progressive lowering of plasma glucose, resulting in significantly (p<0.05) lowered concentrations at 14 days (Fig. 53C). At this time, glycated hemoglobin was also significantly (p<0.01) decreased in N-AcGIP(LysPAL37) treated ob/ob mice (Fig. 53E). These changes were accompanied by a tendency towards elevated insulin concentrations, but these did not achieve statistical significance over the time frame studies (Fig. 53D).
Values are means SEM for 8 mice. *p<0.05, "p<0.01 compared to control. mp<0.01 compared to native GIP.
GIP or N-AcGIP(LysPAL37) had no effect on body weight or food intake (Figs. 53A, 53B). Plasma glucose and insulin concentrations were also unchanged by treatment with native GIP for 14 days (Figs. 53C, 53D). In contrast, daily injection of N-AcGIP(LysPAL37) resulted in a progressive lowering of plasma glucose, resulting in significantly (p<0.05) lowered concentrations at 14 days (Fig. 53C). At this time, glycated hemoglobin was also significantly (p<0.01) decreased in N-AcGIP(LysPAL37) treated ob/ob mice (Fig. 53E). These changes were accompanied by a tendency towards elevated insulin concentrations, but these did not achieve statistical significance over the time frame studies (Fig. 53D).
Effects of long term treatment of ob/ob mice with N-AcGIP (LysPAL37) on glucose tolerance. Figs. 54A through 54D are a set of two line graphs (Figs. 54A, 54C) and two bar graphs (Figs. 54B, 54D) showing the effects of daily N-AcGIP(LysPAL37) administration on glucose tolerance (Figs. 54A, 54B) and plasma insulin response (Figs. 54C, 54D) to glucose. Tests were conducted after 14 daily injections of either N-AcGIP(Ly5PAL37) (12.5 nmoles/kg/day; 41; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; o; white bars).
Glucose (18 mmoles/kg) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels. Values are means SEM for 8 mice. *p<0.05, **p<0.01, ***p<0.001 compared to control. 6p<0.05, mp<0.01, LiMp<0.001 compared to native GIP.
Consistent with effects on glycated hemoglobin, treatment of ob/ob mice for 14 days with N-AcGIP(LysPAL37) resulted in a significant improvement in glucose tolerance (Figs. 54A, 54B). Plasma glucose concentrations throughout the test and the overall 0-60 minutes AUC values were decreased (p<0.01 to p<0.001). This was accompanied by increased insulin concentrations during the latter stages (p<0.05) and a greater (p<0.01) overall AUC insulin response (Figs. 54C, 54D). In contrast, daily administration of native GIP had no effect on glucose tolerance or the plasma insulin response to glucose compared with control ob/ob mice receiving saline injections for 14 days (Fig. 54).
Effects long term treatment of ob/ob mice with N-AcGIP(LysPAL37) on insulin sensitivity, and effects of long term treatment of ob/ob mice with N-AcGIP(LysPAL37) on pancreatic insulin content. Figs. 55A through 55D are a line graph and three bar graphs showing the effects of daily N-AcGIP(LysPAL37) administration on insulin sensitivity (Figs. 55A, 55B) and pancreatic weight (Fig. 55C) and insulin content (Fig.
55D). Observations were conducted after 14 daily injections of either N-AcG1P(LysP AL37) (12.5 nmoles/kg/day; *; black bars), native GIP (12.5 nmoles/kg/day; = ; lined bars) or saline vehicle (control; o; white bars). In Fig. 55A, insulin (50 U/kg) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose AUC values for 0-60 minutes post injection are shown in the right panels. Values are means SEM for 8 mice. *p<0.05, **p<0.01 compared to control. A<0.05, mp<0.01 compared to native GIP.
Insulin sensitivity of the 3 groups of mice after 14 days treatment is shown in Figs. 55A, 55B. Compared with ob/ob mice receiving daily injections of saline or native GIP, N-AcGIP(LysPAL37) prompted a significant improvement of insulin sensitivity. Both the individual glucose concentrations and 0-60 minutes AUC
values were significantly different (p<0.01) from the other two groups. In contrast, daily treatment with native GIP did not affect the characteristic insulin resistance of ob/ob mice (Fig. 55A, 55B).
Treatment of ob/ob mice for 14 days with native GIP or N-AcGIP(LysPAL37) did not affect pancreatic weight compared with saline-treated controls (Figs.
55C, 55D). Similarly, pancreatic insulin content was similar in the GIP and saline treated groups. However, daily administration of N-AcGIP(LysPAL37) significantly increased (p<0.01) insulin content compared with each of the other groups (Figs. 55C, 55D).
Evaluation of GIP receptor desensitization after long term treatment of ob/ob mice with N-AcGIP (LysPAL37). Figs. 56A through 56D are a set of two line graphs (Figs.
56A, 56C) and two bar graphs (Figs. 56B, 56D) showing the retention of glucose lowering (Figs. 56A, 56B) and insulin releasing (Figs. 56C, 56D) activity of N-AcGIP(LysP AL37) and native GIP after daily injection for 14 days. Glucose (18 mmoles/kg) was administered by intraperitoneal injection alone (o; white bars) or in combination with either N-AcGIP(LysPAL37) (0; black bars) or native GIP (A;
lined bars) (both at 25 nmoles/kg) at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels.
Values are means + SEM for 8 mice. *p<0.05, "p<0.01 compared to glucose alone.
Ap<0.05, mp<0.01 compared to native GIP. Figs. 57A through 57D are a set of two line graphs (Figs. 57A, 57C) and two bar graphs (Figs. 57B, 57D) showing the acute glucose lowering (Figs. 57A, 57B) and insulin releasing (Figs. 57C, 57D) effects of N-AcGIP(LysPAL37) after 14 daily injections of either N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; 0; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; o; white bars). N-AcGIP(LysPAL37) (25 nmoles/kg) was administered by intraperitoneal injection with glucose (18 mmoles/kg) at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels. Values are means SEM for 8 mice.
*p<0.05, **p<0.01 compared to mice receiving control injections. 6p<0.05, '6"6'p<0.01 compared to group receiving injections of native GIP.
As shown in Figs. 56A through 56D, treatment of ob/ob mice with N-AcGIP(LysPAL37) for 14 days did not prevent the ability of the peptide to significantly moderate the glycaemic excursion (p<0.01) and enhance plasma insulin concentrations (p<0.01) when administered acutely with intraperitoneal glucose. In contrast, the responses of ob/ob mice to acute administration of native GIP
were almost identical in mice receiving treatment with GIP or saline for 14 days (Figs. 56A
- 56D). To further substantiate the lack of GIP receptor desensitization following chronic treatment with N-AcGIP(LysPAL37), the acute effects of the analogue, administered with glucose, were examined in each of the 3 groups after 14 days treatment with N-AcGIP(LysPAL37), native GIP or saline (Figs. 57A - 57D).
Apart from lower basal values in the former group, the glucose and insulin responses were identical with similar 0-60 minutes AUC measures for both plasma glucose and insulin concentrations.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
Glucose (18 mmoles/kg) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels. Values are means SEM for 8 mice. *p<0.05, **p<0.01, ***p<0.001 compared to control. 6p<0.05, mp<0.01, LiMp<0.001 compared to native GIP.
Consistent with effects on glycated hemoglobin, treatment of ob/ob mice for 14 days with N-AcGIP(LysPAL37) resulted in a significant improvement in glucose tolerance (Figs. 54A, 54B). Plasma glucose concentrations throughout the test and the overall 0-60 minutes AUC values were decreased (p<0.01 to p<0.001). This was accompanied by increased insulin concentrations during the latter stages (p<0.05) and a greater (p<0.01) overall AUC insulin response (Figs. 54C, 54D). In contrast, daily administration of native GIP had no effect on glucose tolerance or the plasma insulin response to glucose compared with control ob/ob mice receiving saline injections for 14 days (Fig. 54).
Effects long term treatment of ob/ob mice with N-AcGIP(LysPAL37) on insulin sensitivity, and effects of long term treatment of ob/ob mice with N-AcGIP(LysPAL37) on pancreatic insulin content. Figs. 55A through 55D are a line graph and three bar graphs showing the effects of daily N-AcGIP(LysPAL37) administration on insulin sensitivity (Figs. 55A, 55B) and pancreatic weight (Fig. 55C) and insulin content (Fig.
55D). Observations were conducted after 14 daily injections of either N-AcG1P(LysP AL37) (12.5 nmoles/kg/day; *; black bars), native GIP (12.5 nmoles/kg/day; = ; lined bars) or saline vehicle (control; o; white bars). In Fig. 55A, insulin (50 U/kg) was administered by intraperitoneal injection at the time indicated by the arrow. Plasma glucose AUC values for 0-60 minutes post injection are shown in the right panels. Values are means SEM for 8 mice. *p<0.05, **p<0.01 compared to control. A<0.05, mp<0.01 compared to native GIP.
Insulin sensitivity of the 3 groups of mice after 14 days treatment is shown in Figs. 55A, 55B. Compared with ob/ob mice receiving daily injections of saline or native GIP, N-AcGIP(LysPAL37) prompted a significant improvement of insulin sensitivity. Both the individual glucose concentrations and 0-60 minutes AUC
values were significantly different (p<0.01) from the other two groups. In contrast, daily treatment with native GIP did not affect the characteristic insulin resistance of ob/ob mice (Fig. 55A, 55B).
Treatment of ob/ob mice for 14 days with native GIP or N-AcGIP(LysPAL37) did not affect pancreatic weight compared with saline-treated controls (Figs.
55C, 55D). Similarly, pancreatic insulin content was similar in the GIP and saline treated groups. However, daily administration of N-AcGIP(LysPAL37) significantly increased (p<0.01) insulin content compared with each of the other groups (Figs. 55C, 55D).
Evaluation of GIP receptor desensitization after long term treatment of ob/ob mice with N-AcGIP (LysPAL37). Figs. 56A through 56D are a set of two line graphs (Figs.
56A, 56C) and two bar graphs (Figs. 56B, 56D) showing the retention of glucose lowering (Figs. 56A, 56B) and insulin releasing (Figs. 56C, 56D) activity of N-AcGIP(LysP AL37) and native GIP after daily injection for 14 days. Glucose (18 mmoles/kg) was administered by intraperitoneal injection alone (o; white bars) or in combination with either N-AcGIP(LysPAL37) (0; black bars) or native GIP (A;
lined bars) (both at 25 nmoles/kg) at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels.
Values are means + SEM for 8 mice. *p<0.05, "p<0.01 compared to glucose alone.
Ap<0.05, mp<0.01 compared to native GIP. Figs. 57A through 57D are a set of two line graphs (Figs. 57A, 57C) and two bar graphs (Figs. 57B, 57D) showing the acute glucose lowering (Figs. 57A, 57B) and insulin releasing (Figs. 57C, 57D) effects of N-AcGIP(LysPAL37) after 14 daily injections of either N-AcGIP(LysPAL37) (12.5 nmoles/kg/day; 0; black bars), native GIP (12.5 nmoles/kg/day; A; lined bars) or saline vehicle (control; o; white bars). N-AcGIP(LysPAL37) (25 nmoles/kg) was administered by intraperitoneal injection with glucose (18 mmoles/kg) at the time indicated by the arrow. Plasma glucose and insulin AUC values for 0-60 minutes post injection are shown in the right panels. Values are means SEM for 8 mice.
*p<0.05, **p<0.01 compared to mice receiving control injections. 6p<0.05, '6"6'p<0.01 compared to group receiving injections of native GIP.
As shown in Figs. 56A through 56D, treatment of ob/ob mice with N-AcGIP(LysPAL37) for 14 days did not prevent the ability of the peptide to significantly moderate the glycaemic excursion (p<0.01) and enhance plasma insulin concentrations (p<0.01) when administered acutely with intraperitoneal glucose. In contrast, the responses of ob/ob mice to acute administration of native GIP
were almost identical in mice receiving treatment with GIP or saline for 14 days (Figs. 56A
- 56D). To further substantiate the lack of GIP receptor desensitization following chronic treatment with N-AcGIP(LysPAL37), the acute effects of the analogue, administered with glucose, were examined in each of the 3 groups after 14 days treatment with N-AcGIP(LysPAL37), native GIP or saline (Figs. 57A - 57D).
Apart from lower basal values in the former group, the glucose and insulin responses were identical with similar 0-60 minutes AUC measures for both plasma glucose and insulin concentrations.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional volumes please contact the Canadian Patent Office.
LA PRESENTE PARTIE DE CETTE DEMANDE OU CE BREVETS
COMPREND PLUS D'UN TOME.
NOTE: Pour les tomes additionels, veillez contacter le Bureau Canadien des Brevets.
JUMBO APPLICATIONS / PATENTS
THIS SECTION OF THE APPLICATION / PATENT CONTAINS MORE
THAN ONE VOLUME.
NOTE: For additional volumes please contact the Canadian Patent Office.
Claims (12)
1. Use of a GIP receptor antagonist to treat insulin resistance, wherein the GIP receptor antagonist comprises a peptide analogue of GIP (1-42) (SEQ ID NO: 1), the peptide analogue being selected from the group consisting of (i) at least 12 amino acid residues from the N-terminal end of GIP (3-42) wherein the N-terminal amino acid of the analogue is the amino acid at position 3; and (ii) at least 12 amino acid residues from the N-terminal end of GIP (1-42) and having an amino acid substitution at Glu3 selected from the group consisting of hydroxyproline, lysine, tyrosine, phenylalanine, and tryptophan.
2. Use of a GIP receptor antagonist to treat obesity, wherein the GIP receptor antagonist comprises a peptide analogue of GIP (1-42) (SEQ ID NO: 1), the peptide analogue being selected from the group consisting of (i) at least 12 amino acid residues from the N-terminal end of GIP (3-42) wherein the N-terminal amino acid of the analogue is the amino acid at position 3; and (ii) at least 12 amino acid residues from the N-terminal end of GIP (1-42) and having an amino acid substitution at Glu3 selected from the group consisting of hydroxyproline, lysine, tyrosine, phenylalanine, and tryptophan.
3. Use of a peptide analogue of GIP (1-42) (SEQ ID NO: 1) to treat insulin resistance, wherein the analogue comprises a base peptide selected from the group consisting of GIP
(1-16), GIP (1-17), GIP (1-18), GIP (1-19), GIP (1-20), GIP (1-21), GIP (1-22), GIP (1-23), GIP (1-24), GIP (1-25), GIP (1-26), GIP (1-27), GIP (1-28), GIP (1-29), GIP (1-30), GIP (1-31), GIP (1-32), GIP (1-33), GIP (1-34), GIP (1-35), GIP (1-36), GIP (1-37), GIP (1-38), GIP
(1-39), GIP (1-40), GIP (1-41) and GIP (1-42);
wherein the base peptide is modified by N-terminal acetylation and by palmitate fatty acid addition at an epsilon amino group of at least one lysine residue.
(1-16), GIP (1-17), GIP (1-18), GIP (1-19), GIP (1-20), GIP (1-21), GIP (1-22), GIP (1-23), GIP (1-24), GIP (1-25), GIP (1-26), GIP (1-27), GIP (1-28), GIP (1-29), GIP (1-30), GIP (1-31), GIP (1-32), GIP (1-33), GIP (1-34), GIP (1-35), GIP (1-36), GIP (1-37), GIP (1-38), GIP
(1-39), GIP (1-40), GIP (1-41) and GIP (1-42);
wherein the base peptide is modified by N-terminal acetylation and by palmitate fatty acid addition at an epsilon amino group of at least one lysine residue.
4. Use of a peptide analogue of GIP (1-42) (SEQ ID NO: 1) to treat obesity, wherein the analogue comprises a base peptide selected from the group consisting of GIP (1-16), GIP
(1-17), GIP (1-18), GIP (1-19), GIP (1-20), GIP (1-21), GIP (1-22), GIP (1-23), GIP (1-24), GIP (1-25), GIP (1-26), GIP (1-27), GIP (1-28), GIP (1-29), GIP (1-30), GIP (1-31), GIP (1-32), GIP (1-33), GIP (1-34), GIP (1-35), GIP (1-36), GIP (1-37), GIP (1-38), GIP (1-39), GIP
(1-40), GIP (1-41) and GIP (1-42);
wherein the base peptide is modified by N-terminal acetylation and by palmitate fatty acid addition at an epsilon amino group of at least one lysine residue.
(1-17), GIP (1-18), GIP (1-19), GIP (1-20), GIP (1-21), GIP (1-22), GIP (1-23), GIP (1-24), GIP (1-25), GIP (1-26), GIP (1-27), GIP (1-28), GIP (1-29), GIP (1-30), GIP (1-31), GIP (1-32), GIP (1-33), GIP (1-34), GIP (1-35), GIP (1-36), GIP (1-37), GIP (1-38), GIP (1-39), GIP
(1-40), GIP (1-41) and GIP (1-42);
wherein the base peptide is modified by N-terminal acetylation and by palmitate fatty acid addition at an epsilon amino group of at least one lysine residue.
5. The use of Claim 1, further comprising modification by fatty acid addition at an epsilon amino group of at least one lysine residue.
6. The use of Claim 5, wherein the modification is the linking of a C-16 palmitate group to the epsilon amino group of a lysine residue.
7. The use of Claim 6, wherein the lysine residue is Lys16.
8. The use of Claim 6, wherein the lysine residue is Lys37.
9. The use of Claim 2, further comprising modification by fatty acid addition at an epsilon amino group of at least one lysine residue.
10. The use of Claim 9, wherein the modification is the linking of a C-16 palmitate group to the epsilon amino group of a lysine residue.
11. The use of Claim 10, wherein the lysine residue is Lys16.
12. The use of Claim 10, wherein the lysine residue is Lys37.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GBGB0404124.0A GB0404124D0 (en) | 2004-02-25 | 2004-02-25 | Antagonists of GIP |
GB0404124.0 | 2004-02-25 | ||
PCT/GB2005/000710 WO2005082928A2 (en) | 2004-02-25 | 2005-02-25 | Peptide analogues of gip for treatment of diabetes, insulin resistance and obesity |
Publications (2)
Publication Number | Publication Date |
---|---|
CA2557151A1 CA2557151A1 (en) | 2005-09-09 |
CA2557151C true CA2557151C (en) | 2015-02-17 |
Family
ID=32050802
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA2557151A Active CA2557151C (en) | 2004-02-25 | 2005-02-25 | Peptide analogues of gip for treatment of diabetes, insulin resistance and obesity |
Country Status (6)
Country | Link |
---|---|
EP (1) | EP1730188A2 (en) |
JP (1) | JP2008500280A (en) |
AU (1) | AU2005217198A1 (en) |
CA (1) | CA2557151C (en) |
GB (1) | GB0404124D0 (en) |
WO (1) | WO2005082928A2 (en) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TWI770085B (en) * | 2017-11-21 | 2022-07-11 | 日商武田藥品工業股份有限公司 | Peptide compound |
Families Citing this family (38)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7572451B2 (en) | 2004-10-25 | 2009-08-11 | Cytos Biotechnology Ag | Gastric inhibitory polypeptide (GIP) antigen arrays and uses thereof |
CA2622069A1 (en) * | 2005-09-08 | 2007-03-15 | Uutech Limited | Treatment of diabetes related obesity |
US20090286722A1 (en) * | 2005-09-08 | 2009-11-19 | Utech Limited | Analogs of Gastric Inhibitory Polypeptide as a Treatment for Age Related Decreased Pancreatic Beta Cell Function |
ES2507098T3 (en) | 2005-11-07 | 2014-10-14 | Indiana University Research And Technology Corporation | Glucagon analogs showing physiological solubility and stability |
AU2007284365A1 (en) * | 2006-08-17 | 2008-02-21 | Amylin Pharmaceuticals, Inc. | DPP-IV resistant GIP hybrid polypeptides with selectable properties |
EP2124974B1 (en) | 2007-01-05 | 2017-03-15 | Indiana University Research and Technology Corporation | Glucagon analogs exhibiting enhanced solubility in physiological ph buffers |
US8454971B2 (en) | 2007-02-15 | 2013-06-04 | Indiana University Research And Technology Corporation | Glucagon/GLP-1 receptor co-agonists |
GB0717388D0 (en) * | 2007-09-07 | 2007-10-17 | Uutech Ltd | Use of GIP for the treatment of disorders associated with dysfunctional synaptic transmission |
WO2009058662A2 (en) | 2007-10-30 | 2009-05-07 | Indiana University Research And Technology Corporation | Glucagon antagonists |
JP5771005B2 (en) | 2007-10-30 | 2015-08-26 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションIndiana University Research And Technology Corporation | Glucagon antagonist and compound showing GLP-1 agonist activity |
WO2009067268A1 (en) * | 2007-11-23 | 2009-05-28 | Michael Rothkopf | Methods of enhancing diabetes resolution |
US8546327B2 (en) | 2008-06-17 | 2013-10-01 | Indiana University Research And Technology Corporation | Glucagon/GLP-1 receptor co-agonists |
JP5753779B2 (en) | 2008-06-17 | 2015-07-22 | インディアナ ユニバーシティー リサーチ アンド テクノロジー コーポレーションIndiana University Research And Technology Corporation | Glucagon analogs with improved solubility and stability in buffers at physiological pH |
PL2300035T3 (en) | 2008-06-17 | 2016-04-29 | Univ Indiana Res & Tech Corp | Gip-based mixed agonists for treatment of metabolic disorders and obesity |
GB0814068D0 (en) * | 2008-08-01 | 2008-09-10 | Univ Ulster | Active immunisation against GIP |
EP2318433A4 (en) * | 2008-08-07 | 2012-08-08 | Ipsen Pharma Sas | Analogues of glucose-dependent insulinotropic polypeptide (gip) modified at n-terminal |
JP2011530507A (en) | 2008-08-07 | 2011-12-22 | イプセン ファルマ ソシエテ パール アクシオン サンプリフィエ | Glucose-dependent insulinotropic polypeptide analog |
MX2011001028A (en) * | 2008-08-07 | 2011-04-26 | Ipsen Pharma Sas | Truncated analogues of glucose-dependent insulinotropic polypeptide. |
EP2987805A3 (en) * | 2008-08-07 | 2016-04-13 | Ipsen Pharma S.A.S. | Analogues of glucose-dependent insulinotropic polypeptide |
KR20110110174A (en) | 2008-12-19 | 2011-10-06 | 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 | Amide based glucagon superfamily peptide prodrugs |
JP5576694B2 (en) * | 2009-04-10 | 2014-08-20 | 花王株式会社 | GIP elevation inhibitor |
MX2011013625A (en) | 2009-06-16 | 2012-01-20 | Univ Indiana Res & Tech Corp | Gip receptor-active glucagon compounds. |
WO2011075393A2 (en) | 2009-12-18 | 2011-06-23 | Indiana University Research And Technology Corporation | Glucagon/glp-1 receptor co-agonists |
RU2012136450A (en) | 2010-01-27 | 2014-03-10 | Индиана Юниверсити Рисерч Энд Текнолоджи Корпорейшн | CONJUGATES GLUCAGON ANTAGONIST - GIP AGONIST AND COMPOSITIONS FOR TREATMENT OF METABOLIC DISORDERS AND OBESITY |
KR20130111923A (en) | 2010-05-13 | 2013-10-11 | 인디애나 유니버시티 리서치 앤드 테크놀로지 코퍼레이션 | Glucagon superfamily peptides exhibiting g protein-coupled receptor activity |
CA2797095A1 (en) | 2010-05-13 | 2011-11-17 | Indiana University Research And Technology Corporation | Glucagon superfamily peptides exhibiting nuclear hormone receptor activity |
US9023986B2 (en) * | 2010-10-25 | 2015-05-05 | Hoffmann-La Roche Inc. | Glucose-dependent insulinotropic peptide analogs |
MA34885B1 (en) | 2010-12-22 | 2014-02-01 | Indiana Unversity Res And Technology Corp | GLUCAGON ANALOGS HAVING A GIP RECEPTOR ACTIVITY |
WO2012167744A1 (en) * | 2011-06-10 | 2012-12-13 | Beijing Hanmi Pharmaceutical Co., Ltd. | Glucose dependent insulinotropic polypeptide analogs, pharmaceutical compositions and use thereof |
CN103748109A (en) | 2011-06-22 | 2014-04-23 | 印第安纳大学研究及科技有限公司 | Glucagon/glp-1 receptor co-agonists |
LT2723367T (en) | 2011-06-22 | 2017-08-25 | Indiana University Research And Technology Corporation | Glucagon/glp-1 receptor co-agonists |
WO2013074910A1 (en) | 2011-11-17 | 2013-05-23 | Indiana University Research And Technology Corporation | Glucagon superfamily peptides exhibiting glucocorticoid receptor activity |
RU2015101697A (en) | 2012-06-21 | 2016-08-10 | Индиана Юниверсити Рисерч Энд Текнолоджи Корпорейшн | GLUCAGON ANALOGUES WITH GIP RECEPTOR ACTIVITY |
BR112017008659A2 (en) | 2014-10-29 | 2018-01-30 | Zealand Pharma As | ? gip agonist methods and compounds? |
JOP20180028A1 (en) | 2017-03-31 | 2019-01-30 | Takeda Pharmaceuticals Co | Peptide compound |
BR112019025195A8 (en) | 2017-05-31 | 2020-07-07 | Univ Copenhagen | long-acting gip peptide analogues |
CN112074531A (en) * | 2018-05-04 | 2020-12-11 | 诺和诺德股份有限公司 | GIP derivatives and uses thereof |
CN113827706B (en) * | 2021-09-27 | 2023-12-26 | 南通大学 | Application of GIP and its derivative peptide in preparing skin wound treating medicine |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PT1171465E (en) | 1999-03-29 | 2004-12-31 | Uutech Ltd | ANALYSIS OF A GASTRIC INHIBITOR AND THEIR USE FOR THE TREATMENT OF DIABETES |
-
2004
- 2004-02-25 GB GBGB0404124.0A patent/GB0404124D0/en not_active Ceased
-
2005
- 2005-02-25 JP JP2007500292A patent/JP2008500280A/en active Pending
- 2005-02-25 AU AU2005217198A patent/AU2005217198A1/en not_active Abandoned
- 2005-02-25 WO PCT/GB2005/000710 patent/WO2005082928A2/en active Application Filing
- 2005-02-25 EP EP05717793A patent/EP1730188A2/en not_active Withdrawn
- 2005-02-25 CA CA2557151A patent/CA2557151C/en active Active
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
TWI770085B (en) * | 2017-11-21 | 2022-07-11 | 日商武田藥品工業股份有限公司 | Peptide compound |
Also Published As
Publication number | Publication date |
---|---|
CA2557151A1 (en) | 2005-09-09 |
AU2005217198A1 (en) | 2005-09-09 |
JP2008500280A (en) | 2008-01-10 |
WO2005082928A2 (en) | 2005-09-09 |
EP1730188A2 (en) | 2006-12-13 |
WO2005082928A3 (en) | 2005-12-01 |
GB0404124D0 (en) | 2004-03-31 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CA2557151C (en) | Peptide analogues of gip for treatment of diabetes, insulin resistance and obesity | |
US7875587B2 (en) | Peptide analogues of GIP for treatment of diabetes, insulin resistance and obesity | |
EP1171465B1 (en) | Analogs of gastric inhibitory peptide and their use for treatment of diabetes | |
JP7211712B2 (en) | glucagon receptor agonist | |
KR102622642B1 (en) | Incretin analogs and uses thereof | |
JP6373270B2 (en) | Exendin-4 derivatives as dual GLP1 / glucagon agonists | |
US5604203A (en) | Analogs of peptide YY and uses thereof | |
Gault et al. | Evidence that the major degradation product of glucose-dependent insulinotropic polypeptide, GIP (3-42), is a GIP receptor antagonist in vivo | |
US20090170762A1 (en) | Treatment of Diabetes Related Obesity | |
US20090286722A1 (en) | Analogs of Gastric Inhibitory Polypeptide as a Treatment for Age Related Decreased Pancreatic Beta Cell Function | |
JP5485876B2 (en) | Unacylated ghrelin as a therapeutic agent in the treatment of metabolic diseases | |
CZ315594A3 (en) | Peptide of glucagon type, insulinotropic derivatives, process of their preparation, pharmaceutical composition containing such compounds and use | |
KR20080045185A (en) | Hybrid polypeptides with selectable properties | |
KR20210005093A (en) | Acylated GLP-1 derivatives | |
KR102230368B1 (en) | Acylated oxyntomodulin analogues | |
KR20120116942A (en) | Polypeptide conjugate | |
JP2023544959A (en) | hAM15-52 analog with improved amylin receptor (hAMY3R) potency | |
WO2024003359A1 (en) | AMYLIN RECEPTOR (hAMY3R) AGONISTS WITH IMPROVED CHEMICAL STABILITY |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
EEER | Examination request |