US20180214514A1 - Compositions and Treatments of Metabolic Disorders Using FGF Binding Protein 3 - Google Patents
Compositions and Treatments of Metabolic Disorders Using FGF Binding Protein 3 Download PDFInfo
- Publication number
- US20180214514A1 US20180214514A1 US15/784,730 US201715784730A US2018214514A1 US 20180214514 A1 US20180214514 A1 US 20180214514A1 US 201715784730 A US201715784730 A US 201715784730A US 2018214514 A1 US2018214514 A1 US 2018214514A1
- Authority
- US
- United States
- Prior art keywords
- fgfbp3
- fgf19
- subject
- amino acid
- protein
- 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.)
- Abandoned
Links
- 102100023599 Fibroblast growth factor-binding protein 3 Human genes 0.000 title claims abstract description 174
- 101710094943 Fibroblast growth factor-binding protein 3 Proteins 0.000 title claims abstract description 174
- 238000011282 treatment Methods 0.000 title claims abstract description 94
- 208000030159 metabolic disease Diseases 0.000 title claims abstract description 33
- 239000000203 mixture Substances 0.000 title description 32
- 238000000034 method Methods 0.000 claims abstract description 107
- 101710153349 Fibroblast growth factor 19 Proteins 0.000 claims description 183
- 102100031734 Fibroblast growth factor 19 Human genes 0.000 claims description 183
- 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 claims description 81
- 239000008103 glucose Substances 0.000 claims description 75
- 210000004027 cell Anatomy 0.000 claims description 48
- 210000004369 blood Anatomy 0.000 claims description 37
- 239000008280 blood Substances 0.000 claims description 37
- 230000037396 body weight Effects 0.000 claims description 22
- 206010012601 diabetes mellitus Diseases 0.000 claims description 14
- 230000004060 metabolic process Effects 0.000 claims description 12
- 230000001965 increasing effect Effects 0.000 claims description 10
- 230000014616 translation Effects 0.000 claims description 9
- 229920002527 Glycogen Polymers 0.000 claims description 8
- 229940096919 glycogen Drugs 0.000 claims description 8
- 230000002440 hepatic effect Effects 0.000 claims description 8
- 230000000291 postprandial effect Effects 0.000 claims description 7
- 201000001421 hyperglycemia Diseases 0.000 claims description 6
- 238000001243 protein synthesis Methods 0.000 claims description 6
- 230000015572 biosynthetic process Effects 0.000 claims description 5
- 208000008589 Obesity Diseases 0.000 claims description 4
- 230000033228 biological regulation Effects 0.000 claims description 4
- 238000004113 cell culture Methods 0.000 claims description 4
- 235000020824 obesity Nutrition 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 2
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 claims 1
- 125000003275 alpha amino acid group Chemical group 0.000 description 78
- 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 75
- 108090000765 processed proteins & peptides Proteins 0.000 description 70
- 241000699670 Mus sp. Species 0.000 description 59
- 108090000623 proteins and genes Proteins 0.000 description 50
- 230000000694 effects Effects 0.000 description 47
- 102000004169 proteins and genes Human genes 0.000 description 39
- 235000018102 proteins Nutrition 0.000 description 36
- 230000014509 gene expression Effects 0.000 description 33
- 235000001014 amino acid Nutrition 0.000 description 31
- 102100027844 Fibroblast growth factor receptor 4 Human genes 0.000 description 30
- 101710182387 Fibroblast growth factor receptor 4 Proteins 0.000 description 30
- 150000001413 amino acids Chemical class 0.000 description 30
- 102000004196 processed proteins & peptides Human genes 0.000 description 26
- 210000004185 liver Anatomy 0.000 description 25
- 230000027455 binding Effects 0.000 description 23
- 230000026731 phosphorylation Effects 0.000 description 23
- 238000006366 phosphorylation reaction Methods 0.000 description 23
- 238000007446 glucose tolerance test Methods 0.000 description 22
- 229920001184 polypeptide Polymers 0.000 description 22
- 241001465754 Metazoa Species 0.000 description 21
- 108010017324 STAT3 Transcription Factor Proteins 0.000 description 21
- 102100024040 Signal transducer and activator of transcription 3 Human genes 0.000 description 21
- 101100390675 Mus musculus Fgf15 gene Proteins 0.000 description 20
- 108091008611 Protein Kinase B Proteins 0.000 description 20
- 102100033810 RAC-alpha serine/threonine-protein kinase Human genes 0.000 description 20
- 102000037865 fusion proteins Human genes 0.000 description 20
- 108020001507 fusion proteins Proteins 0.000 description 20
- 229920001223 polyethylene glycol Polymers 0.000 description 16
- 102000003973 Fibroblast growth factor 21 Human genes 0.000 description 15
- 108090000376 Fibroblast growth factor 21 Proteins 0.000 description 15
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 15
- 230000004044 response Effects 0.000 description 15
- 241000699666 Mus <mouse, genus> Species 0.000 description 14
- 239000002202 Polyethylene glycol Substances 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 14
- 230000002829 reductive effect Effects 0.000 description 14
- 239000000126 substance Substances 0.000 description 14
- 238000006467 substitution reaction Methods 0.000 description 14
- 102000004042 Fibroblast Growth Factor-23 Human genes 0.000 description 13
- 108090000569 Fibroblast Growth Factor-23 Proteins 0.000 description 13
- 102000004889 Interleukin-6 Human genes 0.000 description 13
- 108090001005 Interleukin-6 Proteins 0.000 description 13
- 101710149951 Protein Tat Proteins 0.000 description 13
- 230000004913 activation Effects 0.000 description 13
- 239000007924 injection Substances 0.000 description 13
- 229940090044 injection Drugs 0.000 description 13
- 238000002347 injection Methods 0.000 description 13
- 229940100601 interleukin-6 Drugs 0.000 description 13
- 230000037361 pathway Effects 0.000 description 13
- 101150021185 FGF gene Proteins 0.000 description 12
- 102000019050 90-kDa Ribosomal Protein S6 Kinases Human genes 0.000 description 11
- 108010012196 90-kDa Ribosomal Protein S6 Kinases Proteins 0.000 description 11
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 11
- 239000003937 drug carrier Substances 0.000 description 11
- -1 i.e. Proteins 0.000 description 11
- 101150018665 MAPK3 gene Proteins 0.000 description 10
- 238000007912 intraperitoneal administration Methods 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 10
- 239000000243 solution Substances 0.000 description 10
- 230000001225 therapeutic effect Effects 0.000 description 10
- 102100038637 Cytochrome P450 7A1 Human genes 0.000 description 9
- 101000957672 Homo sapiens Cytochrome P450 7A1 Proteins 0.000 description 9
- 125000000539 amino acid group Chemical group 0.000 description 9
- 238000004458 analytical method Methods 0.000 description 9
- 150000001875 compounds Chemical class 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 230000001976 improved effect Effects 0.000 description 9
- 239000007928 intraperitoneal injection Substances 0.000 description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 9
- 108010076504 Protein Sorting Signals Proteins 0.000 description 8
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 8
- 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 8
- 239000011780 sodium chloride Substances 0.000 description 8
- 235000002639 sodium chloride Nutrition 0.000 description 8
- 208000024891 symptom Diseases 0.000 description 8
- 102000016267 Leptin Human genes 0.000 description 7
- 108010092277 Leptin Proteins 0.000 description 7
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 7
- 238000007792 addition Methods 0.000 description 7
- 230000000692 anti-sense effect Effects 0.000 description 7
- 210000004899 c-terminal region Anatomy 0.000 description 7
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 7
- 230000003828 downregulation Effects 0.000 description 7
- 230000004927 fusion Effects 0.000 description 7
- 230000005764 inhibitory process Effects 0.000 description 7
- 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 7
- 229940039781 leptin Drugs 0.000 description 7
- 238000004519 manufacturing process Methods 0.000 description 7
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 230000009467 reduction Effects 0.000 description 7
- 238000013518 transcription Methods 0.000 description 7
- 230000035897 transcription Effects 0.000 description 7
- 102100035427 Forkhead box protein O1 Human genes 0.000 description 6
- 101000877727 Homo sapiens Forkhead box protein O1 Proteins 0.000 description 6
- 101001123325 Homo sapiens Peroxisome proliferator-activated receptor gamma coactivator 1-beta Proteins 0.000 description 6
- 102000009433 Insulin Receptor Substrate Proteins Human genes 0.000 description 6
- 108010034219 Insulin Receptor Substrate Proteins Proteins 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 102100028961 Peroxisome proliferator-activated receptor gamma coactivator 1-beta Human genes 0.000 description 6
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 6
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 6
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 6
- 239000000872 buffer Substances 0.000 description 6
- 150000002632 lipids Chemical class 0.000 description 6
- 125000001360 methionine group Chemical group N[C@@H](CCSC)C(=O)* 0.000 description 6
- 239000008194 pharmaceutical composition Substances 0.000 description 6
- 238000002360 preparation method Methods 0.000 description 6
- 230000019491 signal transduction Effects 0.000 description 6
- 238000012360 testing method Methods 0.000 description 6
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 5
- ZEOWTGPWHLSLOG-UHFFFAOYSA-N Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F Chemical compound Cc1ccc(cc1-c1ccc2c(n[nH]c2c1)-c1cnn(c1)C1CC1)C(=O)Nc1cccc(c1)C(F)(F)F ZEOWTGPWHLSLOG-UHFFFAOYSA-N 0.000 description 5
- 102100029602 Eukaryotic translation initiation factor 4B Human genes 0.000 description 5
- 101710092092 Eukaryotic translation initiation factor 4B Proteins 0.000 description 5
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 5
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 5
- 102100023593 Fibroblast growth factor receptor 1 Human genes 0.000 description 5
- 101710182386 Fibroblast growth factor receptor 1 Proteins 0.000 description 5
- 108010001483 Glycogen Synthase Proteins 0.000 description 5
- 102000019149 MAP kinase activity proteins Human genes 0.000 description 5
- 108040008097 MAP kinase activity proteins Proteins 0.000 description 5
- 108700027337 Suppressor of Cytokine Signaling 3 Proteins 0.000 description 5
- 102100024283 Suppressor of cytokine signaling 3 Human genes 0.000 description 5
- 239000003613 bile acid Substances 0.000 description 5
- 239000003795 chemical substances by application Substances 0.000 description 5
- 239000002299 complementary DNA Substances 0.000 description 5
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 5
- 238000003119 immunoblot Methods 0.000 description 5
- 239000004615 ingredient Substances 0.000 description 5
- 230000004132 lipogenesis Effects 0.000 description 5
- 238000000746 purification Methods 0.000 description 5
- 102000005962 receptors Human genes 0.000 description 5
- 108020003175 receptors Proteins 0.000 description 5
- 230000011664 signaling Effects 0.000 description 5
- 239000002904 solvent Substances 0.000 description 5
- 230000001629 suppression Effects 0.000 description 5
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- HSINOMROUCMIEA-FGVHQWLLSA-N (2s,4r)-4-[(3r,5s,6r,7r,8s,9s,10s,13r,14s,17r)-6-ethyl-3,7-dihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1h-cyclopenta[a]phenanthren-17-yl]-2-methylpentanoic acid Chemical compound C([C@@]12C)C[C@@H](O)C[C@H]1[C@@H](CC)[C@@H](O)[C@@H]1[C@@H]2CC[C@]2(C)[C@@H]([C@H](C)C[C@H](C)C(O)=O)CC[C@H]21 HSINOMROUCMIEA-FGVHQWLLSA-N 0.000 description 4
- 108010088751 Albumins Proteins 0.000 description 4
- 102000009027 Albumins Human genes 0.000 description 4
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 4
- 101100093804 Caenorhabditis elegans rps-6 gene Proteins 0.000 description 4
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 4
- 108091008794 FGF receptors Proteins 0.000 description 4
- 208000002705 Glucose Intolerance Diseases 0.000 description 4
- 206010018429 Glucose tolerance impaired Diseases 0.000 description 4
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 4
- 102000004877 Insulin Human genes 0.000 description 4
- 108090001061 Insulin Proteins 0.000 description 4
- 238000011529 RT qPCR Methods 0.000 description 4
- 230000001154 acute effect Effects 0.000 description 4
- 125000003295 alanine group Chemical group N[C@@H](C)C(=O)* 0.000 description 4
- 238000010171 animal model Methods 0.000 description 4
- 239000003242 anti bacterial agent Substances 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 235000012000 cholesterol Nutrition 0.000 description 4
- 230000002950 deficient Effects 0.000 description 4
- 230000001419 dependent effect Effects 0.000 description 4
- 239000003085 diluting agent Substances 0.000 description 4
- 208000035475 disorder Diseases 0.000 description 4
- 239000006185 dispersion Substances 0.000 description 4
- 239000012636 effector Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000006870 function Effects 0.000 description 4
- 230000004110 gluconeogenesis Effects 0.000 description 4
- 230000001890 gluconeogenic effect Effects 0.000 description 4
- 230000004153 glucose metabolism Effects 0.000 description 4
- 235000011187 glycerol Nutrition 0.000 description 4
- 238000001114 immunoprecipitation Methods 0.000 description 4
- 229940125396 insulin Drugs 0.000 description 4
- 239000006166 lysate Substances 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000001019 normoglycemic effect Effects 0.000 description 4
- 230000006320 pegylation Effects 0.000 description 4
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 4
- 238000002864 sequence alignment Methods 0.000 description 4
- 230000004936 stimulating effect Effects 0.000 description 4
- 238000003860 storage Methods 0.000 description 4
- 230000002459 sustained effect Effects 0.000 description 4
- 230000003827 upregulation Effects 0.000 description 4
- 229920003169 water-soluble polymer Polymers 0.000 description 4
- 238000001262 western blot Methods 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- 102000014914 Carrier Proteins Human genes 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 102000005233 Eukaryotic Initiation Factor-4E Human genes 0.000 description 3
- 108060002636 Eukaryotic Initiation Factor-4E Proteins 0.000 description 3
- 102000001267 GSK3 Human genes 0.000 description 3
- 108060006662 GSK3 Proteins 0.000 description 3
- 108010010803 Gelatin Proteins 0.000 description 3
- 208000031886 HIV Infections Diseases 0.000 description 3
- 101100012875 Homo sapiens FGFBP3 gene Proteins 0.000 description 3
- 206010022489 Insulin Resistance Diseases 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000005917 acylation reaction Methods 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 108091008324 binding proteins Proteins 0.000 description 3
- 238000004422 calculation algorithm Methods 0.000 description 3
- 230000005754 cellular signaling Effects 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 230000009918 complex formation Effects 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 239000002612 dispersion medium Substances 0.000 description 3
- 102000052178 fibroblast growth factor receptor activity proteins Human genes 0.000 description 3
- 235000021588 free fatty acids Nutrition 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 229920000159 gelatin Polymers 0.000 description 3
- 235000019322 gelatine Nutrition 0.000 description 3
- 235000011852 gelatine desserts Nutrition 0.000 description 3
- 238000003780 insertion Methods 0.000 description 3
- 230000037431 insertion Effects 0.000 description 3
- 238000001990 intravenous administration Methods 0.000 description 3
- 230000001404 mediated effect Effects 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 230000007102 metabolic function Effects 0.000 description 3
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 3
- 238000006386 neutralization reaction Methods 0.000 description 3
- 150000005830 nonesterified fatty acids Chemical class 0.000 description 3
- 239000000546 pharmaceutical excipient Substances 0.000 description 3
- 229920001451 polypropylene glycol Polymers 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 230000000069 prophylactic effect Effects 0.000 description 3
- 241000894007 species Species 0.000 description 3
- 238000007920 subcutaneous administration Methods 0.000 description 3
- 239000003826 tablet Substances 0.000 description 3
- 230000005945 translocation Effects 0.000 description 3
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 3
- 239000003981 vehicle Substances 0.000 description 3
- 102100032645 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase Human genes 0.000 description 2
- 208000004611 Abdominal Obesity Diseases 0.000 description 2
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 2
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 2
- 206010065941 Central obesity Diseases 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 108020004394 Complementary RNA Proteins 0.000 description 2
- 238000011537 Coomassie blue staining Methods 0.000 description 2
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 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 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
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 101710153363 Fibroblast growth factor 15 Proteins 0.000 description 2
- 102000051325 Glucagon Human genes 0.000 description 2
- 108060003199 Glucagon Proteins 0.000 description 2
- 102000003638 Glucose-6-Phosphatase Human genes 0.000 description 2
- 108010086800 Glucose-6-Phosphatase Proteins 0.000 description 2
- 108010070675 Glutathione transferase Proteins 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 102000015779 HDL Lipoproteins Human genes 0.000 description 2
- 108010010234 HDL Lipoproteins Proteins 0.000 description 2
- 102100029100 Hematopoietic prostaglandin D synthase Human genes 0.000 description 2
- 229920002971 Heparan sulfate Polymers 0.000 description 2
- 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 2
- 101000846394 Homo sapiens Fibroblast growth factor 19 Proteins 0.000 description 2
- 101000629597 Homo sapiens Sterol regulatory element-binding protein 1 Proteins 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 108090000144 Human Proteins Proteins 0.000 description 2
- 102000003839 Human Proteins Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000010781 Interleukin-6 Receptors Human genes 0.000 description 2
- 108010038501 Interleukin-6 Receptors Proteins 0.000 description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 102000018042 Peroxisome proliferator-activated receptor gamma coactivator 1-beta Human genes 0.000 description 2
- 108050007135 Peroxisome proliferator-activated receptor gamma coactivator 1-beta Proteins 0.000 description 2
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 108700008625 Reporter Genes Proteins 0.000 description 2
- 229920002684 Sepharose Polymers 0.000 description 2
- 229920002472 Starch Polymers 0.000 description 2
- 108010058254 Steroid 12-alpha-Hydroxylase Proteins 0.000 description 2
- 102100026839 Sterol regulatory element-binding protein 1 Human genes 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 238000001042 affinity chromatography Methods 0.000 description 2
- 150000001299 aldehydes Chemical class 0.000 description 2
- 235000010443 alginic acid Nutrition 0.000 description 2
- 229920000615 alginic acid Polymers 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 238000005804 alkylation reaction Methods 0.000 description 2
- 230000000844 anti-bacterial effect Effects 0.000 description 2
- 229940121375 antifungal agent Drugs 0.000 description 2
- 239000003429 antifungal agent Substances 0.000 description 2
- 125000003118 aryl group Chemical group 0.000 description 2
- 235000010323 ascorbic acid Nutrition 0.000 description 2
- 229960005070 ascorbic acid Drugs 0.000 description 2
- 239000011668 ascorbic acid Substances 0.000 description 2
- 230000000923 atherogenic effect Effects 0.000 description 2
- 239000011230 binding agent Substances 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- 230000036765 blood level Effects 0.000 description 2
- 239000002775 capsule Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 235000014633 carbohydrates Nutrition 0.000 description 2
- 239000013592 cell lysate Substances 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 210000003855 cell nucleus Anatomy 0.000 description 2
- 230000001413 cellular effect Effects 0.000 description 2
- OSASVXMJTNOKOY-UHFFFAOYSA-N chlorobutanol Chemical compound CC(C)(O)C(Cl)(Cl)Cl OSASVXMJTNOKOY-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 238000011284 combination treatment Methods 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 239000008121 dextrose Substances 0.000 description 2
- UQLDLKMNUJERMK-UHFFFAOYSA-L di(octadecanoyloxy)lead Chemical compound [Pb+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O UQLDLKMNUJERMK-UHFFFAOYSA-L 0.000 description 2
- 208000016097 disease of metabolism Diseases 0.000 description 2
- 238000010494 dissociation reaction Methods 0.000 description 2
- 230000005593 dissociations Effects 0.000 description 2
- 231100000673 dose–response relationship Toxicity 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000002708 enhancing effect Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 208000010706 fatty liver disease Diseases 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 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 2
- 229960004666 glucagon Drugs 0.000 description 2
- 230000014101 glucose homeostasis Effects 0.000 description 2
- 229940093181 glucose injection Drugs 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 229920000669 heparin Polymers 0.000 description 2
- 229960002897 heparin Drugs 0.000 description 2
- 238000007417 hierarchical cluster analysis Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 102000047000 human FGF19 Human genes 0.000 description 2
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 2
- 238000001597 immobilized metal affinity chromatography Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000006698 induction Effects 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000007951 isotonicity adjuster Substances 0.000 description 2
- 239000008101 lactose Substances 0.000 description 2
- 239000002502 liposome Substances 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 230000010534 mechanism of action Effects 0.000 description 2
- OSWPMRLSEDHDFF-UHFFFAOYSA-N methyl salicylate Chemical compound COC(=O)C1=CC=CC=C1O OSWPMRLSEDHDFF-UHFFFAOYSA-N 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 108091001476 mouse fibroblast growth factor binding protein 3 Proteins 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 230000003472 neutralizing effect Effects 0.000 description 2
- 239000000346 nonvolatile oil Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 238000003068 pathway analysis Methods 0.000 description 2
- 102000040430 polynucleotide Human genes 0.000 description 2
- 108091033319 polynucleotide Proteins 0.000 description 2
- 239000002157 polynucleotide Substances 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 230000028327 secretion Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000600 sorbitol Substances 0.000 description 2
- 235000010356 sorbitol Nutrition 0.000 description 2
- 230000009870 specific binding Effects 0.000 description 2
- 235000019698 starch Nutrition 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 239000000725 suspension Substances 0.000 description 2
- 238000002560 therapeutic procedure Methods 0.000 description 2
- 230000000699 topical effect Effects 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 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
- MZOFCQQQCNRIBI-VMXHOPILSA-N (3s)-4-[[(2s)-1-[[(2s)-1-[[(1s)-1-carboxy-2-hydroxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-(diaminomethylideneamino)-1-oxopentan-2-yl]amino]-3-[[2-[[(2s)-2,6-diaminohexanoyl]amino]acetyl]amino]-4-oxobutanoic acid Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN MZOFCQQQCNRIBI-VMXHOPILSA-N 0.000 description 1
- IIZPXYDJLKNOIY-JXPKJXOSSA-N 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCC\C=C/C\C=C/C\C=C/C\C=C/CCCCC IIZPXYDJLKNOIY-JXPKJXOSSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 1
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- OBULAGGRIVAQEG-DFGXMLLCSA-N 5-[(3as,4s,6ar)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoic acid;[[(2r,3s,4r,5r)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] phosphono hydrogen phosphate Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21.O[C@@H]1[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O[C@H]1N1C(=O)NC(=O)C=C1 OBULAGGRIVAQEG-DFGXMLLCSA-N 0.000 description 1
- 102000011690 Adiponectin Human genes 0.000 description 1
- 108010076365 Adiponectin Proteins 0.000 description 1
- WPWUFUBLGADILS-WDSKDSINSA-N Ala-Pro Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(O)=O WPWUFUBLGADILS-WDSKDSINSA-N 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- RJUHZPRQRQLCFL-IMJSIDKUSA-N Asn-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(O)=O RJUHZPRQRQLCFL-IMJSIDKUSA-N 0.000 description 1
- IIFDPDVJAHQFSR-WHFBIAKZSA-N Asn-Glu Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CCC(O)=O IIFDPDVJAHQFSR-WHFBIAKZSA-N 0.000 description 1
- IQTUDDBANZYMAR-WDSKDSINSA-N Asn-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC(N)=O IQTUDDBANZYMAR-WDSKDSINSA-N 0.000 description 1
- HSPSXROIMXIJQW-BQBZGAKWSA-N Asp-His Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 HSPSXROIMXIJQW-BQBZGAKWSA-N 0.000 description 1
- 241000416162 Astragalus gummifer Species 0.000 description 1
- 241000702009 Bacillus phage SPR Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 208000031648 Body Weight Changes Diseases 0.000 description 1
- 108010074051 C-Reactive Protein Proteins 0.000 description 1
- 102100032752 C-reactive protein Human genes 0.000 description 1
- 101150075266 CYP7A1 gene Proteins 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 241000282465 Canis Species 0.000 description 1
- 241000283707 Capra Species 0.000 description 1
- 241000282693 Cercopithecidae Species 0.000 description 1
- 229920002261 Corn starch Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 229920002307 Dextran Polymers 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000016942 Elastin Human genes 0.000 description 1
- 108010014258 Elastin Proteins 0.000 description 1
- 206010014486 Elevated triglycerides Diseases 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 208000004930 Fatty Liver Diseases 0.000 description 1
- 241000282324 Felis Species 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 102000044168 Fibroblast Growth Factor Receptor Human genes 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 241000233866 Fungi Species 0.000 description 1
- 101800001586 Ghrelin Proteins 0.000 description 1
- 102400000442 Ghrelin-28 Human genes 0.000 description 1
- PABVKUJVLNMOJP-WHFBIAKZSA-N Glu-Cys Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(O)=O PABVKUJVLNMOJP-WHFBIAKZSA-N 0.000 description 1
- BCCRXDTUTZHDEU-VKHMYHEASA-N Gly-Ser Chemical compound NCC(=O)N[C@@H](CO)C(O)=O BCCRXDTUTZHDEU-VKHMYHEASA-N 0.000 description 1
- 108010051975 Glycogen Synthase Kinase 3 beta Proteins 0.000 description 1
- 102100022975 Glycogen synthase kinase-3 alpha Human genes 0.000 description 1
- 102100038104 Glycogen synthase kinase-3 beta Human genes 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102000008055 Heparan Sulfate Proteoglycans Human genes 0.000 description 1
- 206010019708 Hepatic steatosis Diseases 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 101001050473 Homo sapiens Intelectin-1 Proteins 0.000 description 1
- 108010070875 Human Immunodeficiency Virus tat Gene Products Proteins 0.000 description 1
- 102000008100 Human Serum Albumin Human genes 0.000 description 1
- 108091006905 Human Serum Albumin Proteins 0.000 description 1
- 208000031226 Hyperlipidaemia Diseases 0.000 description 1
- 206010020772 Hypertension Diseases 0.000 description 1
- 208000003623 Hypoalbuminemia Diseases 0.000 description 1
- WMDZARSFSMZOQO-DRZSPHRISA-N Ile-Phe Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 WMDZARSFSMZOQO-DRZSPHRISA-N 0.000 description 1
- 108060003951 Immunoglobulin Proteins 0.000 description 1
- 206010056997 Impaired fasting glucose Diseases 0.000 description 1
- 102000003746 Insulin Receptor Human genes 0.000 description 1
- 108010001127 Insulin Receptor Proteins 0.000 description 1
- 102100023353 Intelectin-1 Human genes 0.000 description 1
- 240000007049 Juglans regia Species 0.000 description 1
- 235000009496 Juglans regia Nutrition 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical group CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 108090001090 Lectins Proteins 0.000 description 1
- 102000004856 Lectins Human genes 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- 108091006025 MBP-tagged proteins Proteins 0.000 description 1
- PEEHTFAAVSWFBL-UHFFFAOYSA-N Maleimide Chemical compound O=C1NC(=O)C=C1 PEEHTFAAVSWFBL-UHFFFAOYSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 241000124008 Mammalia Species 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 244000246386 Mentha pulegium Species 0.000 description 1
- 235000016257 Mentha pulegium Nutrition 0.000 description 1
- 235000004357 Mentha x piperita Nutrition 0.000 description 1
- IMTUWVJPCQPJEE-IUCAKERBSA-N Met-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN IMTUWVJPCQPJEE-IUCAKERBSA-N 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 238000006957 Michael reaction Methods 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- 101000756628 Mus musculus Actin, cytoplasmic 1 Proteins 0.000 description 1
- 101100440912 Mus musculus Cyp7a1 gene Proteins 0.000 description 1
- 101000878182 Mus musculus Fibroblast growth factor 15 Proteins 0.000 description 1
- 101001076414 Mus musculus Interleukin-6 Proteins 0.000 description 1
- 101100287085 Mus musculus Irs2 gene Proteins 0.000 description 1
- 101100409193 Mus musculus Ppargc1b gene Proteins 0.000 description 1
- 101100310576 Mus musculus Socs3 gene Proteins 0.000 description 1
- 125000000534 N(2)-L-lysino group Chemical group [H]OC(=O)[C@@]([H])(N([H])[*])C([H])([H])C([H])([H])C(C([H])([H])N([H])[H])([H])[H] 0.000 description 1
- 108091028043 Nucleic acid sequence Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 102000001406 Perilipin Human genes 0.000 description 1
- 108060006002 Perilipin Proteins 0.000 description 1
- 241000009328 Perro Species 0.000 description 1
- FADYJNXDPBKVCA-STQMWFEESA-N Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 FADYJNXDPBKVCA-STQMWFEESA-N 0.000 description 1
- 108091000080 Phosphotransferase Proteins 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- 229920002732 Polyanhydride Polymers 0.000 description 1
- 206010036049 Polycystic ovaries Diseases 0.000 description 1
- 229920000954 Polyglycolide Polymers 0.000 description 1
- 229920001710 Polyorthoester Polymers 0.000 description 1
- 239000004372 Polyvinyl alcohol Substances 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 238000011530 RNeasy Mini Kit Methods 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 108700005075 Regulator Genes Proteins 0.000 description 1
- 102000003861 Ribosomal protein S6 Human genes 0.000 description 1
- 108090000221 Ribosomal protein S6 Proteins 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- UJTZHGHXJKIAOS-WHFBIAKZSA-N Ser-Gln Chemical compound OC[C@H](N)C(=O)N[C@H](C(O)=O)CCC(N)=O UJTZHGHXJKIAOS-WHFBIAKZSA-N 0.000 description 1
- LZLREEUGSYITMX-JQWIXIFHSA-N Ser-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CO)N)C(O)=O)=CNC2=C1 LZLREEUGSYITMX-JQWIXIFHSA-N 0.000 description 1
- ILVGMCVCQBJPSH-WDSKDSINSA-N Ser-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CO ILVGMCVCQBJPSH-WDSKDSINSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- DWAQJAXMDSEUJJ-UHFFFAOYSA-M Sodium bisulfite Chemical compound [Na+].OS([O-])=O DWAQJAXMDSEUJJ-UHFFFAOYSA-M 0.000 description 1
- 108010074436 Sterol Regulatory Element Binding Protein 1 Proteins 0.000 description 1
- 102000008078 Sterol Regulatory Element Binding Protein 1 Human genes 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 108090000054 Syndecan-2 Proteins 0.000 description 1
- DSGIVWSDDRDJIO-ZXXMMSQZSA-N Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O DSGIVWSDDRDJIO-ZXXMMSQZSA-N 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 229920001615 Tragacanth Polymers 0.000 description 1
- 101710132316 Transactivation protein Proteins 0.000 description 1
- 102000040945 Transcription factor Human genes 0.000 description 1
- 108091023040 Transcription factor Proteins 0.000 description 1
- 108060008682 Tumor Necrosis Factor Proteins 0.000 description 1
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 1
- JAQGKXUEKGKTKX-HOTGVXAUSA-N Tyr-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 JAQGKXUEKGKTKX-HOTGVXAUSA-N 0.000 description 1
- LEHOTFFKMJEONL-UHFFFAOYSA-N Uric Acid Chemical compound N1C(=O)NC(=O)C2=C1NC(=O)N2 LEHOTFFKMJEONL-UHFFFAOYSA-N 0.000 description 1
- TVWHNULVHGKJHS-UHFFFAOYSA-N Uric acid Natural products N1C(=O)NC(=O)C2NC(=O)NC21 TVWHNULVHGKJHS-UHFFFAOYSA-N 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 239000003070 absorption delaying agent Substances 0.000 description 1
- 201000010272 acanthosis nigricans Diseases 0.000 description 1
- 150000001242 acetic acid derivatives Chemical class 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 230000002730 additional effect Effects 0.000 description 1
- 239000002671 adjuvant Substances 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- OFHCOWSQAMBJIW-AVJTYSNKSA-N alfacalcidol Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)C[C@H](O)C1=C OFHCOWSQAMBJIW-AVJTYSNKSA-N 0.000 description 1
- 239000000783 alginic acid Substances 0.000 description 1
- 229960001126 alginic acid Drugs 0.000 description 1
- 150000004781 alginic acids Chemical class 0.000 description 1
- 230000029936 alkylation Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 238000005571 anion exchange chromatography Methods 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003385 bacteriostatic effect Effects 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 210000000941 bile Anatomy 0.000 description 1
- 229920000249 biocompatible polymer Polymers 0.000 description 1
- 229940088623 biologically active substance Drugs 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N bis-tris Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 230000004579 body weight change Effects 0.000 description 1
- DQXBYHZEEUGOBF-UHFFFAOYSA-N but-3-enoic acid;ethene Chemical compound C=C.OC(=O)CC=C DQXBYHZEEUGOBF-UHFFFAOYSA-N 0.000 description 1
- 210000004900 c-terminal fragment Anatomy 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 230000023852 carbohydrate metabolic process Effects 0.000 description 1
- 238000005277 cation exchange chromatography Methods 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
- 230000004700 cellular uptake Effects 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 235000010980 cellulose Nutrition 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000002738 chelating agent Substances 0.000 description 1
- 239000012707 chemical precursor Substances 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 229960004926 chlorobutanol Drugs 0.000 description 1
- 150000001860 citric acid derivatives Chemical class 0.000 description 1
- 238000007621 cluster analysis Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 235000019868 cocoa butter Nutrition 0.000 description 1
- 229940110456 cocoa butter Drugs 0.000 description 1
- 229940075614 colloidal silicon dioxide Drugs 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- 239000008120 corn starch Substances 0.000 description 1
- 238000012937 correction Methods 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 229940127089 cytotoxic agent Drugs 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- 238000001212 derivatisation Methods 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- UGMCXQCYOVCMTB-UHFFFAOYSA-K dihydroxy(stearato)aluminium Chemical compound CCCCCCCCCCCCCCCCCC(=O)O[Al](O)O UGMCXQCYOVCMTB-UHFFFAOYSA-K 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 238000003182 dose-response assay Methods 0.000 description 1
- 230000007783 downstream signaling Effects 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 229920002549 elastin Polymers 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 238000012869 ethanol precipitation Methods 0.000 description 1
- 239000005038 ethylene vinyl acetate Substances 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 230000029142 excretion Effects 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 230000004129 fatty acid metabolism Effects 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 235000013355 food flavoring agent Nutrition 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 235000012631 food intake Nutrition 0.000 description 1
- 235000003599 food sweetener Nutrition 0.000 description 1
- 238000003304 gavage Methods 0.000 description 1
- 239000007903 gelatin capsule Substances 0.000 description 1
- BGHSOEHUOOAYMY-JTZMCQEISA-N ghrelin Chemical compound C([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)CN)C1=CC=CC=C1 BGHSOEHUOOAYMY-JTZMCQEISA-N 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 230000010030 glucose lowering effect Effects 0.000 description 1
- 229930195712 glutamate Natural products 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-L glutamate group Chemical group N[C@@H](CCC(=O)[O-])C(=O)[O-] WHUUTDBJXJRKMK-VKHMYHEASA-L 0.000 description 1
- 108010049611 glycogen synthase kinase 3 alpha Proteins 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 206010073071 hepatocellular carcinoma Diseases 0.000 description 1
- 231100000844 hepatocellular carcinoma Toxicity 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 229920001519 homopolymer Polymers 0.000 description 1
- 235000001050 hortel pimenta Nutrition 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 238000004191 hydrophobic interaction chromatography Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000012872 hydroxylapatite chromatography Methods 0.000 description 1
- 230000003345 hyperglycaemic effect Effects 0.000 description 1
- 230000000910 hyperinsulinemic effect Effects 0.000 description 1
- 208000010522 hyperproinsulinemia Diseases 0.000 description 1
- 208000006575 hypertriglyceridemia Diseases 0.000 description 1
- 238000003384 imaging method Methods 0.000 description 1
- 102000018358 immunoglobulin Human genes 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000012405 in silico analysis Methods 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910052738 indium Inorganic materials 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 239000003701 inert diluent Substances 0.000 description 1
- 230000002757 inflammatory effect Effects 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 239000007972 injectable composition Substances 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000001361 intraarterial administration Methods 0.000 description 1
- 230000031146 intracellular signal transduction Effects 0.000 description 1
- 230000004068 intracellular signaling Effects 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 238000007913 intrathecal administration Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000005304 joining Methods 0.000 description 1
- 230000007775 late Effects 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 239000002523 lectin Substances 0.000 description 1
- 230000037356 lipid metabolism Effects 0.000 description 1
- 230000003520 lipogenic effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 210000005228 liver tissue Anatomy 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 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 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 235000011147 magnesium chloride Nutrition 0.000 description 1
- 229940057948 magnesium stearate Drugs 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000004962 mammalian cell Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 235000010270 methyl p-hydroxybenzoate Nutrition 0.000 description 1
- 229960001047 methyl salicylate Drugs 0.000 description 1
- LXCFILQKKLGQFO-UHFFFAOYSA-N methylparaben Chemical group COC(=O)C1=CC=C(O)C=C1 LXCFILQKKLGQFO-UHFFFAOYSA-N 0.000 description 1
- 238000002493 microarray Methods 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 230000003278 mimic effect Effects 0.000 description 1
- 239000002480 mineral oil Substances 0.000 description 1
- 235000010446 mineral oil Nutrition 0.000 description 1
- 230000001483 mobilizing effect Effects 0.000 description 1
- 230000003990 molecular pathway Effects 0.000 description 1
- 230000032393 negative regulation of gluconeogenesis Effects 0.000 description 1
- 239000002687 nonaqueous vehicle Substances 0.000 description 1
- 230000009871 nonspecific binding Effects 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 230000005937 nuclear translocation Effects 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 235000021062 nutrient metabolism Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000002688 persistence Effects 0.000 description 1
- 239000008024 pharmaceutical diluent Substances 0.000 description 1
- 229960003742 phenol Drugs 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 229940080469 phosphocellulose Drugs 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 230000000865 phosphorylative effect Effects 0.000 description 1
- 102000020233 phosphotransferase Human genes 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000004962 physiological condition Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920000191 poly(N-vinyl pyrrolidone) Polymers 0.000 description 1
- 229920001200 poly(ethylene-vinyl acetate) Polymers 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001583 poly(oxyethylated polyols) Polymers 0.000 description 1
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 1
- 239000008389 polyethoxylated castor oil Substances 0.000 description 1
- 239000004633 polyglycolic acid Substances 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 229920005862 polyol Polymers 0.000 description 1
- 150000003077 polyols Chemical class 0.000 description 1
- 229920002451 polyvinyl alcohol Polymers 0.000 description 1
- 230000010077 post-prandial secretion Effects 0.000 description 1
- 230000003389 potentiating effect Effects 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 201000009104 prediabetes syndrome Diseases 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- QELSKZZBTMNZEB-UHFFFAOYSA-N propylparaben Chemical compound CCCOC(=O)C1=CC=C(O)C=C1 QELSKZZBTMNZEB-UHFFFAOYSA-N 0.000 description 1
- 229960003415 propylparaben Drugs 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000010188 recombinant method Methods 0.000 description 1
- 230000018406 regulation of metabolic process Effects 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 230000029058 respiratory gaseous exchange Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 1
- 229940081974 saccharin Drugs 0.000 description 1
- 235000019204 saccharin Nutrition 0.000 description 1
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 1
- 230000008313 sensitization Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 235000010267 sodium hydrogen sulphite Nutrition 0.000 description 1
- 239000001488 sodium phosphate Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 235000011008 sodium phosphates Nutrition 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-N sodium;5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,6-trione Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)NC1=O QGMRQYFBGABWDR-UHFFFAOYSA-N 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 231100000240 steatosis hepatitis Toxicity 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 235000000346 sugar Nutrition 0.000 description 1
- 150000005846 sugar alcohols Polymers 0.000 description 1
- 150000008163 sugars Chemical class 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- 229910052717 sulfur Inorganic materials 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
- 208000011580 syndromic disease Diseases 0.000 description 1
- 230000009885 systemic effect Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 235000012222 talc Nutrition 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 229940033134 talc Drugs 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000002123 temporal effect Effects 0.000 description 1
- RTKIYNMVFMVABJ-UHFFFAOYSA-L thimerosal Chemical compound [Na+].CC[Hg]SC1=CC=CC=C1C([O-])=O RTKIYNMVFMVABJ-UHFFFAOYSA-L 0.000 description 1
- 229940033663 thimerosal Drugs 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 125000000341 threoninyl group Chemical group [H]OC([H])(C([H])([H])[H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 230000006016 thyroid dysfunction Effects 0.000 description 1
- 230000009261 transgenic effect Effects 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 229940116269 uric acid Drugs 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 125000002987 valine group Chemical group [H]N([H])C([H])(C(*)=O)C([H])(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 235000020234 walnut Nutrition 0.000 description 1
- 239000011534 wash buffer Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000008215 water for injection Substances 0.000 description 1
- 230000003442 weekly effect Effects 0.000 description 1
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 229910000368 zinc sulfate Inorganic materials 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
Definitions
- a computer readable text file entitled “SequenceListing.txt,” created on or about 16 Oct. 2017 with a file size of about 9 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
- the invention relates to methods of treating a metabolic disorder in a subject, the method comprising administering fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- FGFBP3 fibroblast growth factor binding protein 3
- the invention also relates to methods of treating a metabolic disorder in a subject, the method comprising administering a complex of fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23 (FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- FGF19 fibroblast growth factor 19
- FGF21 fibroblast growth factor 21
- FGF23 fibroblast growth factor 23
- FGFBP3 fibroblast growth factor binding protein 3
- Fibroblast growth factor 19 and other members of the FGF19 family (i.e. FGF21 and FGF23, the so-called “endocrine FGFs”) are involved in the regulation of metabolism. FGF19 and FGF21 have also been recently described as a sensitizer to insulin. In addition, some members of the FGF19 family interact with the co-receptor klotho to affect metabolism.
- FGFBP FGF-binding proteins
- the invention relates to methods of treating a metabolic disorder in a subject, the method comprising administering fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- FGFBP3 fibroblast growth factor binding protein 3
- the invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering FGFBP3 to a subject in need of lowering of blood glucose levels.
- the invention also relates to methods of lowering a subject's body weight, the method comprising administering FGFBP3 to a subject that is in need of lowering its body weight.
- the invention also relates to methods of lowering a subject's atherogenic serum lipids, the method comprising administering FGFBP3 to a subject that is in need of lowering atherogenic lipids.
- the invention also relates to methods of treating a metabolic disorder in a subject, the method comprising administering a complex of fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23 (FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- FGF19 fibroblast growth factor 19
- FGF21 fibroblast growth factor 21
- FGF23 fibroblast growth factor 23
- FGFBP3 fibroblast growth factor binding protein 3
- the invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering a complex of FGF19, FGF21 or FGF23 and FGFBP3 to a subject in need of lowering of blood glucose levels.
- the invention also relates to methods of lowering a subject's body weight, the method comprising administering a complex of FGF19, FGF21 or FGF23 and FGFBP3 to a subject that is in need of lowering its body weight.
- FIG. 1 depicts the effects of a single dose of FGFBP3 alone or FGF19+FGFBP3 treatments on glucose metabolism in fed ob/ob mice that are diabetic.
- the Left Panel shows that upon intraperitoneal injection of FGFBP3 alone, glucose levels fell from a diabetic level to roughly normal levels (100 to 150 mg/dl) beginning within 2 hours after the first treatment. Levels stayed close to normal range (compared to controls) for 24 hours following injection.
- a comparison with an injection of FGF19+FGFBP3 shows that FGFBP3 alone had the same absolute effect as the combination with FGF19.
- BP3 showed a greater relative effect (Right Panel) on lowering glucose levels when normalized to account for different baseline glucose levels.
- FIG. 2 depicts the effects of BP3 on glycemia in fed ob/ob mice.
- Blood glucose levels after treatment with a single intraperitoneal injection of BP3 or control protein ( MBP) ⁇ pretreatment with an anti FGF15 antibody.
- Mean ⁇ SEM; n ⁇ 5 mice/group. ns, non significant; ***, P ⁇ 0.0001 vs. control ( MBP).
- B. Blood glucose levels in fed ob/ob mice 24 hours after a single intraperitoneal administration of increasing doses of BP3, FGF19 or control ( MBP). The values are calculated relative to baseline levels of blood glucose (mg/dl).
- Mean ⁇ SEM; n ⁇ 5 mice/group. ***, P ⁇ 0.0001 vs. control ( MBP).
- FIG. 3 depicts the ability of BP3 to inhibit gluconeogenesis through IRS2/AKT and IL6/STAT3-dependent downregulation of G6PC by modulating endogenous FGF15 activity.
- B AKT and STAT3 phosphorylation in ob/ob liver lysates from each experimental group were assayed by immunoprecipitation and western blot with phosphospecific antibodies.
- AKT and STAT3 were also determined by immunoblotting with specific antibodies. The numbers below the blots indicate the fold-change, corrected for the total protein expression. The blots are representative of three independent experiments.
- C Schematic summary of the FGFR4-FGF19-BP3 regulatory pathways in liver tissue. Sensitization of FGFR4/FGF19 pathway by BP3 results in a downstream activation of IRS2/AKT and IL6/STAT3 signaling pathways, leading to the inhibition of gluconeogenesis, through G6PC downregulation. Likewise, activation of STAT3 results in an inhibition of PPARGC1B and SREBF1, leading to the inhibition of lipogenesis.
- Activation of FGFR4/FGF19 by BP3 also results in a suppression of bile acid biosynthesis through the downregulation of CYP7A1 gene.
- FIG. 4 depicts the driver pathways of BP3 effects.
- Ingenuity pathway analysis of the differentially expressed liver genes in ob/ob mice treated with BP3, anti FGF15, BP3+anti FGF15 (striped bar) or FGF19 and normalized to the control group ( MBP).
- FIG. 5 depicts the ability of BP3 to reduce blood glucose levels in fed, healthy, non-diabetic C57BL mice.
- C C.
- C57BL mice were administered MBP or BP3 (0.8 mg/kg) by intraperitoneal injection for four hours.
- AKT and STAT3 phosphorylation in liver lysates were assayed by immunoprecipitation and western blot with specific antibodies.
- the expression of total AKT and STAT3 were also determined by immunoblotting with specific antibodies.
- the numbers below the blots indicate the fold-change, corrected for the total protein expression, relative to the control group.
- the blots are representative of three independent experiments.
- D Ingenuity pathway analysis of the differentially expressed genes in C57BL mouse livers treated with BP3 and normalized to the MBP control group.
- Z-score predicting the activation of signaling pathways based on Ingenuity upstream regulator analysis. Z-scores smaller (inhibited) or greater (activated) than 2 were considered biologically significant and are represented by dashed lines.
- FIG. 6 depicts the ability of BP3 to selectively bind to FGFR4.
- A Binding of BP3 or MBP (ctrl) to immobilized FGFRs was measured by direct ELISA with an anti MBP antibody. Mean ⁇ SEM of one of three independent experiments done in duplicate. ***, P ⁇ 0.0001 BP3 (black bars) vs. MBP ctrl (white bars).
- B SPR sensorgrams illustrating the binding kinetics of BP3 to immobilized FGFR4 and FGFR1. The concentration of the BP3 analyte was varied from 4 to 1 nM. RU, response units.
- C Binding of increasing concentrations of BP3 or MBP to immobilized FGFR4 measured by direct ELISA with an anti MBP antibody. Mean ⁇ SEM of one of three independent experiments done in duplicate
- FIG. 7 depicts that the C-terminal 66-amino acid long FGF-binding domain of BP3 (“C66”) is sufficient to reduce hyperglycemia in diabetic mice and to stabilize FGFR4/FGF19 complex formation.
- C66 C-terminal 66-amino acid long FGF-binding domain of BP3
- A Coomassie blue staining of MBP-tagged C66 fusion protein purified by amylose affinity chromatography. The arrow indicates a band of an apparent molecular mass of 52 kDa.
- B SPR sensorgrams illustrating the binding of C66 to immobilized FGF19, FGF2 and FGFR4. RU, response units.
- C Schematic representation of human BP3. The numbers correspond to the human BP3 amino acid sequence (upper panel).
- A Five treatments with FGF19 reduced blood glucose levels at 180 minutes after the beginning of the glucose tolerance test, and the curve returned to the baseline 9 days after receiving no treatment.
- B Five treatments with FGF19+BP3 improved the glucose tolerance at 60, 120, and 180 minutes with a sustained effect for 9 days.
- A The glucose levels were not changed 2 days after a single dose of FGF19 alone.
- Filled Squares Baseline, Open Squares: single dose treatment.
- B In the FGF19+BP3 group, the glucose levels were significantly reduced at 15, 30 and 60 minutes post-injection of glucose.
- C The area under the curve of the glucose tolerance test (AUC) was reduced 2 days after a single treatment in FGF19+BP3 group.
- Filled Squares Baseline, Open Squares: Single treatment. * denotes P ⁇ 0.05, ** P ⁇ 0.01.
- FIG. 10 depicts the effects of acute single doses of BP3, FGF19, and the complex of BP3+FGF19 in ob/ob mice in response to a glucose tolerance test.
- FIGS. 3A, 3B show the effect when the test was conducted two hours after treatment.
- FIG. 3C shows the effect when the test was performed 24 hours after treatment. Even 7 days of dosing of BP3 alone with a single daily dose of BP3 lacked an effect on the baseline blood glucose or on the IPGTT test ( FIG. 3D ).
- FIG. 11 depicts the body weight changes in ob/ob mice following single or multiple treatments with FGF19 or FGF19+BP3.
- B The percentage changes in body weight is also different between the two groups immediately after 5 daily treatments (6.04 ⁇ 2.33% vs. ⁇ 3.40 ⁇ 2.50%). Filled Squares: single treatment; Open Squares: 5 treatments (one dose/day). * denotes P ⁇ 0.05.
- FIG. 12 depicts the presence of BP3 enhancing the ability of FGF19 to induce phosphorylation of Erk1/2 in HepG2 cells.
- HepG2 cells were treated with FGF19 ⁇ BP3 or the negative control protein MBP.
- pERK1/2 was measured in cell lysates at different times after treatment.
- NEFA non-esterified fatty acids
- the invention relates to methods of treating a metabolic disorder in a subject, the methods comprising administering a fibroblast growth factor binding protein 3 (FGFBP3) or an appropriate variant thereof to a subject in need of treatment of a metabolic disorder.
- the invention also relates to methods of treating a metabolic disorder in a subject, the methods comprising administering a complex of fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23 (FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3) or variant thereof to a subject in need of treatment of a metabolic disorder.
- FGF19 fibroblast growth factor 19
- FGF21 fibroblast growth factor 21
- FGF23 fibroblast growth factor 23
- FGFBP3 fibroblast growth factor binding protein 3
- the term “subject” is used interchangeably with the term “patient” and is also used to include an animal, in particular a mammal, and even more particularly a non-human or human primate or dog or cat to give examples
- the fibroblast growth factor binding protein 3 (herein referred to interchangeably as BP3 or FGFBP3) is a secreted protein that binds to human FGF19, FGF21 and FGF23.
- Fibroblast growth factor 19 (FGF19) is the signature member of the FGF19 family of proteins that is involved in nutrient metabolism.
- FGF19 is a protein of 216 amino acids, with the signal peptide being amino acids 1-24.
- “fibroblast growth factor 19” or “FGF19” can mean the full length FGF19 with or without the N-terminus signal sequence.
- the mouse ortholog to human FGF19 is known as FGF15 and is 218 amino acids in length, including the 25-amino acid signal sequence at the N-terminus.
- fibroblast growth factor 15 or “FGF15” or “mFGF15” is used to indicate any ortholog to hFGF19 and can include the full length amino acid sequence, with or without the N-terminus signal sequence. It is understood that a reference to “FGF19” or “hFGF19” herein will also include a reference to its art-accepted orthologs, such as mouse FGF15.
- concentration of FGF19 or mFGF15
- FGF19 is synthesized in the distal small intestine in response to uptake of bile acids via the nuclear bile receptor and controlled by food intake.
- FGF21 expression in the liver and fat tissues is also regulated by the feeding or starving status and function in a temporal cascade with insulin, glucagon and other hormones to regulate responses to nutrition (Potthoff, et al., Genes and Development 2012).
- FGFBP3 is believed to act as a co-receptor with FGFR4.
- the full length amino acid sequence of human FGFBP3 is shown below as SEQ ID NO:1.
- the full length amino acid sequence of human FGFBP3, without the 26 amino-acid signal sequence, is shown below as SEQ ID NO:2.
- the C-terminus of FGFBP3 is shown below as SEQ ID NO:3.
- FGFBP3 means a peptide that comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof that still retains activity similar to the wild-type FGFBP3.
- amino acid sequence of SEQ ID NOs:1 and 2 are just two embodiments of the term FGFBP3 as it is used herein. “Variants” of FGFBP3 are discussed below.
- the present invention is directed to methods that include administration of FGFBP3.
- the FGFBP3 can, but need not, be specifically interacting with FGF19, i.e., specifically binding to one another.
- Other functions of FGFBP3 include but are not limited to the ability to interact with other members of the family of FGF19 proteins such as FGF21 and FGF23.
- FGFBP3 may exert its effect by interacting with FGF21 and/or FGF23.
- the present invention is also directed to methods that include administration of a complex of FGF19 and FGFBP3.
- the term “complex” as it relates to FGF19 and FGFBP3 means the presence of both FGFBP3 and FGF19.
- the FGF19 and FGFBP3 can, but need not, specifically interact, i.e., specifically bind to one another.
- the FGF19 and FGFBP3 within the complex are specifically bound to one another.
- the FGF19 and FGFBP3 within the complex are not necessarily specifically binding to one another.
- full length FGFBP3 (a peptide amino acid sequence of SEQ ID NO:1) is administered.
- the FGFBP3 comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1.
- the FGFBP3 consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1.
- full length FGFBP3 (a peptide amino acid sequence of SEQ ID NO:1) is complexed with FGF19.
- the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1.
- the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1.
- full length FGFBP3 without the signal sequence (a peptide amino acid sequence of SEQ ID NO:2) is administered.
- the FGFBP3 peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2.
- the FGFBP3 peptide consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2.
- full length FGFBP3 without the signal sequence (a peptide amino acid sequence of SEQ ID NO:2) is complexed with FGF19.
- the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2.
- the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2.
- the C-terminal FGFBP3 (a peptide amino acid sequence of SEQ ID NO:3) is administered.
- the FGFBP3 peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3.
- the FGFBP3 peptide consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3.
- the C-terminal FGFBP3 (a peptide amino acid sequence of SEQ ID NO:3) is complexed with FGF19.
- the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3.
- the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3.
- the C-terminal FGFBP3 “C66” peptide (a peptide amino acid sequence of SEQ ID NO:4) is administered.
- the FGFBP3 peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4.
- the FGFBP3 peptide consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4.
- the C-terminal FGFBP3 “C66” peptide (a peptide amino acid sequence of SEQ ID NO:4) is complexed with FGF19.
- the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4.
- the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4.
- an “isolated polypeptide” is intended to mean a polypeptide that has been completely or partially removed from its native environment. For example, polypeptides that have been removed or purified from cells are considered isolated. In addition, recombinantly produced polypeptides molecules contained in host cells are considered isolated for the purposes of the present invention. Moreover, a peptide that is found in a cell, tissue or matrix in which it is not normally expressed or found is also considered as “isolated” for the purposes of the present invention. Similarly, polypeptides that have been synthesized are considered to be isolated polypeptides.
- “Purified,” on the other hand is well understood in the art and generally means that the peptides are substantially free of cellular material, cellular components, chemical precursors or other chemicals beyond, perhaps, buffer or solvent. “Substantially free” is not intended to mean that other components beyond the novel peptides are undetectable.
- FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 3.
- the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 3.
- FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 4.
- the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 4.
- FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 1.
- the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 1.
- FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 2.
- the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 2.
- a metabolic disorder can be any disorder associated with metabolism, and examples include but are not limited to, obesity, central obesity, insulin resistance, glucose intolerance, abnormal glycogen metabolism, type 2 diabetes, hyperlipidemia, hypoalbuminemia, hypertriglyceridemia, metabolic syndrome, syndrome X, a fatty liver, fatty liver disease, polycystic ovarian syndrome, and acanthosis nigricans.
- the methods are directed towards treating at least one component of postprandial metabolism, such as, but not limited to hepatic glycogen synthesis, protein synthesis and clearance of plasma glucose.
- trait and “phenotype” are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of or susceptibility to a disorder.
- phenotype are used herein to refer to symptoms of a metabolic disorder, or a susceptibility to an metabolic disorder.
- traits of metabolic disorders include but are not limited to high total cholesterol, low high-density lipoprotein (HDL) cholesterol, impaired fasting glucose levels, insulin resistance, hyperproinsulinemia, central obesity, elevated triglyceride levels, postprandial glucose levels, elevated uric acid levels, thyroid dysfunction, increased body-mass index (BMI), hypertension, impaired glucose tolerance, alterations in hormone and peptide levels (e.g., leptin, ghrelin, obstatin, adiponectin, perilipin, omentin), interactions with substances involved in insulin signaling, lipid, amino acid and glucose metabolism, life expectancy, increased systemic inflammatory state (e.g., as reflected in levels of C-reactive protein, interleukin-6, and TNF-alpha), depression, and sleep disordered breathing.
- HDL high-density lipoprotein
- the peptide variants described herein are functional and capable of altering a subject's response in a glucose tolerance test when administered alone or in complex with FGF19.
- the FGFBP3 variants of the present invention alone or in complex with FGF19, have enhanced ability to alter a subject's response in a glucose tolerance test compared to wild-type FGFBP3.
- the FGFBP3 variants of the present invention also have enhanced stability compared to the wild-type FGFBP3 regardless of their association with FGF19.
- a polypeptide having an amino acid sequence at least, for example, about 95% “identical” to a reference an amino acid sequence is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence.
- up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence.
- These modifications of the reference sequence may occur at the N-terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
- identity is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using well known techniques. While there are several methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo (1988) J. Applied Math. 48, 1073).
- Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux (1984) Nucleic Acids Research 12, 387), BLASTP, ExPASy, BLASTN, FASTA (Atschul (1990) J. Mol. Biol. 215, 403) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels (2011) Current Protocols in Protein Science, Vol. 1, John Wiley & Sons.
- the algorithm used to determine identity between two or more polypeptides is BLASTP.
- the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag (1990) Comp. App. Biosci. 6, 237-245).
- FASTDB sequence alignment the query and reference sequences are amino sequences. The result of sequence alignment is in percent identity.
- the reference sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, but not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the reference sequence when calculating percent identity.
- the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence.
- the results of the FASTDB sequence alignment determine matching/alignment.
- the alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
- This corrected score can be used for the purposes of determining how alignments “correspond” to each other, as well as percentage identity. Residues of the reference sequence that extend past the N- or C-termini of the query sequence may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
- a 90 amino acid residue query sequence is aligned with a 100 residue reference sequence to determine percent identity.
- the deletion occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus.
- the 10 unpaired residues represent 10% of the reference sequence (number of residues at the N- and C-termini not matched/total number of residues in the reference sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched (100% alignment) the final percent identity would be 90% (100% alignment ⁇ 10% unmatched overhang).
- a 90 residue query sequence is compared with a 100 reference sequence, except that the deletions are internal deletions.
- the percent identity calculated by FASTDB is not manually corrected, since there are no residues at the N- or C-termini of the subject sequence that are not matched/aligned with the query.
- a 110 amino acid query sequence is aligned with a 100 residue reference sequence to determine percent identity. The addition in the query occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment may not show a match/alignment of the first 10 residues at the N-terminus. If the remaining 100 amino acid residues of the query sequence have 95% identity to the entire length of the reference sequence, the N-terminal addition of the query would be ignored and the percent identity of the query to the reference sequence would be 95%.
- the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within the reference protein, e.g., wild-type FGFBP3, and those positions in the variant or ortholog of FGFBP3 that align with the positions with the reference protein.
- the amino acid sequence of a subject FGFBP3 is aligned with the amino acid sequence of a reference FGFBP3, e.g., SEQ ID NO: 2
- the amino acids in the subject sequence that “correspond to” certain enumerated positions of the reference sequence are those that align with these positions of the reference sequence, e.g., SEQ ID NO: 2, but are not necessarily in these exact numerical positions of the reference sequence.
- the invention further embraces other species, preferably mammalian, homologs with amino acid sequences that correspond to FGFBP3.
- Species homologs sometimes referred to as “orthologs,” in general, share at least 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the human version of the full length binding proteins or the full length binding proteins without the signal sequence.
- Such corresponding sequences account for FGFBP3 from across a variety of species, such as canine, feline, mouse, rat, rabbit, monkey, etc.
- FGFBP3 with an additional methionine residue at position ⁇ 1 are contemplated, as are variants with additional methionine and lysine residues at positions ⁇ 2 and ⁇ 1 (Met ⁇ 2 -Lys ⁇ 1 -peptide).
- Variants of FGFBP3 with additional Met, Met-Lys, or Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.
- variants resulting from insertion of the polynucleotide encoding FGFBP3 into an expression vector system are also contemplated.
- variants usually insertions
- the invention provides deletion variants wherein one or more amino acid residues in FGFBP3 are removed.
- Deletions can be effected at one or both termini of the FGFBP3, or with removal of one or more non-terminal amino acid residues of the FGFBP3.
- Deletion variants therefore, include all fragments of the FGFBP3.
- substitution variants include those polypeptides wherein one or more amino acid residues of FGFBP3 are removed and replaced with alternative residues.
- substitution variants include those polypeptides wherein one or more amino acid residues of FGFBP3 are removed and replaced with alternative residues.
- two variants of SEQ ID NOs: 1 or 2 are known to exist and the invention contemplates the use of these known variants in the methods described herein.
- a variant of FGFBP3 wherein Alanine at position 107 of SEQ ID NO:1 is replaced with Threonine (A107T) is included in the methods of the present invention.
- Another variant of FGFBP3 wherein Glutamate at position 206 of SEQ ID NO:1 is replaced with Valine (E206V) is included in the methods of the present invention.
- positions 107 and 206 of SEQ ID NO:1 correspond to positions 81 and 180 of SEQ ID NO:2, and position 206 of SEQ ID NO:1 also corresponds to position 14 of SEQ ID NO:4.
- the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in the tables below. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in below.
- conservative amino acids can be grouped as described in Lehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp. 71-77, as set forth below.
- peptides or polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues.
- the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties.
- Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs.
- the invention further embraces FGFBP3 or variants thereof that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol.
- compositions in which the FGFBP3 or variants thereof is linked to a polymer are included within the scope of the present invention.
- the polymer may be water soluble to prevent precipitation of the protein in an aqueous environment, such as a physiological environment.
- Suitable water-soluble polymers may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol.
- PEG polyethylene glycol
- monomethoxypolyethylene glycol dextran, cellulose, or other carbohydrate based polymers
- poly-(N-vinyl pyrrolidone) polyethylene glycol polypropylene
- the selected polymer is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled.
- Polymers may be of any molecular weight, and may be branched or unbranched, and mixtures of such polymers may also be used. When the chemically modified NgR polymer is destined for therapeutic use, pharmaceutically acceptable polymers will be selected for use.
- Pegylation of FGFBP3 or variants thereof may be carried out by any of the pegylation reactions known in the art.
- the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer).
- a preferred water-soluble polymer for pegylation of polypeptides is polyethylene glycol (PEG), including, but not limited to bi-functional PEGs.
- PEG polyethylene glycol
- polyethylene glycol is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl—ClO) alkoxy- or aryloxy-polyethylene glycol.
- FGFBP3 or variants thereof may be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule.
- Methods for preparing pegylated FGFBP3 or variants thereof will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby FGFBP3 or variants thereof becomes attached to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result.
- Pegylated and other polymer-modified FGFBP3 or variants thereof may generally be used in the methods of the current invention.
- the chemically-derivatized polymer-modified FGFBP3 or variants thereof disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to the nonderivatized molecules.
- the modified FGFBP3 or variants thereof, alone or in complex, may be employed alone, together, or in combination with other pharmaceutical compositions.
- cytokines, growth factors, antibiotics, anti-inflammatories and/or chemotherapeutic agents may be co-administered as is appropriate for the indication being treated.
- compositions comprising purified polypeptides, alone or in complex, of the invention.
- compositions include but are not limited to a pharmaceutically acceptable, i.e., sterile and non-toxic, liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient or medium. Any diluent known in the art may be used.
- Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil and cocoa butter.
- the invention provides fusion proteins comprising at least a first and a second fusion peptide.
- the fusion partners are, generally speaking, covalently bonded to one another via a typical amine bond between the fusion peptides, thus creating one contiguous amino acid chain.
- Types of fusion proteins provided by the present invention include but are not limited to, fusions with secretion signals and other heterologous functional regions.
- a region of additional amino acids, particularly charged amino acids may be added to the N-terminus of the FGFBP3 or variant thereof to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
- Additional fusion proteins include fusions for enhancing translocation of the protein across cell membranes.
- Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1).
- HIV-1 Tat transactivation protein is efficiently taken up by cells, and it has been demonstrated that low concentrations (nM) are sufficient to transactivate a reporter gene expressed from the HIV-1 promoter.
- Exogenous Tat protein is able to translocate through the plasma membrane and reach the nucleus to transactivate the viral genome. Tat peptide-mediated cellular uptake and nuclear translocation have been demonstrated in several systems.
- Tat-derived peptide (residues 37-72 of Tat) to several proteins results in their internalization in several cell lines or tissues (Fawell (1994) Proc. Natl. Acad. Sci. USA 91, 664-668.
- Tat-NLS- ⁇ -Gal a fusion protein consisting of Tat amino acids 48-59 fused by their amino-terminus to ⁇ -galactosidase amino acids 9-1023 translocates to the cell nucleus in an ATP-dependent, cytosolic factor-independent manner.
- the fusion proteins of the present invention may comprise all or a portion of HIV-Tat, such as any sequential residues of the Tat protein basic peptide motif 37-72 (37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72 (SEQ ID NO: 5).
- the minimum number of amino acid residues can be in the range of from about three to about six.
- the Tat portion of the fusion protein is from about three to about five contiguous amino acids in length.
- the Tat portion of the fusion protein is about four amino acids in length, i.e., the minimal requirement for one alpha helical turn.
- the Tat portion of the fusion protein comprises Tat protein residues 48-57 (GRKKRRQRRR) (SEQ ID NO: 6).
- a region may be added to facilitate purification.
- “histidine tags” (“his tags”) or “lysine tags” may be appended to the first fusion peptide.
- histidine tags include, but are not limited to hexaH, heptaH and hexaHN.
- lysine tags include, but are not limited to pentaL, heptaL and FLAG.
- Such regions may be removed prior to final preparation of the FGFBP3 or variant thereof.
- Other examples of a second fusion peptide include, but are not limited to, glutathione S-transferase (GST) and alkaline phosphatase (AP).
- peptide moieties to proteins, whether to engender secretion or excretion, to improve stability and to facilitate purification or translocation, among others, is a familiar and routine technique in the art and may include modifying amino acids at the terminus to accommodate the tags.
- the N-terminus amino acid may be modified to, for example, arginine and/or serine to accommodate a tag.
- the amino acid residues of the C-terminus may also be modified to accommodate tags.
- One particularly useful fusion protein comprises a heterologous region from immunoglobulin that can be used solubilize proteins.
- EP A0464 533 discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
- the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thereby results, for example, in improved pharmacokinetic properties (EP A0232 262).
- the fusion proteins of the current invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, e.g., immobilized metal affinity chromatography (IMAC), hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) may also be employed for purification.
- Well-known techniques for refolding protein may be employed to regenerate active conformation when the fusion protein is denatured during isolation and/or purification.
- Fusion proteins of the present invention include, but are not limited to, products of chemical synthetic procedures and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the fusion proteins of the present invention may be glycosylated or may be non-glycosylated. In addition, fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
- the FGFBP3 or variant, alone or in complex, thereof can be prepared as a pharmaceutical composition.
- one or more cofactors may also be added to the FGFBP3 or variant thereof, or to the complex of FGFBP3 or variant thereof and FGF19, to form a composition.
- Cofactors that may be added include, but are not limited to, heparin, hyaluronic acid, a fibronectin, an elastin, a laminin, albumin, a proteoglycan, collagen, gelatin, a divalent cation, calcium chloride, zinc sulfate, magnesium chloride, sodium bicarbonate, sodium chloride, sodium acetate, or sodium phosphate.
- a protein or a protein fragment may be added as a cofactor to the FGFBP3 or variant thereof. In other embodiments, a protein or a protein fragment may be added as a cofactor to the complex of FGFBP3 or variant thereof and FGF19.
- pharmaceutically acceptable carrier or “pharmaceutical carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
- the nature of the pharmaceutical carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington: The Science and Practice of Pharmacy (2010), Lippincott Williams & Wilkins. Examples of such pharmaceutical carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin.
- Liposomes and non-aqueous vehicles such as fixed oils may also be used.
- the use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
- routes of administration include oral and parenteral (e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal and rectal administration).
- Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include, but are not limited to, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
- the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
- the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
- compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
- suitable pharmaceutical carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF) or phosphate buffered saline (PBS).
- the compositions must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
- the pharmaceutical carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
- the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
- Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
- isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
- Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound/composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
- dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
- methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- Oral compositions generally include an inert diluent or an edible pharmaceutical carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition.
- the tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature, such as but not limited to a binder, such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch, a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide, a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate or flavoring.
- a binder such as microcrystalline cellulose, gum tragacanth or gelatin
- an excipient such as starch or lactose
- a disintegrating agent such as alginic acid, Primogel or corn starch
- a lubricant such as magnesium stearate or Sterotes
- a glidant such as colloidal silicon dioxide
- the active is prepared with pharmaceutical carriers that will protect the active against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
- a controlled release formulation including implants and microencapsulated delivery systems.
- Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art.
- the materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc.
- Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
- compositions can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
- Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the active calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
- the specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
- compositions can be included in a container, pack or dispenser together with instructions for administration.
- the dosage of the FGFBP3 and/or the dosage of the FGFBP3-FGF19 complex will depend on the disorder or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound.
- the FGFBP3 or variant thereof, or the complex can be administered at a dose of between about 0.005 mg/kg of body weight to 500 mg/kg of body weight. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
- the proper dosage depends on various factors such as the type of disorder being treated, the particular composition being used and the size and physiological condition of the patient.
- Therapeutically effective doses for the compositions described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC 50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
- the invention also relates to methods of altering intracellular signaling of a cell, comprising contacting cells with FGFBP3 or a variant thereof, or comprising contacting cells with the complex of FGFBP3 or a variant thereof plus FGF19, wherein the cell possesses a receptor that specifically binds to or associates with FGFBP3.
- the receptor is the fibroblast growth factor receptor 4 (FGFR4).
- FGFR4 fibroblast growth factor receptor 4
- FIG. 12 demonstrates that administration of the complex of FGFBP3 or a variant thereof and FGF19 results in phosphorylation of Erk1/2.
- the present invention provides for methods of stimulating phosphorylation of Erk1/2 in a cell comprising contacting the cell(s) with a complex of FGF19 and FGFBP3 or a variant thereof. Additional methods of the present invention comprise assessing the levels of Erk1/2 phosphorylation, both before and after contacting the cell(s) with the complexes of the present invention and determining the increase or decrease of Erk1/2 phosphorylation in response to the complexes of the present invention.
- one embodiment of the present invention comprises methods of stimulating phosphorylation of p90RSK, rpS6 and/or eIF4B in cells. These methods of phosphorylating p90RSK, rpS6 and/or eIF4B comprise contacting the cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, in an amount sufficient to stimulate phosphorylation thereof.
- GSK3 kinases glycogen synthase kinase 3 ⁇ and 3 ⁇
- the GSK3 kinases are also inhibited or inactivated when they themselves are phosphorylated.
- phosphorylated p90RSK inhibits or inactivates the GSK3 kinases which block the inhibition of GS. Once the inhibition of GS is removed, GS is activated and, in turn, can trigger production of glycogen.
- one embodiment of the present invention is directed to methods of increasing glycogen production in a subject in need thereof, with the methods comprising administering a FGFBP3 or a variant thereof, alone or in complex with FGF19, in a subject in need thereof in an amount sufficient to stimulate production of glycogen.
- Phosphorylated p90RSK also stimulates protein synthesis at least in the liver. Accordingly, one embodiment of the present invention is directed towards increasing protein synthesis in a subject in need thereof, with the methods comprising administering FGFBP3 or a variant, alone or in complex with FGF19, thereof to the subject in an amount sufficient to stimulate protein synthesis.
- a liver-synthesized protein is albumin.
- one specific embodiment of the present invention is directed towards increasing production of albumin in a subject in need thereof, with the methods comprising administering FGFBP3 or a variant thereof, alone or in complex with FGF19, to the subject in an amount sufficient to stimulate production of albumin.
- the present invention provides methods of stimulating promoter activity in a cell or population of cells, where the promoter is responsive to activated Erk1/2 or p90RSK with the methods comprising contacting the cell(s) with FGFBP3 or a variant thereof, alone or in complex with FGF19.
- FGFBP3 FGFBP3 or a variant thereof
- FGF19 FGF19
- One of skill in the art would be aware of promoters that respond to activated Erk1/2 or p90RSK.
- the activity of a variant of FGFBP3, alone or in complex with FGF19, with respect to stimulating Erk1/2-responsive promoters or p90RSK-responsive promoters may or may not be altered relative to the variant's ability to complex with FGF19.
- Additional methods of the present invention comprise assessing the activity of an Erk1/2-responsive promoter both before and after contacting the cell(s) with the FGFBP3 or variant thereof, alone or in complex with FGF19, of the present invention and determining the increase or decrease of the promoter in response to the FGFBP3 or variant thereof, alone or in complex with FGF19, of the present invention.
- the present invention also provides methods of altering the activity or expression of cell signaling molecules in a cell or population of cells in which there is a need to alter the expression or activity thereof. For example, contacting the cell or cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, causes a reduction in the activity and/or expression of the CYP7A1 enzyme (Cholesterol 7 ⁇ -hydrolase), a reduction in the activity or expression of glucose-6-phosphatase (G6PC), and/or a reduction in the activity or expression of peroxisome proliferator-activated receptor- ⁇ coactivator-1 ⁇ (PPARGC1B).
- the CYP7A1 enzyme Choesterol 7 ⁇ -hydrolase
- G6PC glucose-6-phosphatase
- PPARGC1B peroxisome proliferator-activated receptor- ⁇ coactivator-1 ⁇
- contacting the cell or cells with FGFBP3 or a variant thereof, alone or in complex with FGF19 causes an increase in the activity and/or expression of interleukin-6 (IL-6), an increase in the activity and/or expression of insulin receptor substrate (IRS2) and/or an increase in the activity and/or expression of suppressor of cytokine signaling 3 (SOCS3).
- the methods comprise contacting a cell or cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, to a cell or cell in need thereof to alter the phosphorylation state of cell signaling molecules such as but not limited to AKT, STAT3 and forkhead box O1 (FoxO1).
- contacting the cells with FGFBP3 or a variant thereof, alone or in complex with FGF19 will cause an increase in levels of phosphorylated AKT, STAT3 and/or FoxO1.
- contacting when used in connection with the methods of the present invention means bringing the compounds or compositions of the present invention in proximity to the target cells such that a specific binding event or a biological effect is possible.
- contacting can include adding the FGFBP3 in culture medium and applying the culture medium to cells in culture.
- contacting would also include administration of the FGFBP3, or pharmaceutical compositions thereof, of the present invention to cells in an intact organism. Compositions for administering the FGFBP3 of the present invention have been described herein.
- administering are used to mean introducing FGFBP3 or variant thereof, alone or in complex with FGF19, of the present invention into a subject.
- administration is for the purpose of treatment, the composition is provided at, or after the onset of, a symptom or condition in need of treatment.
- the therapeutic administration of this composition serves to attenuate any symptom, or prevent additional symptoms from arising.
- administration is for the purposes of preventing a condition from arising (“prophylactic administration”), the composition is provided in advance of any visible or detectable symptom.
- the prophylactic administration of the composition serves to attenuate subsequently arising symptoms or prevent symptoms from arising altogether.
- the route of administration of the composition includes, but is not limited to, topical, transdermal, intranasal, vaginal, rectal, oral, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal as previously disclosed herein.
- the methods would also include coadministering one or more substances in addition to the composition the present invention.
- coadminister indicates that each of at least two substances, with one of the substances being FGFBP3 or a variant thereof, alone or in complex with FGF19, is administered during a time frame wherein the respective periods of biological activity or effects of each of the substances overlap.
- the term includes sequential as well as coextensive administration of the FGFBP3 of the present invention with another substance.
- coadministration of more than one substance can be for therapeutic and/or prophylactic purposes. If more than one substance is coadministered, the routes of administration of the two or more substances need not be the same.
- the invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering FGFBP3 to a subject in need of lowering of blood glucose levels.
- the subject is screened prior to administration of the FGFBP3.
- the invention also relates to methods of lowering a subject's body weight, the method comprising administering FGFBP3 or a variant thereof to a subject that is in need of lowering its body weight.
- the subject is screened prior to administration of the FGFBP3.
- the invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering a complex of FGF19 and FGFBP3 to a subject in need of lowering of blood glucose levels.
- the subject is screened prior to administration of the complex.
- the invention also relates to methods of lowering a subject's body weight, the method comprising administering a complex of FGF19 and FGFBP3 to a subject that is in need of lowering its body weight.
- the subject is screened prior to administration of the complex.
- Human BP3 cDNA (the amino acid sequence of SEQ ID NO:2 and corresponding to amino acids 27-258 of SEQ ID NO:1), and the C-terminal hBP3 region (SEQ ID NO:4 and corresponding to amino acids 167-232 of SEQ ID NO:2), were subcloned into a pMAL-p2X vector (New England BioLabs, Ipswich, Mass.) and MBP-tagged recombinant proteins (hBP3 and C66, respectively) were generated as has been previously described in Xie, B., et al., J. Biol. Chem. 281, 1137-1144 (2006). MBP and MBP-BP3 are referred to herein as “control” and “BP3”, respectively.
- Recombinant proteins were purified by fast protein liquid chromatography (FPLC). Briefly, bacterial cell lysates were loaded onto an MBPTrapTM HP columns (Dextrin Sepharose) (GE Healthcare Life Sciences, Piscataway, N.J.) and MBP-tagged proteins eluted with 20 ml of a gradient of 0-10 mM Maltose in Column Buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1 mM EDTA). Positive fractions were then loaded onto HiTrapTM Heparin HP columns (GE Healthcare Life Sciences) and proteins eluted with 20 ml of a gradient of 0-1.5M NaCl in Column Buffer.
- FPLC fast protein liquid chromatography
- Eluted proteins were analyzed by immunoblotting with an anti hBP3 rabbit polyclonal antibody (Abgent, San Diego, Calif.) or with an anti MBP mouse monoclonal antibody (New England BioLabs). Eluted hBP3 was resolved on a 4-12% Bis-Tris gel (Life Technologies, Carlsbad, Calif.), visualized by Coomassie Blue staining and the bands excised from the gel. Mass spectrometry analysis was conducted as described previously in Zhang, W. et al., J. Biol. Chem., 283:28329-28337 (2008).
- MBP control or BP3 MBP-tagged recombinant protein
- Detection was carried out with 100 ⁇ /well of an anti MBP mouse monoclonal antibody (New England BioLabs) and with an affinity-purified goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (GE Healthcare Life Sciences) (1:1,000 dilution in PBS).
- HRP horseradish peroxidase
- Bound proteins were detected by western blot analysis with 1 ⁇ /ml of an anti FGFR4 (LD1; Genentech, South San Francisco, Calif.), anti MBP (New England BioLabs) or anti FGF19 (Abnova, Walnut, Calif.) mouse monoclonal antibodies.
- FGFR4 LD1; Genentech, South San Francisco, Calif.
- MBP New England BioLabs
- FGF19 Abnova, Walnut, Calif.
- Biacore T200 instrument (GE Healthcare) was used for surface plasmon resonance measurements. Human recombinant FGFR4 or FGFR1 Fc chimera in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% P-20) were immobilized on a flow cell of a CM-5 sensor chip (GE Healthcare, Piscataway, N.J.) via amine coupling. A blank flow cell was used as a negative control for non-specific binding to the sensor surface.
- FGF19 (47.1 and 23.55 nM), C66 (245 nM) in HBS-P buffer, alone or in combination, or BP3 (4, 2, or 1 nM) were injected over the immobilized receptor with a flow rate of 10 ⁇ L/min for 60 seconds and the resulting maximum responses were obtained.
- Dissociation constants were calculated from the association and dissociation rates of the proteins after washing. The experiments were performed in triplicate.
- mice Six to nine week-old female ob/ob or C57BL mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). Animals were maintained in a normal light-cycle room and were provided with rodent chow and water ad libitum. Mice were treated with a single intraperitoneal injection of recombinant proteins and blood glucose levels were determined with a portable glucose meter (Contour, Bayer, Whippany, N.J.) at different times after treatment (0-48 hours). Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Georgetown University.
- Total AKT and STAT3 in the liver lysates were detected using an anti AKT and STAT3 rabbit polyclonal antibodies, respectively.
- ERK1/2 phosphorylation studies in HepG2 cells were performed by immunoblotting for phospho ERK1/2 and anti ERK1/2 rabbit polyclonal antibodies, as described in Tassi, E., et al., J. Biol. Chem., 276:40247-40253 (2001). All antibodies were purchased from Cell Signaling (Danvers, Mass.). Detection of MBP and MBP-BP3 in mouse sera was carried out by immunoprecipitation and immunoblot with anti MBP magnetic beads and anti MBP rabbit polyclonal antibody (New England BioLabs), respectively.
- RNA Liver total RNA was reverse transcribed into complementary RNA (cRNA), biotin-UTP labeled, and hybridized to the Illumina mouseRef-8v2.0 Expression BeadChip (Illumina Inc., San Diego, Calif.).
- cDNA was synthesized from 1 ⁇ g of total RNA using the iScriptTM cDNA Synthesis Kit, according to the manufacturer's protocol (Bio-Rad Laboratories, Hercules, Calif.).
- Real-time PCR was performed in a Realplex2 (Eppendorf, Hauppauge, N.Y.) using the iQ SYBR Green Supermix (Bio-Rad Laboratories) under the following conditions: 95° C. for 3 minutes, followed by 40 cycles (95° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 40 seconds).
- mice ⁇ -actin sense 5′-GGCGCTTTTGACTCAGGATTTAA-3′ SEQ ID NO: 7
- antisense 5′-CCTCAGCCACATTTGTAGAACTTT-3′ SEQ ID NO: 8
- mouse CYP7A1 sense 5′-CCCACAGTTAATGCACTTGGATCCTG-3′ (SEQ ID NO: 9) and antisense 5′-GGGCATGTAGAAATACTTCAGCTTGTTTCC-3′ (SEQ ID NO: 10
- mouse SOCS3 sense 5′-TCTTTGTCGGAAGACTGTCAACGG-3′ (SEQ ID NO: 11) and antisense 5′-CATCATACTGATCCAGGAACTCCCGA-3′ (SEQ ID NO: 12)
- mouse IL6 sense 5′-GTCACTTTGAGATCTACTCGGCAAACC-3′ (SEQ ID NO: 13) and antisense 5′-TCTGACCACAGTGAGGAATGTCCA-3′ (SEQ ID NO: 14); mouse G6PC
- IPA Ingenuity Pathways Analysis
- Ob/ob mice show pathologically increased glucose levels and exhibit glucose intolerance in traditional glucose tolerance tests. They are a leptin-deficient diabetes model.
- the substances were administered by single intraperitoneal (i.p.) injection.
- i.p. single intraperitoneal
- glucose levels were measured while the animals were fed ad libitum.
- Two series of experiments were performed to investigate acute and sustained effects with the different treatments.
- blood glucose levels blood was sampled from the tail tip at 0, 2, 3, 4 and 24 hours after protein injection. Blood glucose levels were determined with a portable glucose meter (Contour®, Bayer HealthCare).
- FIG. 1 show that the effects of FGFBP3 alone or FGF19+FGFBP3 treatments on glucose metabolism in ob/ob mice.
- Left Panel shows that upon intraperitoneal injection of either FGFBP3 alone or FGF19+FGFBP3, glucose levels fell to roughly normal levels (100 to 150 mg/dl) within 2 hours after the first treatment. Levels stayed close to normal range (compared to controls) for 24 hours following injection.
- Right Panel shows that FGFBP3 had a greater effect on lowering glucose levels when normalized to account for different baseline glucose levels.
- mice with an anti-FGF15 antibody and BP3 did not result in measurably different glucose levels compared to mice treated with the control protein only. Moreover, the effect of the antibody pretreatment did not significantly alter hyperglycemic levels when co-injected with a control protein at all time points ( FIG. 2A ).
- a dose-response curve for BP3 was established and compared that to exogenous FGF19.
- the administration of a single high dose of FGF19 (1 mg/kg) did not impact hyperglycemia in the ob/ob mice, whereas BP3 treatment resulted in a dose-dependent decrease of blood glucose with an ED50 below 0.2 mg/kg ( FIG. 2B ).
- the effect of BP3 in starved animals, which have reduced FGF15 levels was tested. In this setting, BP3 effects on glucose were absent and comparable to those of a single treatment of FGF19 or of the control protein. Normoglycemic levels were achieved when starved animals were co-treated with a combination of BP3 and exogenous FGF19 ( FIG. 2C ).
- CYP7A1 mRNA was reduced 32-fold after administration of exogenous FGF19 suggesting that control of CYP7A1 transcription in the liver in response to the postprandial secretion by ileal entherocytes of FGF15/19 activates FGFR4 in the hepatocytes, thereby resulting in the repression of bile acid (BA) biosynthesis is intact.
- Administration of exogenous FGF19 significantly reduced CYP7A1 transcription by 32 fold versus control mice, thus indicating the efficacy of FGF19 treatment.
- BP3 treatment resulted in a more potent downregulation of CYP7A1, whose transcription was found reduced by 110 fold when compared to the expression levels observed in control mice.
- G6PC glucose-6-phosphatase
- IFS2 insulin receptor substrate 2
- BP3 treatment also resulted in a marked upregulation of both interleukin 6 (IL6) and its downstream effector, suppressor of cytokine signaling 3 (SOCS3), which in turn blocks gluconeogenesis through G6PC suppression.
- IL6 interleukin 6
- SOCS3 suppressor of cytokine signaling 3
- FGF19/15 can negatively modulate lipogenesis in mouse livers by inhibiting peroxisome proliferator-activated receptor- ⁇ coactivator-1 ⁇ (PPARGC1B) expression.
- PPARGC1B peroxisome proliferator-activated receptor- ⁇ coactivator-1 ⁇
- BP3 treatment significantly enhanced this effect ( FIG. 3A ).
- a single administration of FGF19 was not sufficient to alter the expression of these gluconeogenic or lipogenic genes, neutralization of endogenous FGF15 reverted BP3-induced gene regulation to levels indistinguishable from those observed in control livers.
- BP3's ability to modulate FGF15/19-induced downstream activity was examined. Specifically, the phosphorylation state of candidate signaling molecules upstream of gluconeogenesis and lipogenesis in the same ob/ob mouse livers was analyzed. Protein kinase B (AKT) is a critical effector kinase downstream of IRS2 whose phosphorylation results in a negative regulation of gluconeogenic genes, such as G6PC.
- Administration of BP3, but not of FGF19 induced a two-fold increase of AKT phosphorylation over the basal status in control levels, without affecting AKT expression.
- activation of AKT by BP3 was reduced to baseline intensities by an anti FGF15 antibody ( FIG. 3B ). The addition of exogenous FGF19 to BP3 treatment evoked a comparable AKT phosphorylation to that induced by BP3 only.
- FIG. 3C The graphic model depicted in FIG. 3C summarizes the molecular pathways utilized by BP3 to modulate BA biosynthesis, gluconeogenesis and lipogenesis in mouse livers.
- the Ingenuity Pathway Analysis software was used to integrate gene expression with key biochemical networks in response to the different experimental treatments ( FIG. 4 ).
- Analysis of activated pathways revealed a significant activation of FGF2 and FGF19-induced signaling after BP3 treatment.
- the IL6/STAT3 signaling pathway showed the highest induction upon BP3 administration, with z-scores of 7.98 and 6.15, respectively.
- IL6R IL6 receptor
- AKT Activated AKT phosphorylates the downstream forkhead box O1 (FoxO1) transcription factor, which results in the suppression of gluconeogenic gene transcription, such as G6PC.
- FoxO1 downstream forkhead box O1
- Both AKT and FoxO1 pathways are concomitantly activated with BP3 treatment, whereas treatment with FGF19 alone or an anti-FGF15 antibody maintains these pathways in an inhibitory state.
- BP3 treatment induced the inhibition of sterol regulatory element-binding protein 1c (SREBF1) signaling pathway, a PPARGC1B downstream effector molecule, thus indicating a reduction of lipogenesis.
- SREBF1 sterol regulatory element-binding protein 1c
- FGF19 can promote initiation of protein translation in mouse livers by inducing eukaryotic initiation factor 4E (EIF4E) phosphorylation.
- EIF4E eukaryotic initiation factor 4E
- the EIF4E pathway was highly activated in response to BP3 administration, whereas FGF19 alone failed to activate it.
- BP3 treatment on ad libitum fed normoglycemic wild type C57BL mice was examined using a similar protocol as above. Similar to the results seen in the ob/ob mice, a single intraperitoneal dose of BP3, but not of a control protein, was sufficient to significantly reduce plasma glucose levels ( FIG. 5A ). Similarly to what observed in ob/ob mouse livers, BP3 treatment of wild type mice induced a marked downregulation of hepatic CYP7A1, CYP8B1, G6PC, and PPARGC1B, and an upregulation of IL6 and SOCS3 ( FIG.
- FGF19 exhibits a high affinity for FGFR4, and BP3 can enhance FGFR4/FGF19 complex formation.
- the data herein shows that BP3 binds to FGFR4, but not to other FGFRs ( FIG. 6A ), and contains a high-affinity binding site for FGFR4, as determined in a dose response assay by surface plasmon resonance (SPR) and ELISA ( FIG. 6B-C ).
- SPR surface plasmon resonance
- ELISA FIG. 6B-C
- a 66 amino acid-long BP3 C-terminal fragment has been previously identified as the FGF2 binding domain. This binding domain was purified, and an MBP-tagged C66 fusion protein (referred as C66) ( FIG. 7A ) and used as a tool for binding studies.
- C66 Whilst C66 retained its ability to bind to FGF2, its binding to FGF19 was even higher. The C66 fragment, however, did not bind to FGFR4, indicating that the FGF-binding domain of BP3 is not sufficient to elicit BP3 binding to FGFR4 ( FIG. 7B ). The contribution of C66 to FGFR4/FGF19 complex formation was assessed in vitro. C66 significantly enhanced FGF19 binding to immobilized FGFR4, but immobilized FGFR1 did not display any binding to FGF19 or C66, either alone or in combination, which is similar to the binding characteristics of full length BP3 ( FIG. 7C ).
- the FGF-binding domain of BP3 is sufficient to increase FGFR4/FGF19 binding affinity and to reduce hyperglycemia in diabetic mice to the same extent as full-length BP3. Moreover, binding of BP3 to FGFR4 is not required for BP3 regulation of glucose homeostasis.
- mice Five week old female ob/ob mice, obtained from Jackson Laboratories, were randomly assigned to treatment groups with FGF19 alone, or FGF19 in combination with BP3. The substances were administered in the morning by single intraperitoneal (i.p.) injection. To assess potential therapeutic effects on the metabolic profile with FGF19 or FGF19+BP3 treatments, glucose tolerance tests were performed and changes in body weights were measured. Two series of experiments were performed to investigate acute and sustained effects with the different treatments.
- mice were fasted overnight (14 h) before they were subjected to a standard glucose tolerance test (GTT).
- GTT is used to evaluate the ability of an organism to metabolize exogenous glucose.
- the GTT is used to uncover patients with latent diabetes or patients at risk for diabetes, e.g. during pregnancy and is performed after a fasting period by oral administration of a glucose containing drink.
- oral GTT is performed by gavage of a glucose solution, i.e. orally administration. Intraperitoneal injection of a sterile glucose solution was chosen because this allows for a tighter control of dosing of the glucose.
- the animals were weighed to determine the amount of glucose to inject.
- the glucose tolerance test was performed in a quiet room and handling was kept down to a minimum to reduce stress during the procedure.
- a bolus of glucose (1 g/kg) was injected into the intraperitoneal cavity (30% D-glucose:H 2 O solution) and blood was sampled from the tail tip at 0, 15, 30, 60, 120 and 180 minutes after glucose injection. Blood glucose levels were determined with a portable glucose meter (Contour®, Bayer HealthCare).
- IPGTT intraperitoneal glucose tolerance test
- the glucose tolerance test (IPGTT) was seen improved at 60, 120 and 180 minutes post-injection of glucose (p ⁇ 0.05).
- the respective blood glucose levels were significantly lower than those of the control group. This effect was sustained even 9 days after the last of 5 doses of FGF19+BP3 ( FIG. 8B ).
- the results from the glucose tolerance test shown as the area under the curve of the blood glucose levels after glucose injection (AUC), was reduced by 26% compared with baseline in the FGF19 group and by 33% in the FGF19+BP3 group.
- the FGF19+BP3 group displayed an improved AUC compared with baseline even 9 days after completed treatment (p ⁇ 0.01), whereas no such effect was observed in mice receiving FGF19 alone ( FIG. 8C ).
- FIG. 10A , B The effects of acute single doses of BP3 or FGF19 alone, and the combination of BP3+FGF19 were tested in ob/ob mice by IPGTT described above ( FIG. 10A , B). Mice were starved overnight (14 hours) and then treated for two hours by i.p. injection of vehicle (filled squares, FIG. 10A ,B) or FGF19 alone or BP3+FGF19 (open squares FIG. 10A ,B). The IPGTT test was initiated two hours after treatments. The combination of BP3+FGF19 induced a striking effect and normalized the baseline blood glucose and the IPGTT blood glucose curve ( FIG. 10A ). This effect was still present 24 hours after the single dose of BP3+FGF19 ( FIG.
- FIG. 10C FGF19 alone showed no significant effect in the test ( FIG. 10B ). Also, treatment of animals with BP3 alone (without FGF19) showed no significant effect, and even 7 days of dosing of BP3 with a single daily dose of BP3 lacked an effect on the baseline blood glucose or on the IPGTT test ( FIG. 10D ).
- BP3 enhances and prolongs the effects of FGF19 on glucose homeostasis, i.e., there is an improvement of the glucose tolerance, from either single or multiple doses.
- the combination of FGF19+BP3 also reduced the body weight per mouse after 1 dose/day for 5 days, while the animals in FGF19 treatment group gained weight.
- the combination of BP3 and FGF19 has an unexpectedly better therapeutic effect on obesity than FGF19 alone.
- FGFBP3 enhanced the effects of FGF19, i.e., a molecule that that will not be retained by the extracellular matrix and that administration of a complex of FGF19 and FGFBP3 would affect metabolism more quickly, (i.e. after a single dose), for a longer period (i.e for up to two days after a single dose) and more profoundly (i.e. better efficacy in normalizing glucose tolerance) than FGF19 alone.
- HepG2 cells hepatocellular carcinoma cells
- FGF19 hepatocellular carcinoma cells
- FIG. 12 shows that the presence of BP3 enhanced the ability of FGF19 to induce phosphorylation of Erk1/2.
- FGFBP3 was expressed in ob/ob, leptin deficient mice that develop metabolic disease.
- mouse FGFBP3 was used to avoid immune rejection of a human protein.
- Exogenously introduced FGFBP3 expression was achieved by injecting mice twice weekly with a liposomally packaged FGFBP3 expression plasmid to achieve uptake and expression of the exogenously introduce FGFBP3 gene. After 21 ⁇ 2 weeks of treatments the content of blood in mice was analyzed as shown in FIG. 13 .
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Veterinary Medicine (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Zoology (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Marine Sciences & Fisheries (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Abstract
Description
- Part of the work performed during development of this invention utilized U.S. Government funds through National Institutes of Health Grant Nos. RO1 CA71508 and POI HL068686. The U.S. Government has certain rights in this invention.
- A computer readable text file, entitled “SequenceListing.txt,” created on or about 16 Oct. 2017 with a file size of about 9 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
- The invention relates to methods of treating a metabolic disorder in a subject, the method comprising administering fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- The invention also relates to methods of treating a metabolic disorder in a subject, the method comprising administering a complex of fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23 (FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- Fibroblast growth factor 19 (FGF19) and other members of the FGF19 family (i.e. FGF21 and FGF23, the so-called “endocrine FGFs”) are involved in the regulation of metabolism. FGF19 and FGF21 have also been recently described as a sensitizer to insulin. In addition, some members of the FGF19 family interact with the co-receptor klotho to affect metabolism.
- Previous studies indicate that FGF-binding proteins (FGFBP) can enhance the effects of FGF by mobilizing FGFs from their storage depots found in extracellular glycosaminoglycans or heparansulfate proteoglycans. In contrast to most other FGFs, members of the FGF19 family of proteins, i.e., FGF19, FGF21 and FGF23, show only very little binding to glycosaminoglycans or heparansulfates in the extracellular matrix. The most recently discovered member of the FGFBP3 family also appears to mobilize FGFs from such storage depots and bind to FGFs including FGF19 family members.
- The invention relates to methods of treating a metabolic disorder in a subject, the method comprising administering fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- The invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering FGFBP3 to a subject in need of lowering of blood glucose levels.
- The invention also relates to methods of lowering a subject's body weight, the method comprising administering FGFBP3 to a subject that is in need of lowering its body weight.
- The invention also relates to methods of lowering a subject's atherogenic serum lipids, the method comprising administering FGFBP3 to a subject that is in need of lowering atherogenic lipids.
- The invention also relates to methods of treating a metabolic disorder in a subject, the method comprising administering a complex of fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23 (FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3) to a subject in need of treatment of a metabolic disorder.
- The invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering a complex of FGF19, FGF21 or FGF23 and FGFBP3 to a subject in need of lowering of blood glucose levels.
- The invention also relates to methods of lowering a subject's body weight, the method comprising administering a complex of FGF19, FGF21 or FGF23 and FGFBP3 to a subject that is in need of lowering its body weight.
-
FIG. 1 depicts the effects of a single dose of FGFBP3 alone or FGF19+FGFBP3 treatments on glucose metabolism in fed ob/ob mice that are diabetic. The Left Panel, shows that upon intraperitoneal injection of FGFBP3 alone, glucose levels fell from a diabetic level to roughly normal levels (100 to 150 mg/dl) beginning within 2 hours after the first treatment. Levels stayed close to normal range (compared to controls) for 24 hours following injection. A comparison with an injection of FGF19+FGFBP3 (both panels), shows that FGFBP3 alone had the same absolute effect as the combination with FGF19. BP3 showed a greater relative effect (Right Panel) on lowering glucose levels when normalized to account for different baseline glucose levels. -
FIG. 2 depicts the effects of BP3 on glycemia in fed ob/ob mice. Blood glucose levels after treatment with a single intraperitoneal injection of BP3 or control protein (=MBP)±pretreatment with an anti FGF15 antibody. Mean±SEM; n≥5 mice/group. ns, non significant; ***, P<0.0001 vs. control (=MBP). B. Blood glucose levels in fed ob/ob mice 24 hours after a single intraperitoneal administration of increasing doses of BP3, FGF19 or control (=MBP). The values are calculated relative to baseline levels of blood glucose (mg/dl). Mean±SEM; n≥5 mice/group. ***, P<0.0001 vs. control (=MBP). C. Blood glucose of short-term starved ob/ob mice 2 hours after treatment with a single intraperitoneal injection of BP3 (0.8 mg/kg), FGF19 (1 mg/kg), BP3+FGF19, and control (=MBP; 1 mg/kg). Mean±SEM; n=4 mice/group. **, P<0.001. -
FIG. 3 depicts the ability of BP3 to inhibit gluconeogenesis through IRS2/AKT and IL6/STAT3-dependent downregulation of G6PC by modulating endogenous FGF15 activity. A. Changes in hepatic gene expression determined by cDNA array (left) versus qRT-PCR (right). Values are calculated as fold of control treatment levels. Mean±SEM, n=3/group. ns, non significant; *, P<0.05; **, P<0.001; ***, P<0.0001. B. AKT and STAT3 phosphorylation in ob/ob liver lysates from each experimental group were assayed by immunoprecipitation and western blot with phosphospecific antibodies. The expression of AKT and STAT3 were also determined by immunoblotting with specific antibodies. The numbers below the blots indicate the fold-change, corrected for the total protein expression. The blots are representative of three independent experiments. C. Schematic summary of the FGFR4-FGF19-BP3 regulatory pathways in liver tissue. Sensitization of FGFR4/FGF19 pathway by BP3 results in a downstream activation of IRS2/AKT and IL6/STAT3 signaling pathways, leading to the inhibition of gluconeogenesis, through G6PC downregulation. Likewise, activation of STAT3 results in an inhibition of PPARGC1B and SREBF1, leading to the inhibition of lipogenesis. Activation of FGFR4/FGF19 by BP3 also results in a suppression of bile acid biosynthesis through the downregulation of CYP7A1 gene. D. Hierarchical cluster analysis of gene expression in livers of fed ob/ob mice treated with recombinant human BP3, FGF19, anti FGF15, anti FGF15+BP3 or MBP control for 4 hours. The cluster analysis shows a separation of BP3 treatment from all other conditions (n=3 independent samples per group). -
FIG. 4 depicts the driver pathways of BP3 effects. Ingenuity pathway analysis of the differentially expressed liver genes in ob/ob mice treated with BP3, anti FGF15, BP3+anti FGF15 (striped bar) or FGF19 and normalized to the control group (=MBP). Upper panel: Z-score predicting the activation of signaling pathways based on Ingenuity upstream regulator analysis. Lower panel: Z-score predicting the activation of metabolic functions identified by Ingenuity global function analysis. Z-scores smaller (inhibited) or greater (activated) than 2 were considered biologically significant and are represented by dashed lines. The respective P-values for the Z-scores are stated on the right. -
FIG. 5 depicts the ability of BP3 to reduce blood glucose levels in fed, healthy, non-diabetic C57BL mice. A. Blood glucose levels of fed C57BL mice treated with a single intraperitoneal injection of BP3 or MBP control (0.8 mg/kg). Blood glucose was measured at 2 and 4 hours after administration. Mean±SEM; n=3. *, P<0.05; **, P<0.001; ***, P<0.0001. B. Changes in hepatic gene expression determined by qRT-PCR (right) versus cDNA array (Illumina) (left). Values are calculated as fold of MBP control levels. Mean±SEM, n=2. *, P<0.05; **, P<0.001; ***, P<0.0001. C. C57BL mice were administered MBP or BP3 (0.8 mg/kg) by intraperitoneal injection for four hours. AKT and STAT3 phosphorylation in liver lysates were assayed by immunoprecipitation and western blot with specific antibodies. The expression of total AKT and STAT3 were also determined by immunoblotting with specific antibodies. The numbers below the blots indicate the fold-change, corrected for the total protein expression, relative to the control group. The blots are representative of three independent experiments. D. Ingenuity pathway analysis of the differentially expressed genes in C57BL mouse livers treated with BP3 and normalized to the MBP control group. Z-score predicting the activation of signaling pathways based on Ingenuity upstream regulator analysis. Z-scores smaller (inhibited) or greater (activated) than 2 were considered biologically significant and are represented by dashed lines. -
FIG. 6 depicts the ability of BP3 to selectively bind to FGFR4. A. Binding of BP3 or MBP (ctrl) to immobilized FGFRs was measured by direct ELISA with an anti MBP antibody. Mean±SEM of one of three independent experiments done in duplicate. ***, P<0.0001 BP3 (black bars) vs. MBP ctrl (white bars). B: SPR sensorgrams illustrating the binding kinetics of BP3 to immobilized FGFR4 and FGFR1. The concentration of the BP3 analyte was varied from 4 to 1 nM. RU, response units. C: Binding of increasing concentrations of BP3 or MBP to immobilized FGFR4 measured by direct ELISA with an anti MBP antibody. Mean±SEM of one of three independent experiments done in duplicate -
FIG. 7 depicts that the C-terminal 66-amino acid long FGF-binding domain of BP3 (“C66”) is sufficient to reduce hyperglycemia in diabetic mice and to stabilize FGFR4/FGF19 complex formation. A. Coomassie blue staining of MBP-tagged C66 fusion protein purified by amylose affinity chromatography. The arrow indicates a band of an apparent molecular mass of 52 kDa. B. SPR sensorgrams illustrating the binding of C66 to immobilized FGF19, FGF2 and FGFR4. RU, response units. C. Schematic representation of human BP3. The numbers correspond to the human BP3 amino acid sequence (upper panel). Equilibrium binding of FGF19, C66 or their combination to immobilized FGFR1 or FGFR4 was analyzed by SPR (lower panel). Mean±SEM of three independent experiments. Data are represented as percent difference to FGF19 binding to FGFR1 or FGFR4. ***, P<0.0001, FGF19+C66 vs. FGF19. D. Effect of C66 on glycemia in ob/ob mice. Blood glucose levels of fed ob/ob mice treated with a single intraperitoneal injection of C66, BP3 or MBP control (0.8 mg/kg). Blood glucose was measured at 2 and 4 hours after administration. Mean±SEM; n=3-11 mice/group. *, P<0.05; **, P<0.001; ***, P<0.0001. top asterisks: BP3 vs. MBP; bottom asterisks: C66 vs. MBP. E. Changes in hepatic gene expression determined by qRT-PCR in fed ob/ob mice treated with MBP and C66 for 4 hours. Values are calculated as fold of MBP control levels. Mean±SEM, n=3/group. *, P<0.05; **, P<0.001. -
FIG. 8 depicts the effects of multiple FGF19 or FGF19+BP3 treatments on glucose blood levels in ob/ob mice after a bolus injection of glucose (=glucose tolerance test). A, Five treatments with FGF19 reduced blood glucose levels at 180 minutes after the beginning of the glucose tolerance test, and the curve returned to thebaseline 9 days after receiving no treatment. Filled Squares: Baseline; Open Squares: FGF19 (one dose per day for 5 days); Open Circles: FGF19 (one dose per day for 5 days+9 days of no treatment). B, Five treatments with FGF19+BP3 improved the glucose tolerance at 60, 120, and 180 minutes with a sustained effect for 9 days. Filled Squares: Baseline; Open Squares: FGF19+BP3 (one dose per day for 5 days); Open Circles:FGF 19+BP3 (one dose per day for 5 days+9 days of no treatment). C, The area under the curve of the glucose tolerance test (AUC) was improved after 5 treatments in both groups. After another 9 days without any treatment, only the FGF19+BP3 group exhibited an improved glucose tolerance test. Solid Bars: Baseline, Open Bars: FGF19 or FGF19+BP3 (one dose per day for 5 days); Hatched Bars, FGF19 or FGF19+BP3 (one dose per day for 5 days+9 days of no treatment). * denotes P<0.01. -
FIG. 9 depicts the effects of a single dose of FGF19 or FGF19+BP3 treatment on glucose blood levels in ob/ob mice after a bolus injection of glucose (=glucose tolerance test). A, The glucose levels were not changed 2 days after a single dose of FGF19 alone. Filled Squares: Baseline, Open Squares: single dose treatment. B, In the FGF19+BP3 group, the glucose levels were significantly reduced at 15, 30 and 60 minutes post-injection of glucose. C, The area under the curve of the glucose tolerance test (AUC) was reduced 2 days after a single treatment in FGF19+BP3 group. Filled Squares: Baseline, Open Squares: Single treatment. * denotes P<0.05, ** P<0.01. -
FIG. 10 depicts the effects of acute single doses of BP3, FGF19, and the complex of BP3+FGF19 in ob/ob mice in response to a glucose tolerance test.FIGS. 3A, 3B show the effect when the test was conducted two hours after treatment.FIG. 3C shows the effect when the test was performed 24 hours after treatment. Even 7 days of dosing of BP3 alone with a single daily dose of BP3 lacked an effect on the baseline blood glucose or on the IPGTT test (FIG. 3D ). -
FIG. 11 depicts the body weight changes in ob/ob mice following single or multiple treatments with FGF19 or FGF19+BP3. A, The changes in body weight were significantly different between the FGF19 group and the combination group immediately after 5 treatments (2.2±0.8 g vs. −1.0±0.7 g), and a clear trend was also observed 2 days after a single treatment (p=0.055). Filled Squares: single treatment, Open Squares: 5 treatments at one dose/day. B, The percentage changes in body weight is also different between the two groups immediately after 5 daily treatments (6.04±2.33% vs. −3.40±2.50%). Filled Squares: single treatment; Open Squares: 5 treatments (one dose/day). * denotes P<0.05. -
FIG. 12 depicts the presence of BP3 enhancing the ability of FGF19 to induce phosphorylation of Erk1/2 in HepG2 cells. HepG2 cells were treated with FGF19±BP3 or the negative control protein MBP. pERK1/2 was measured in cell lysates at different times after treatment. -
FIG. 13 depicts a significant reduction of non-esterified fatty acids (NEFA) in the serum (p=0.025) of transgenic animals expressing mouse FGFBP3. No significant changes in the other lipids were noted. In addition, the mice remained hyperinsulinemic, which is a known phenotype for ob/ob mice. FGFBP3 alone did not change glucagon or insulin levels significantly. The reduction of NEFA after BP3 expression indicates an improved metabolic disease state of the animals. - The invention relates to methods of treating a metabolic disorder in a subject, the methods comprising administering a fibroblast growth factor binding protein 3 (FGFBP3) or an appropriate variant thereof to a subject in need of treatment of a metabolic disorder. The invention also relates to methods of treating a metabolic disorder in a subject, the methods comprising administering a complex of fibroblast growth factor 19 (FGF19), fibroblast growth factor 21 (FGF21) or fibroblast growth factor 23 (FGF23), plus fibroblast growth factor binding protein 3 (FGFBP3) or variant thereof to a subject in need of treatment of a metabolic disorder. As used herein, the term “subject” is used interchangeably with the term “patient” and is also used to include an animal, in particular a mammal, and even more particularly a non-human or human primate or dog or cat to give examples.
- The fibroblast growth factor binding protein 3 (herein referred to interchangeably as BP3 or FGFBP3) is a secreted protein that binds to human FGF19, FGF21 and FGF23. Fibroblast growth factor 19 (FGF19) is the signature member of the FGF19 family of proteins that is involved in nutrient metabolism. FGF19 is a protein of 216 amino acids, with the signal peptide being amino acids 1-24. As used herein, “
fibroblast growth factor 19” or “FGF19” can mean the full length FGF19 with or without the N-terminus signal sequence. The mouse ortholog to human FGF19 is known as FGF15 and is 218 amino acids in length, including the 25-amino acid signal sequence at the N-terminus. As used herein, “fibroblast growth factor 15” or “FGF15” or “mFGF15” is used to indicate any ortholog to hFGF19 and can include the full length amino acid sequence, with or without the N-terminus signal sequence. It is understood that a reference to “FGF19” or “hFGF19” herein will also include a reference to its art-accepted orthologs, such as mouse FGF15. In general the concentration of FGF19 (or mFGF15) is upregulated after feeding and binds preferentially to FGF Receptor 4 (FGFR4). Specifically, FGF19 is synthesized in the distal small intestine in response to uptake of bile acids via the nuclear bile receptor and controlled by food intake. FGF21 expression in the liver and fat tissues is also regulated by the feeding or starving status and function in a temporal cascade with insulin, glucagon and other hormones to regulate responses to nutrition (Potthoff, et al., Genes and Development 2012). - FGFBP3 is believed to act as a co-receptor with FGFR4. The full length amino acid sequence of human FGFBP3 is shown below as SEQ ID NO:1. The full length amino acid sequence of human FGFBP3, without the 26 amino-acid signal sequence, is shown below as SEQ ID NO:2. The C-terminus of FGFBP3 is shown below as SEQ ID NO:3.
- As used herein, “FGFBP3” means a peptide that comprises the amino acid sequence of SEQ ID NO:3 or a variant thereof that still retains activity similar to the wild-type FGFBP3. Thus, the amino acid sequence of SEQ ID NOs:1 and 2 are just two embodiments of the term FGFBP3 as it is used herein. “Variants” of FGFBP3 are discussed below.
-
(SEQ ID NO: 1) MTPPKLRASL SPSLLLLLSG CLLAAARREK GAASNVAEPV PGPTGGSSGR FLSPEQHACS 60 WQLLLPAPEA AAGSELALRC QSPDGARHQC AYRGHPERCA AYAARRAHFW KQVLGGLRKK 120 RRPCHDPAPL QARLCAGKKG HGAELRLVPR ASPPARPTVA GFAGESKPRA RNRGRTRERA 180 SGPAAGTPPP QSAPPKENPS ERKTNEGKRK AALVPNEERP MGTGPDPDGL DGNAELTETY 240 CAEKWHSLCN FFVNFWNG 258 (SEQ ID NO: 2) RREK GAASNVAEPV PGPTGGSSGR FLSPEQHACS WQLLLPAPEA AAGSELALRC QSPDGARHQC 64 AYRGHPERCA AYAARRAHFW KQVLGGLRKK RRPCHDPAPL QARLCAGKKG HGAELRLVPR 124 ASPPARPTVA GFAGESKPRA RNRGRTRERA SGPAAGTPPP QSAPPKENPS ERKTNEGKRK 184 AALVPNEERP MGTGPDPDGL DGNAELTETY CAEKWHSLCN FFVNFWNG 232 (SEQ ID NO: 3) LDGNAELTET YCAEKWHSLC NFFVNFWNG 29 (SEQ ID NO: 4) APPKENPSER KTNEGKRKAA LVPNEERPMG TGPDPDGLDG NAELTETYCA EKWHSLCNFF 60 VNFWNG 66 - The present invention is directed to methods that include administration of FGFBP3. The FGFBP3 can, but need not, be specifically interacting with FGF19, i.e., specifically binding to one another. Other functions of FGFBP3 include but are not limited to the ability to interact with other members of the family of FGF19 proteins such as FGF21 and FGF23. FGFBP3 may exert its effect by interacting with FGF21 and/or FGF23.
- The present invention is also directed to methods that include administration of a complex of FGF19 and FGFBP3. As used herein, the term “complex” as it relates to FGF19 and FGFBP3 means the presence of both FGFBP3 and FGF19. The FGF19 and FGFBP3 can, but need not, specifically interact, i.e., specifically bind to one another. In one embodiment, the FGF19 and FGFBP3 within the complex are specifically bound to one another. In another embodiment, the FGF19 and FGFBP3 within the complex are not necessarily specifically binding to one another.
- Accordingly, in some embodiment of the methods of the present invention, full length FGFBP3 (a peptide amino acid sequence of SEQ ID NO:1) is administered. In select of these embodiments, the FGFBP3 comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1. In additional embodiments, the FGFBP3 consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1.
- In other embodiments, full length FGFBP3 (a peptide amino acid sequence of SEQ ID NO:1) is complexed with FGF19. In select of these embodiments, the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1. In additional embodiments, the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:1.
- In additional embodiments of the methods of the present invention, full length FGFBP3 without the signal sequence (a peptide amino acid sequence of SEQ ID NO:2) is administered. In select of these embodiments, the FGFBP3 peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2. In additional embodiments, the FGFBP3 peptide consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2.
- In other embodiments, full length FGFBP3 without the signal sequence (a peptide amino acid sequence of SEQ ID NO:2) is complexed with FGF19. In select of these embodiments, the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2. In additional embodiments, the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:2.
- In still additional embodiment of the methods of the present invention, the C-terminal FGFBP3 (a peptide amino acid sequence of SEQ ID NO:3) is administered. In select of these embodiments, the FGFBP3 peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3. In additional embodiments, the FGFBP3 peptide consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3.
- In still additional embodiment of the methods of the present invention, the C-terminal FGFBP3 (a peptide amino acid sequence of SEQ ID NO:3) is complexed with FGF19. In select of these embodiments, the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3. In additional embodiments, the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:3.
- In still additional embodiment of the methods of the present invention, the C-terminal FGFBP3 “C66” peptide (a peptide amino acid sequence of SEQ ID NO:4) is administered. In select of these embodiments, the FGFBP3 peptide comprises an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4. In additional embodiments, the FGFBP3 peptide consists of an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4.
- In still additional embodiment of the methods of the present invention, the C-terminal FGFBP3 “C66” peptide (a peptide amino acid sequence of SEQ ID NO:4) is complexed with FGF19. In select of these embodiments, the FGFBP3 in the complex comprises a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4. In additional embodiments, the FGFBP3 in the complex consists of a peptide with an amino acid sequence that is 100% identical to the amino acid sequence of SEQ ID NO:4.
- The terms “peptide,” “polypeptide” and “protein” are used interchangeably herein. As used herein, an “isolated polypeptide” is intended to mean a polypeptide that has been completely or partially removed from its native environment. For example, polypeptides that have been removed or purified from cells are considered isolated. In addition, recombinantly produced polypeptides molecules contained in host cells are considered isolated for the purposes of the present invention. Moreover, a peptide that is found in a cell, tissue or matrix in which it is not normally expressed or found is also considered as “isolated” for the purposes of the present invention. Similarly, polypeptides that have been synthesized are considered to be isolated polypeptides. “Purified,” on the other hand is well understood in the art and generally means that the peptides are substantially free of cellular material, cellular components, chemical precursors or other chemicals beyond, perhaps, buffer or solvent. “Substantially free” is not intended to mean that other components beyond the novel peptides are undetectable.
- The invention also relates to the use of variants of FGFBP3 that still retain their ability to specifically interact, at least partially, with FGF19. In one embodiment, FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 3. In another embodiment, the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 3.
- In one embodiment, FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 4. In another embodiment, the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 4.
- In one embodiment, FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 1. In another embodiment, the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 1.
- In one embodiment, FGFBP3 variants comprise an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 2. In another embodiment, the FGFBP3 variant consists of a peptide with an amino acid sequence that is at least 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequences of SEQ ID NO: 2.
- As used herein, a metabolic disorder can be any disorder associated with metabolism, and examples include but are not limited to, obesity, central obesity, insulin resistance, glucose intolerance, abnormal glycogen metabolism,
type 2 diabetes, hyperlipidemia, hypoalbuminemia, hypertriglyceridemia, metabolic syndrome, syndrome X, a fatty liver, fatty liver disease, polycystic ovarian syndrome, and acanthosis nigricans. In one embodiment, the methods are directed towards treating at least one component of postprandial metabolism, such as, but not limited to hepatic glycogen synthesis, protein synthesis and clearance of plasma glucose. - The terms “trait” and “phenotype” are used interchangeably herein and refer to any visible, detectable or otherwise measurable property of an organism such as symptoms of or susceptibility to a disorder. Typically the terms “trait” or “phenotype” are used herein to refer to symptoms of a metabolic disorder, or a susceptibility to an metabolic disorder. Examples of traits of metabolic disorders include but are not limited to high total cholesterol, low high-density lipoprotein (HDL) cholesterol, impaired fasting glucose levels, insulin resistance, hyperproinsulinemia, central obesity, elevated triglyceride levels, postprandial glucose levels, elevated uric acid levels, thyroid dysfunction, increased body-mass index (BMI), hypertension, impaired glucose tolerance, alterations in hormone and peptide levels (e.g., leptin, ghrelin, obstatin, adiponectin, perilipin, omentin), interactions with substances involved in insulin signaling, lipid, amino acid and glucose metabolism, life expectancy, increased systemic inflammatory state (e.g., as reflected in levels of C-reactive protein, interleukin-6, and TNF-alpha), depression, and sleep disordered breathing.
- In additional embodiments, the peptide variants described herein are functional and capable of altering a subject's response in a glucose tolerance test when administered alone or in complex with FGF19. In some embodiments, the FGFBP3 variants of the present invention, alone or in complex with FGF19, have enhanced ability to alter a subject's response in a glucose tolerance test compared to wild-type FGFBP3. In some embodiments, the FGFBP3 variants of the present invention also have enhanced stability compared to the wild-type FGFBP3 regardless of their association with FGF19.
- A polypeptide having an amino acid sequence at least, for example, about 95% “identical” to a reference an amino acid sequence, e.g., SEQ ID NO: 1, is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to about five modifications per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a peptide having an amino acid sequence at least about 95% identical to a reference amino acid sequence, up to about 5% of the amino acid residues of the reference sequence may be deleted or substituted with another amino acid or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the N-terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
- As used herein, “identity” is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. “Identity” per se has an art-recognized meaning and can be calculated using well known techniques. While there are several methods to measure identity between two polynucleotide or polypeptide sequences, the term “identity” is well known to skilled artisans (Carillo (1988) J. Applied Math. 48, 1073). Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux (1984)
Nucleic Acids Research 12, 387), BLASTP, ExPASy, BLASTN, FASTA (Atschul (1990) J. Mol. Biol. 215, 403) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels (2011) Current Protocols in Protein Science, Vol. 1, John Wiley & Sons. - In one embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is BLASTP. In another embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag (1990) Comp. App. Biosci. 6, 237-245). In a FASTDB sequence alignment, the query and reference sequences are amino sequences. The result of sequence alignment is in percent identity. In one embodiment, parameters that may be used in a FASTDB alignment of amino acid sequences to calculate percent identity include, but are not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject amino sequence, whichever is shorter.
- If the reference sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, but not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the reference sequence when calculating percent identity. For query sequences truncated at the N- or C-termini, relative to the reference sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment. The alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score can be used for the purposes of determining how alignments “correspond” to each other, as well as percentage identity. Residues of the reference sequence that extend past the N- or C-termini of the query sequence may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
- For example, a 90 amino acid residue query sequence is aligned with a 100 residue reference sequence to determine percent identity. The deletion occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N-terminus. The 10 unpaired residues represent 10% of the reference sequence (number of residues at the N- and C-termini not matched/total number of residues in the reference sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched (100% alignment) the final percent identity would be 90% (100% alignment−10% unmatched overhang). In another example, a 90 residue query sequence is compared with a 100 reference sequence, except that the deletions are internal deletions. In this case the percent identity calculated by FASTDB is not manually corrected, since there are no residues at the N- or C-termini of the subject sequence that are not matched/aligned with the query. In still another example, a 110 amino acid query sequence is aligned with a 100 residue reference sequence to determine percent identity. The addition in the query occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment may not show a match/alignment of the first 10 residues at the N-terminus. If the remaining 100 amino acid residues of the query sequence have 95% identity to the entire length of the reference sequence, the N-terminal addition of the query would be ignored and the percent identity of the query to the reference sequence would be 95%.
- As used herein, the terms “correspond(s) to” and “corresponding to,” as they relate to sequence alignment, are intended to mean enumerated positions within the reference protein, e.g., wild-type FGFBP3, and those positions in the variant or ortholog of FGFBP3 that align with the positions with the reference protein. Thus, when the amino acid sequence of a subject FGFBP3 is aligned with the amino acid sequence of a reference FGFBP3, e.g., SEQ ID NO: 2, the amino acids in the subject sequence that “correspond to” certain enumerated positions of the reference sequence are those that align with these positions of the reference sequence, e.g., SEQ ID NO: 2, but are not necessarily in these exact numerical positions of the reference sequence. Methods for aligning sequences for determining corresponding amino acids between sequences are described herein.
- The invention further embraces other species, preferably mammalian, homologs with amino acid sequences that correspond to FGFBP3. Species homologs, sometimes referred to as “orthologs,” in general, share at least 35%, 40%, 45%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the human version of the full length binding proteins or the full length binding proteins without the signal sequence. Such corresponding sequences account for FGFBP3 from across a variety of species, such as canine, feline, mouse, rat, rabbit, monkey, etc.
- FGFBP3 with an additional methionine residue at position −1 (Met−1-peptide) are contemplated, as are variants with additional methionine and lysine residues at positions −2 and −1 (Met−2-Lys−1-peptide). Variants of FGFBP3 with additional Met, Met-Lys, or Lys residues (or one or more basic residues in general) are particularly useful for enhanced recombinant protein production in bacterial host cells.
- Variants resulting from insertion of the polynucleotide encoding FGFBP3 into an expression vector system are also contemplated. For example, variants (usually insertions) may arise from when the amino terminus and/or the carboxy terminus of FGFBP3 is/are fused to another polypeptide.
- In another aspect, the invention provides deletion variants wherein one or more amino acid residues in FGFBP3 are removed. Deletions can be effected at one or both termini of the FGFBP3, or with removal of one or more non-terminal amino acid residues of the FGFBP3. Deletion variants, therefore, include all fragments of the FGFBP3.
- Within the confines of the disclosed percent identity, the invention also relates to substitution variants of disclosed polypeptides of the invention. Substitution variants include those polypeptides wherein one or more amino acid residues of FGFBP3 are removed and replaced with alternative residues. For example two variants of SEQ ID NOs: 1 or 2 are known to exist and the invention contemplates the use of these known variants in the methods described herein. Specifically, a variant of FGFBP3 wherein Alanine at position 107 of SEQ ID NO:1 is replaced with Threonine (A107T) is included in the methods of the present invention. Another variant of FGFBP3 wherein Glutamate at position 206 of SEQ ID NO:1 is replaced with Valine (E206V) is included in the methods of the present invention. Of course, positions 107 and 206 of SEQ ID NO:1 correspond to
positions 81 and 180 of SEQ ID NO:2, and position 206 of SEQ ID NO:1 also corresponds to position 14 of SEQ ID NO:4. In one aspect, the substitutions are conservative in nature; however, the invention embraces substitutions that are also non-conservative. Conservative substitutions for this purpose may be defined as set out in the tables below. Amino acids can be classified according to physical properties and contribution to secondary and tertiary protein structure. A conservative substitution is recognized in the art as a substitution of one amino acid for another amino acid that has similar properties. Exemplary conservative substitutions are set out in below. -
TABLE I Conservative Substitutions Side Chain Characteristic Amino Acid Aliphatic Non-polar Gly, Ala, Pro, Iso, Leu, Val Polar-uncharged Cys, Ser, Thr, Met, Asn, Gln Polar-charged Asp, Glu, Lys, Arg Aromatic His, Phe, Trp, Tyr Other Asn, Gln, Asp, Glu - Alternatively, conservative amino acids can be grouped as described in Lehninger (1975) Biochemistry, Second Edition; Worth Publishers, pp. 71-77, as set forth below.
-
TABLE II Conservative Substitutions Side Chain Characteristic Amino Acid Non-polar (hydrophobic) Aliphatic: Ala, Leu, Iso, Val, Pro Aromatic: Phe, Trp Sulfur-containing: Met Borderline: Gly Uncharged-polar Hydroxyl: Ser, Thr, Tyr Amides: Asn, Gln Sulfhydryl: Cys Borderline: Gly Positively Charged (Basic): Lys, Arg, His Negatively Charged (Acidic) Asp, Glu - And still other alternative, exemplary conservative substitutions are set out below.
-
TABLE III Conservative Substitutions Original Residue Exemplary Substitution Ala (A) Val, Leu, Ile Arg (R) Lys, Gln, Asn Asn (N) Gln, His, Lys, Arg Asp (D) Glu Cys (C) Ser Gln (Q) Asn Glu (E) Asp His (H) Asn, Gln, Lys, Arg Ile (I) Leu, Val, Met, Ala, Phe Leu (L) Ile, Val, Met, Ala, Phe Lys (K) Arg, Gln, Asn Met (M) Leu, Phe, Ile Phe (F) Leu, Val, Ile, Ala Pro (P) Gly Ser (S) Thr Thr (T) Ser Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser Val (V) Ile, Leu, Met, Phe, Ala - It should be understood that the definition of peptides or polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or substitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs. Similarly, the invention further embraces FGFBP3 or variants thereof that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol.
- Compositions in which the FGFBP3 or variants thereof is linked to a polymer are included within the scope of the present invention. The polymer may be water soluble to prevent precipitation of the protein in an aqueous environment, such as a physiological environment. Suitable water-soluble polymers may be selected from the group consisting of, for example, polyethylene glycol (PEG), monomethoxypolyethylene glycol, dextran, cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, polypropylene glycol homopolymers, a polypropylene oxide/ethylene oxide copolymer polyoxyethylated polyols (e.g., glycerol) and polyvinyl alcohol. The selected polymer is usually modified to have a single reactive group, such as an active ester for acylation or an aldehyde for alkylation, so that the degree of polymerization may be controlled. Polymers may be of any molecular weight, and may be branched or unbranched, and mixtures of such polymers may also be used. When the chemically modified NgR polymer is destined for therapeutic use, pharmaceutically acceptable polymers will be selected for use.
- Pegylation of FGFBP3 or variants thereof may be carried out by any of the pegylation reactions known in the art. In one method, the pegylation is carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water-soluble polymer). A preferred water-soluble polymer for pegylation of polypeptides is polyethylene glycol (PEG), including, but not limited to bi-functional PEGs. As used herein, “polyethylene glycol” is meant to encompass any of the forms of PEG that have been used to derivatize other proteins, such as mono (Cl—ClO) alkoxy- or aryloxy-polyethylene glycol.
- Chemical derivatization of FGFBP3 or variants thereof may be performed under any suitable conditions used to react a biologically active substance with an activated polymer molecule. Methods for preparing pegylated FGFBP3 or variants thereof will generally comprise the steps of (a) reacting the polypeptide with polyethylene glycol, such as a reactive ester or aldehyde derivative of PEG, under conditions whereby FGFBP3 or variants thereof becomes attached to one or more PEG groups, and (b) obtaining the reaction products. It will be apparent to one of ordinary skill in the art to select the optimal reaction conditions or the acylation reactions based on known parameters and the desired result.
- Pegylated and other polymer-modified FGFBP3 or variants thereof may generally be used in the methods of the current invention. The chemically-derivatized polymer-modified FGFBP3 or variants thereof disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half-life, as compared to the nonderivatized molecules. The modified FGFBP3 or variants thereof, alone or in complex, may be employed alone, together, or in combination with other pharmaceutical compositions. For example, cytokines, growth factors, antibiotics, anti-inflammatories and/or chemotherapeutic agents may be co-administered as is appropriate for the indication being treated.
- The present invention provides compositions comprising purified polypeptides, alone or in complex, of the invention. Examples of compositions include but are not limited to a pharmaceutically acceptable, i.e., sterile and non-toxic, liquid, semisolid, or solid diluent that serves as a pharmaceutical vehicle, excipient or medium. Any diluent known in the art may be used. Exemplary diluents include, but are not limited to, water, saline solutions, polyoxyethylene sorbitan monolaurate, magnesium stearate, methyl- and propylhydroxybenzoate, talc, alginates, starches, lactose, sucrose, dextrose, sorbitol, mannitol, glycerol, calcium phosphate, mineral oil and cocoa butter.
- In one embodiment, the invention provides fusion proteins comprising at least a first and a second fusion peptide. The fusion partners are, generally speaking, covalently bonded to one another via a typical amine bond between the fusion peptides, thus creating one contiguous amino acid chain. Types of fusion proteins provided by the present invention include but are not limited to, fusions with secretion signals and other heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the FGFBP3 or variant thereof to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
- Additional fusion proteins include fusions for enhancing translocation of the protein across cell membranes. For example, Tat is an 86-amino acid protein involved in the replication of human immunodeficiency virus type 1 (HIV-1). The HIV-1 Tat transactivation protein is efficiently taken up by cells, and it has been demonstrated that low concentrations (nM) are sufficient to transactivate a reporter gene expressed from the HIV-1 promoter. Exogenous Tat protein is able to translocate through the plasma membrane and reach the nucleus to transactivate the viral genome. Tat peptide-mediated cellular uptake and nuclear translocation have been demonstrated in several systems. Chemically coupling a Tat-derived peptide (residues 37-72 of Tat) to several proteins results in their internalization in several cell lines or tissues (Fawell (1994) Proc. Natl. Acad. Sci. USA 91, 664-668.
- It is well-known that a region of the Tat protein centered on a cluster of basic amino acids is responsible for this translocation activity. A synthetic peptide consisting of the Tat basic amino acids 48-60 with a cysteine residue at the C-terminus coupled to fluorescein maleimide translocates to the cell nucleus as determined by fluorescence microscopy. In addition, a fusion protein (Tat-NLS-β-Gal) consisting of Tat amino acids 48-59 fused by their amino-terminus to β-galactosidase amino acids 9-1023 translocates to the cell nucleus in an ATP-dependent, cytosolic factor-independent manner. Accordingly, the fusion proteins of the present invention may comprise all or a portion of HIV-Tat, such as any sequential residues of the Tat protein basic peptide motif 37-72 (37-CFITKALGISYGRKKRRQRRRPPQGSQTHQVSLSKQ-72 (SEQ ID NO: 5). The minimum number of amino acid residues can be in the range of from about three to about six. In one embodiment, the Tat portion of the fusion protein is from about three to about five contiguous amino acids in length. In another embodiment, the Tat portion of the fusion protein is about four amino acids in length, i.e., the minimal requirement for one alpha helical turn. In another embodiment, the Tat portion of the fusion protein comprises Tat protein residues 48-57 (GRKKRRQRRR) (SEQ ID NO: 6).
- In additional embodiments of fusion proteins, a region may be added to facilitate purification. For example, “histidine tags” (“his tags”) or “lysine tags” may be appended to the first fusion peptide. Examples of histidine tags include, but are not limited to hexaH, heptaH and hexaHN. Examples of lysine tags include, but are not limited to pentaL, heptaL and FLAG. Such regions may be removed prior to final preparation of the FGFBP3 or variant thereof. Other examples of a second fusion peptide include, but are not limited to, glutathione S-transferase (GST) and alkaline phosphatase (AP).
- The addition of peptide moieties to proteins, whether to engender secretion or excretion, to improve stability and to facilitate purification or translocation, among others, is a familiar and routine technique in the art and may include modifying amino acids at the terminus to accommodate the tags. For example in SEQ ID NOs: 1, 2, 3 or 4, the N-terminus amino acid may be modified to, for example, arginine and/or serine to accommodate a tag. Of course, the amino acid residues of the C-terminus may also be modified to accommodate tags. One particularly useful fusion protein comprises a heterologous region from immunoglobulin that can be used solubilize proteins. For example, EP A0464 533 discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thereby results, for example, in improved pharmacokinetic properties (EP A0232 262). On the other hand, for some uses, it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described.
- The fusion proteins of the current invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, e.g., immobilized metal affinity chromatography (IMAC), hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography (“HPLC”) may also be employed for purification. Well-known techniques for refolding protein may be employed to regenerate active conformation when the fusion protein is denatured during isolation and/or purification.
- Fusion proteins of the present invention include, but are not limited to, products of chemical synthetic procedures and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the fusion proteins of the present invention may be glycosylated or may be non-glycosylated. In addition, fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
- The FGFBP3 or variant, alone or in complex, thereof can be prepared as a pharmaceutical composition. For example, one or more cofactors may also be added to the FGFBP3 or variant thereof, or to the complex of FGFBP3 or variant thereof and FGF19, to form a composition. Cofactors that may be added include, but are not limited to, heparin, hyaluronic acid, a fibronectin, an elastin, a laminin, albumin, a proteoglycan, collagen, gelatin, a divalent cation, calcium chloride, zinc sulfate, magnesium chloride, sodium bicarbonate, sodium chloride, sodium acetate, or sodium phosphate. In some embodiments, a protein or a protein fragment may be added as a cofactor to the FGFBP3 or variant thereof. In other embodiments, a protein or a protein fragment may be added as a cofactor to the complex of FGFBP3 or variant thereof and FGF19.
- As used herein, “pharmaceutically acceptable carrier” or “pharmaceutical carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The nature of the pharmaceutical carrier or other ingredients will depend on the specific route of administration and particular embodiment of the invention to be administered. Examples of techniques and protocols that are useful in this context are, inter alia, found in Remington: The Science and Practice of Pharmacy (2010), Lippincott Williams & Wilkins. Examples of such pharmaceutical carriers or diluents include, but are not limited to, water, saline, Ringer's solution, dextrose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.
- A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration. Examples of routes of administration include oral and parenteral (e.g., intravenous, intradermal, subcutaneous, inhalation, transdermal (topical), transmucosal and rectal administration). Solutions or suspensions used for parenteral, intradermal or subcutaneous application can include, but are not limited to, a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents, antibacterial agents such as benzyl alcohol or methyl parabens, antioxidants such as ascorbic acid or sodium bisulfite, chelating agents such as ethylenediaminetetraacetic acid, buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
- Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable pharmaceutical carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF) or phosphate buffered saline (PBS). In all cases, the compositions must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The pharmaceutical carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it may be desirable to include isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.
- Sterile injectable solutions can be prepared by incorporating the active compound/composition in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, methods of preparation are vacuum drying and freeze-drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
- Oral compositions generally include an inert diluent or an edible pharmaceutical carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches or capsules. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like may contain any of the following ingredients, or compounds of a similar nature, such as but not limited to a binder, such as microcrystalline cellulose, gum tragacanth or gelatin, an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel or corn starch, a lubricant such as magnesium stearate or Sterotes, a glidant such as colloidal silicon dioxide, a sweetening agent such as sucrose or saccharin, or a flavoring agent such as peppermint, methyl salicylate or flavoring.
- In one embodiment, the active is prepared with pharmaceutical carriers that will protect the active against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. These compositions can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of the active calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the active compound and the particular therapeutic effect to be achieved.
- The pharmaceutical compositions can be included in a container, pack or dispenser together with instructions for administration.
- The dosage of the FGFBP3 and/or the dosage of the FGFBP3-FGF19 complex will depend on the disorder or condition to be treated and other clinical factors such as weight and condition of the human or animal and the route of administration of the compound. For treating human or animals, the FGFBP3 or variant thereof, or the complex, can be administered at a dose of between about 0.005 mg/kg of body weight to 500 mg/kg of body weight. Therapy is typically administered at lower dosages and is continued until the desired therapeutic outcome is observed.
- Methods of determining the dosages of composition to be administered to a patient and modes of administering compositions to an organism are disclosed in, for example, WO 96/22976. Those skilled in the art will appreciate that such descriptions are applicable to the present invention and can be easily adapted to it.
- The proper dosage depends on various factors such as the type of disorder being treated, the particular composition being used and the size and physiological condition of the patient. Therapeutically effective doses for the compositions described herein can be estimated initially from cell culture and animal models. For example, a dose can be formulated in animal models to achieve a circulating concentration range that initially takes into account the IC50 as determined in cell culture assays. The animal model data can be used to more accurately determine useful doses in humans.
- The invention also relates to methods of altering intracellular signaling of a cell, comprising contacting cells with FGFBP3 or a variant thereof, or comprising contacting cells with the complex of FGFBP3 or a variant thereof plus FGF19, wherein the cell possesses a receptor that specifically binds to or associates with FGFBP3. In one embodiment, the receptor is the fibroblast growth factor receptor 4 (FGFR4). The specific binding of the FGFBP3 or the complex to a receptor will, in turn, initiate the intracellular signaling cascade that is normally associated with FGFBP3. For example,
FIG. 12 demonstrates that administration of the complex of FGFBP3 or a variant thereof and FGF19 results in phosphorylation of Erk1/2. Accordingly, the present invention provides for methods of stimulating phosphorylation of Erk1/2 in a cell comprising contacting the cell(s) with a complex of FGF19 and FGFBP3 or a variant thereof. Additional methods of the present invention comprise assessing the levels of Erk1/2 phosphorylation, both before and after contacting the cell(s) with the complexes of the present invention and determining the increase or decrease of Erk1/2 phosphorylation in response to the complexes of the present invention. - Recently, it was shown that FGF19 alone was able to induce phosphorylation of the p90 ribosomal S6 kinase (p90RSK), which is a downstream target of phosphorylated ERK1/2. In turn, phosphorylated p90RSK is known to phosphorylate both ribosomal protein S6 (rpS6) and the eukaryotic translation initiation factor 4B (eIF4B). It was also recently shown that FGF19 alone was able to induce phosphorylation of both rpS6 and eIF4B. Thus one embodiment of the present invention comprises methods of stimulating phosphorylation of p90RSK, rpS6 and/or eIF4B in cells. These methods of phosphorylating p90RSK, rpS6 and/or eIF4B comprise contacting the cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, in an amount sufficient to stimulate phosphorylation thereof.
- One target of a phosphorylated p90RSK is glycogen synthase kinase 3α and 3β (GSK3 kinases), which, when phosphorylated, are responsible for inhibition of glycogen synthase (GS). The GSK3 kinases are also inhibited or inactivated when they themselves are phosphorylated. Specifically, phosphorylated p90RSK inhibits or inactivates the GSK3 kinases which block the inhibition of GS. Once the inhibition of GS is removed, GS is activated and, in turn, can trigger production of glycogen. Thus one embodiment of the present invention is directed to methods of increasing glycogen production in a subject in need thereof, with the methods comprising administering a FGFBP3 or a variant thereof, alone or in complex with FGF19, in a subject in need thereof in an amount sufficient to stimulate production of glycogen.
- Phosphorylated p90RSK also stimulates protein synthesis at least in the liver. Accordingly, one embodiment of the present invention is directed towards increasing protein synthesis in a subject in need thereof, with the methods comprising administering FGFBP3 or a variant, alone or in complex with FGF19, thereof to the subject in an amount sufficient to stimulate protein synthesis. One example of a liver-synthesized protein is albumin. Accordingly, one specific embodiment of the present invention is directed towards increasing production of albumin in a subject in need thereof, with the methods comprising administering FGFBP3 or a variant thereof, alone or in complex with FGF19, to the subject in an amount sufficient to stimulate production of albumin.
- Likewise, the present invention provides methods of stimulating promoter activity in a cell or population of cells, where the promoter is responsive to activated Erk1/2 or p90RSK with the methods comprising contacting the cell(s) with FGFBP3 or a variant thereof, alone or in complex with FGF19. One of skill in the art would be aware of promoters that respond to activated Erk1/2 or p90RSK. The activity of a variant of FGFBP3, alone or in complex with FGF19, with respect to stimulating Erk1/2-responsive promoters or p90RSK-responsive promoters may or may not be altered relative to the variant's ability to complex with FGF19. One of skill in the art can readily determine if a promoter is more or less activated over control groups using well known techniques such as transcription of reporter genes, ELISA assays, etc. Additional methods of the present invention comprise assessing the activity of an Erk1/2-responsive promoter both before and after contacting the cell(s) with the FGFBP3 or variant thereof, alone or in complex with FGF19, of the present invention and determining the increase or decrease of the promoter in response to the FGFBP3 or variant thereof, alone or in complex with FGF19, of the present invention.
- The present invention also provides methods of altering the activity or expression of cell signaling molecules in a cell or population of cells in which there is a need to alter the expression or activity thereof. For example, contacting the cell or cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, causes a reduction in the activity and/or expression of the CYP7A1 enzyme (Cholesterol 7α-hydrolase), a reduction in the activity or expression of glucose-6-phosphatase (G6PC), and/or a reduction in the activity or expression of peroxisome proliferator-activated receptor-γ coactivator-1β (PPARGC1B). In another example, contacting the cell or cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, causes an increase in the activity and/or expression of interleukin-6 (IL-6), an increase in the activity and/or expression of insulin receptor substrate (IRS2) and/or an increase in the activity and/or expression of suppressor of cytokine signaling 3 (SOCS3). In other embodiments, the methods comprise contacting a cell or cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, to a cell or cell in need thereof to alter the phosphorylation state of cell signaling molecules such as but not limited to AKT, STAT3 and forkhead box O1 (FoxO1). Specifically, contacting the cells with FGFBP3 or a variant thereof, alone or in complex with FGF19, will cause an increase in levels of phosphorylated AKT, STAT3 and/or FoxO1.
- As used herein, “contacting,” when used in connection with the methods of the present invention means bringing the compounds or compositions of the present invention in proximity to the target cells such that a specific binding event or a biological effect is possible. Thus, contacting can include adding the FGFBP3 in culture medium and applying the culture medium to cells in culture. Of course, contacting would also include administration of the FGFBP3, or pharmaceutical compositions thereof, of the present invention to cells in an intact organism. Compositions for administering the FGFBP3 of the present invention have been described herein.
- As used herein, “administering,” and “administer” are used to mean introducing FGFBP3 or variant thereof, alone or in complex with FGF19, of the present invention into a subject. When administration is for the purpose of treatment, the composition is provided at, or after the onset of, a symptom or condition in need of treatment. The therapeutic administration of this composition serves to attenuate any symptom, or prevent additional symptoms from arising. When administration is for the purposes of preventing a condition from arising (“prophylactic administration”), the composition is provided in advance of any visible or detectable symptom. The prophylactic administration of the composition serves to attenuate subsequently arising symptoms or prevent symptoms from arising altogether. The route of administration of the composition includes, but is not limited to, topical, transdermal, intranasal, vaginal, rectal, oral, subcutaneous intravenous, intraarterial, intramuscular, intraosseous, intraperitoneal, epidural and intrathecal as previously disclosed herein.
- Furthermore, the methods would also include coadministering one or more substances in addition to the composition the present invention. The term “coadminister” indicates that each of at least two substances, with one of the substances being FGFBP3 or a variant thereof, alone or in complex with FGF19, is administered during a time frame wherein the respective periods of biological activity or effects of each of the substances overlap. Thus the term includes sequential as well as coextensive administration of the FGFBP3 of the present invention with another substance. And similar to administering the compositions of the present invention, coadministration of more than one substance can be for therapeutic and/or prophylactic purposes. If more than one substance is coadministered, the routes of administration of the two or more substances need not be the same.
- The invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering FGFBP3 to a subject in need of lowering of blood glucose levels. In one embodiment, the subject is screened prior to administration of the FGFBP3.
- The invention also relates to methods of lowering a subject's body weight, the method comprising administering FGFBP3 or a variant thereof to a subject that is in need of lowering its body weight. In one embodiment, the subject is screened prior to administration of the FGFBP3.
- The invention also relates to methods of lowering blood glucose levels in a subject, the method comprising administering a complex of FGF19 and FGFBP3 to a subject in need of lowering of blood glucose levels. In one embodiment, the subject is screened prior to administration of the complex.
- The invention also relates to methods of lowering a subject's body weight, the method comprising administering a complex of FGF19 and FGFBP3 to a subject that is in need of lowering its body weight. In one embodiment, the subject is screened prior to administration of the complex.
- The following examples are illustrative and are not intended to limit the scope of the invention described herein.
- Materials and Methods
- Human BP3 cDNA, (the amino acid sequence of SEQ ID NO:2 and corresponding to amino acids 27-258 of SEQ ID NO:1), and the C-terminal hBP3 region (SEQ ID NO:4 and corresponding to amino acids 167-232 of SEQ ID NO:2), were subcloned into a pMAL-p2X vector (New England BioLabs, Ipswich, Mass.) and MBP-tagged recombinant proteins (hBP3 and C66, respectively) were generated as has been previously described in Xie, B., et al., J. Biol. Chem. 281, 1137-1144 (2006). MBP and MBP-BP3 are referred to herein as “control” and “BP3”, respectively. Recombinant proteins were purified by fast protein liquid chromatography (FPLC). Briefly, bacterial cell lysates were loaded onto an MBPTrap™ HP columns (Dextrin Sepharose) (GE Healthcare Life Sciences, Piscataway, N.J.) and MBP-tagged proteins eluted with 20 ml of a gradient of 0-10 mM Maltose in Column Buffer (20 mM Tris HCl pH 7.4, 200 mM NaCl, 1 mM EDTA). Positive fractions were then loaded onto HiTrap™ Heparin HP columns (GE Healthcare Life Sciences) and proteins eluted with 20 ml of a gradient of 0-1.5M NaCl in Column Buffer. Eluted proteins were analyzed by immunoblotting with an anti hBP3 rabbit polyclonal antibody (Abgent, San Diego, Calif.) or with an anti MBP mouse monoclonal antibody (New England BioLabs). Eluted hBP3 was resolved on a 4-12% Bis-Tris gel (Life Technologies, Carlsbad, Calif.), visualized by Coomassie Blue staining and the bands excised from the gel. Mass spectrometry analysis was conducted as described previously in Zhang, W. et al., J. Biol. Chem., 283:28329-28337 (2008).
- MaxiSorp™ microtiter plates (Sigma Aldrich, St. Louis, Mo.) were coated with 100 μl/well of recombinant proteins [human recombinant FGF2 (Life Technologies), human recombinant FGF19, or human recombinant FGFR4 Fc Chimera (R&D Systems Minneapolis, Minn.); 7.5 μg/ml] and incubated overnight at 4° C. Plates were washed thrice between each incubation step with washing buffer [1× Phosphate buffered saline (PBS) with 0.2
% Tween 20, pH 7.4 (PBST)]. Blocking was carried out with 100 μl/well of 5% dry milk diluted in PBST for 1 hour at room temperature. Subsequently, plates were incubated for 2 hours at room temperature with 100 μl/well of an MBP-tagged recombinant protein (MBP control or BP3) at a fixed concentration (1 μg/ml) or in serial dilutions. Detection was carried out with 100 μ/well of an anti MBP mouse monoclonal antibody (New England BioLabs) and with an affinity-purified goat anti-mouse horseradish peroxidase (HRP)-conjugated antibody (GE Healthcare Life Sciences) (1:1,000 dilution in PBS). The reactions were visualized with the aid of 1-Step Turbo TMB (Thermo Scientific, Pittsburgh, Pa.), according to the manufacturer's protocol, and read with an Ultramark Microplate Imaging System (Bio-Rad Laboratories, Hercules, Calif.) at 450 nm absorbance. - MaxiSorp™ microtiter plates were coated with 0.75 mg of recombinant FGFR4 and incubated overnight at 4° C. lates were washed thrice between each incubation step with PBS. Blocking was carried out with 100 μ/well of 5% dry milk diluted in PBS for 1 hour at room temperature. Subsequently, plates were incubated for 2 hours at room temperature with 100 μ/well of FGF19 (2 μ/ml)±BP3 or MBP control (1 μ/ml). Bound proteins were detected by western blot analysis with 1 μ/ml of an anti FGFR4 (LD1; Genentech, South San Francisco, Calif.), anti MBP (New England BioLabs) or anti FGF19 (Abnova, Walnut, Calif.) mouse monoclonal antibodies.
- Biacore T200 instrument (GE Healthcare) was used for surface plasmon resonance measurements. Human recombinant FGFR4 or FGFR1 Fc chimera in HBS-P buffer (0.01 M HEPES pH 7.4, 0.15 M NaCl, 0.05% P-20) were immobilized on a flow cell of a CM-5 sensor chip (GE Healthcare, Piscataway, N.J.) via amine coupling. A blank flow cell was used as a negative control for non-specific binding to the sensor surface. FGF19 (47.1 and 23.55 nM), C66 (245 nM) in HBS-P buffer, alone or in combination, or BP3 (4, 2, or 1 nM) were injected over the immobilized receptor with a flow rate of 10 μL/min for 60 seconds and the resulting maximum responses were obtained. Dissociation constants were calculated from the association and dissociation rates of the proteins after washing. The experiments were performed in triplicate.
- Six to nine week-old female ob/ob or C57BL mice were purchased from The Jackson Laboratory (Bar Harbor, Me.). Animals were maintained in a normal light-cycle room and were provided with rodent chow and water ad libitum. Mice were treated with a single intraperitoneal injection of recombinant proteins and blood glucose levels were determined with a portable glucose meter (Contour, Bayer, Whippany, N.J.) at different times after treatment (0-48 hours). Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee of Georgetown University.
- Immunoprecipitation and Western Blot analyses were performed as described earlier in Tassi, E., et al., Am. J. Pathol., 179:2220-2232 (2011). Briefly, livers from ob/ob or C57BL mice were homogenized in 1 mL of lysis buffer with a MagNa lyser homogenizer (Roche, Indianapolis, Ind.). 5 mg of total lysates were immunoprecipitated with 10 μl of sepharose-conjugated anti-AKT or phospho STAT3 antibodies and immunoblotted with an anti-phospho AKT or anti STAT3 rabbit polyclonal antibodies, respectively. Total AKT and STAT3 in the liver lysates (50 μg) were detected using an anti AKT and STAT3 rabbit polyclonal antibodies, respectively. ERK1/2 phosphorylation studies in HepG2 cells were performed by immunoblotting for phospho ERK1/2 and anti ERK1/2 rabbit polyclonal antibodies, as described in Tassi, E., et al., J. Biol. Chem., 276:40247-40253 (2001). All antibodies were purchased from Cell Signaling (Danvers, Mass.). Detection of MBP and MBP-BP3 in mouse sera was carried out by immunoprecipitation and immunoblot with anti MBP magnetic beads and anti MBP rabbit polyclonal antibody (New England BioLabs), respectively.
- Total RNA was isolated from ob/ob or C57BL mouse livers or WAT using RNeasy Mini kit or RNeasy Lipid Tissue kit (Qiagen, Valencia, Calif.), according to the manufacturer's instructions.
- Liver total RNA was reverse transcribed into complementary RNA (cRNA), biotin-UTP labeled, and hybridized to the Illumina mouseRef-8v2.0 Expression BeadChip (Illumina Inc., San Diego, Calif.).
- cDNA was synthesized from 1 μg of total RNA using the iScript™ cDNA Synthesis Kit, according to the manufacturer's protocol (Bio-Rad Laboratories, Hercules, Calif.). Real-time PCR was performed in a Realplex2 (Eppendorf, Hauppauge, N.Y.) using the iQ SYBR Green Supermix (Bio-Rad Laboratories) under the following conditions: 95° C. for 3 minutes, followed by 40 cycles (95° C. for 20 seconds, 60° C. for 30 seconds, and 72° C. for 40 seconds). The following PCR primers were used: mouse β-
actin sense 5′-GGCGCTTTTGACTCAGGATTTAA-3′ (SEQ ID NO: 7),antisense 5′-CCTCAGCCACATTTGTAGAACTTT-3′ (SEQ ID NO: 8);mouse CYP7A1 sense 5′-CCCACAGTTAATGCACTTGGATCCTG-3′ (SEQ ID NO: 9) andantisense 5′-GGGCATGTAGAAATACTTCAGCTTGTTTCC-3′ (SEQ ID NO: 10);mouse SOCS3 sense 5′-TCTTTGTCGGAAGACTGTCAACGG-3′ (SEQ ID NO: 11) andantisense 5′-CATCATACTGATCCAGGAACTCCCGA-3′ (SEQ ID NO: 12);mouse IL6 sense 5′-GTCACTTTGAGATCTACTCGGCAAACC-3′ (SEQ ID NO: 13) andantisense 5′-TCTGACCACAGTGAGGAATGTCCA-3′ (SEQ ID NO: 14);mouse G6PC 5′-CGACTCGCTATCTCCAAGTGA-3′ (SEQ ID NO: 15) andantisense 5′-GTTGAACCAGTCTCCGACCA (SEQ ID NO: 16);mouse PPARGC1B 5′-TCCTGTAAAAGCCCGGAGTAT-3′ (SEQ ID NO: 17) andantisense 5′-GCTCTGGTAGGGGCAGTGA-3′ (SEQ ID NO: 18);mouse IRS2 5′-ACCGACTTGGTCAGCGAAG-3′ (SEQ ID NO: 19) andantisense 5′-CACGAGCCCGTAGTTGTCAT-3′ (SEQ ID NO: 20). - The web-based Ingenuity Pathways Analysis (IPA) (Ingenuity Systems®, www.ingenuity.com) was used to identify functional networks and pathways analyses. Activation z-score was calculated as a measure of functional and translational activation in Functions and Upstream regulators analysis. z-scores greater than 2 or smaller than −2 were considered significant.
- Six to nine week old female ob/ob mice, obtained from Jackson Laboratories, were randomly assigned to treatment groups with FGFBP3 alone, or FGF19 in combination with BP3. Ob/ob mice show pathologically increased glucose levels and exhibit glucose intolerance in traditional glucose tolerance tests. They are a leptin-deficient diabetes model.
- The substances were administered by single intraperitoneal (i.p.) injection. To assess potential therapeutic effects on the metabolic profile with FGFBP3 or FGF19+FGFBP3 treatments, glucose levels were measured while the animals were fed ad libitum. Two series of experiments were performed to investigate acute and sustained effects with the different treatments.
- Before starting the experiments, the animals were weighed to determine the amount of proteins (FGFBP3 or others) to inject. Contrary to alternative approaches that use glucose challenge after prolonged fasting (=glucose tolerance test), the animals were not fasted and were fed ad libitum. This was done to mimic the natural setting. For the measurements of blood glucose levels, blood was sampled from the tail tip at 0, 2, 3, 4 and 24 hours after protein injection. Blood glucose levels were determined with a portable glucose meter (Contour®, Bayer HealthCare).
- The results in
FIG. 1 show that the effects of FGFBP3 alone or FGF19+FGFBP3 treatments on glucose metabolism in ob/ob mice. Left Panel, shows that upon intraperitoneal injection of either FGFBP3 alone or FGF19+FGFBP3, glucose levels fell to roughly normal levels (100 to 150 mg/dl) within 2 hours after the first treatment. Levels stayed close to normal range (compared to controls) for 24 hours following injection. Right Panel, shows that FGFBP3 had a greater effect on lowering glucose levels when normalized to account for different baseline glucose levels. - Treatment of the mice with an anti-FGF15 antibody and BP3 did not result in measurably different glucose levels compared to mice treated with the control protein only. Moreover, the effect of the antibody pretreatment did not significantly alter hyperglycemic levels when co-injected with a control protein at all time points (
FIG. 2A ). - A dose-response curve for BP3 was established and compared that to exogenous FGF19. The administration of a single high dose of FGF19 (1 mg/kg) did not impact hyperglycemia in the ob/ob mice, whereas BP3 treatment resulted in a dose-dependent decrease of blood glucose with an ED50 below 0.2 mg/kg (
FIG. 2B ). To evaluate if the BP3 effect was impacted by the feeding status of the animals, the effect of BP3 in starved animals, which have reduced FGF15 levels, was tested. In this setting, BP3 effects on glucose were absent and comparable to those of a single treatment of FGF19 or of the control protein. Normoglycemic levels were achieved when starved animals were co-treated with a combination of BP3 and exogenous FGF19 (FIG. 2C ). - Gene expression patterns in livers of ob/ob mice using the treatment cohort above were assayed. Organs were harvested after four hours when the glucose lowering effect of BP3 had reached normoglycemic levels and included the following treatment groups: control protein (MBP), FGF19, BP3 alone, anti-FGF15 antibody alone, or BP3 plus anti-FGF15. As a signature gene of the FGFR4/FGF19 signaling axis CYP7A1 (Cholesterol 7α-hydrolase) was first probed for expression. CYP7A1 mRNA was reduced 32-fold after administration of exogenous FGF19 suggesting that control of CYP7A1 transcription in the liver in response to the postprandial secretion by ileal entherocytes of FGF15/19 activates FGFR4 in the hepatocytes, thereby resulting in the repression of bile acid (BA) biosynthesis is intact. Administration of exogenous FGF19 significantly reduced CYP7A1 transcription by 32 fold versus control mice, thus indicating the efficacy of FGF19 treatment. BP3 treatment, however, resulted in a more potent downregulation of CYP7A1, whose transcription was found reduced by 110 fold when compared to the expression levels observed in control mice. Moreover, the reduction of endogenous FGF15 levels by a specific neutralizing antibody blunted BP3-induced CYP7A1 inhibition, thereby indicating that BP3 can modulate FGF15/19-dependent gene regulation (
FIG. 3A ). - Interestingly, one of the most prominently downregulated genes in livers from BP3 treated mice was glucose-6-phosphatase (G6PC), a key gluconeogenic enzyme, which was reduced about 130 fold when compared to control levels. In addition, there were corresponding increases of G6PC upstream regulatory genes, such as insulin receptor substrate 2 (IRS2). BP3 treatment also resulted in a marked upregulation of both interleukin 6 (IL6) and its downstream effector, suppressor of cytokine signaling 3 (SOCS3), which in turn blocks gluconeogenesis through G6PC suppression. Lastly, it is known that FGF19/15 can negatively modulate lipogenesis in mouse livers by inhibiting peroxisome proliferator-activated receptor-γ coactivator-1β (PPARGC1B) expression. Here, BP3 treatment significantly enhanced this effect (
FIG. 3A ). In these experimental settings, whilst a single administration of FGF19 was not sufficient to alter the expression of these gluconeogenic or lipogenic genes, neutralization of endogenous FGF15 reverted BP3-induced gene regulation to levels indistinguishable from those observed in control livers. These microarray data were validated with qRT-PCR, and it was observed that the results were highly consistent with those obtained in the cDNA array, as shown in the right panel ofFIG. 3A . - BP3's ability to modulate FGF15/19-induced downstream activity was examined. Specifically, the phosphorylation state of candidate signaling molecules upstream of gluconeogenesis and lipogenesis in the same ob/ob mouse livers was analyzed. Protein kinase B (AKT) is a critical effector kinase downstream of IRS2 whose phosphorylation results in a negative regulation of gluconeogenic genes, such as G6PC. Administration of BP3, but not of FGF19, induced a two-fold increase of AKT phosphorylation over the basal status in control levels, without affecting AKT expression. In addition, activation of AKT by BP3 was reduced to baseline intensities by an anti FGF15 antibody (
FIG. 3B ). The addition of exogenous FGF19 to BP3 treatment evoked a comparable AKT phosphorylation to that induced by BP3 only. - Several reports have described the contribution of interleukin-6 (IL6) pathway activation in improving insulin sensitivity through the activation of STAT3, with resulting G6PC downregulation. The status of endogenous STAT3 phosphorylation was examined in the same ob/ob mouse livers. Treatment with BP3, but not with FGF19, resulted in a 3.9-fold increase of STAT3 phosphorylation over baseline levels, whereas total STAT3 expression remained unchanged. Moreover, neutralization of endogenous FGF15 by a specific antibody blunted BP3-induced STAT3 phosphorylation (
FIG. 3B ). The graphic model depicted inFIG. 3C summarizes the molecular pathways utilized by BP3 to modulate BA biosynthesis, gluconeogenesis and lipogenesis in mouse livers. - To further compare the expression pattern of the hepatic genes between different treatments, a sample dendrogram was generated by hierarchical cluster analysis. The analysis revealed a clear separation between the BP3 treated liver samples and control groups, indicating that global gene expression in the former experimental group was significantly altered (
FIG. 3D ). It is noteworthy that the transcript pattern from livers co-treated with BP3 and a neutralizing anti FGF15 antibody clustered together with those of the control group, and they also co-clustered with transcripts from FGF19 or anti FGF15 treated mouse livers. - The Ingenuity Pathway Analysis software was used to integrate gene expression with key biochemical networks in response to the different experimental treatments (
FIG. 4 ). An overall analysis of metabolic functions indicated that carbohydrate, fatty acid and protein metabolism were significantly activated in response of BP3 treatment, but not of FGF19 alone (FIG. 4 , bottom). Analysis of activated pathways revealed a significant activation of FGF2 and FGF19-induced signaling after BP3 treatment. Moreover, major biochemical effector molecules activated by the engagement of FGF/FGFR axes, such as p38, ERK1/2 and JNK, were significantly activated. Amongst all others, the IL6/STAT3 signaling pathway showed the highest induction upon BP3 administration, with z-scores of 7.98 and 6.15, respectively. Commensurate with an upregulation of IL6, IL6 receptor (IL6R)-induced downstream signaling was also enhanced. - Activated AKT phosphorylates the downstream forkhead box O1 (FoxO1) transcription factor, which results in the suppression of gluconeogenic gene transcription, such as G6PC. Both AKT and FoxO1 pathways are concomitantly activated with BP3 treatment, whereas treatment with FGF19 alone or an anti-FGF15 antibody maintains these pathways in an inhibitory state. Commensurate with BP3-mediated suppression of PPARGC1B transcription via STAT3 activation (
FIG. 3A ), BP3 treatment induced the inhibition of sterol regulatory element-binding protein 1c (SREBF1) signaling pathway, a PPARGC1B downstream effector molecule, thus indicating a reduction of lipogenesis. - It has been reported that FGF19 can promote initiation of protein translation in mouse livers by inducing eukaryotic initiation factor 4E (EIF4E) phosphorylation. Here, the EIF4E pathway was highly activated in response to BP3 administration, whereas FGF19 alone failed to activate it.
- It is also noteworthy that BP3 treatment in leptin deficient ob/ob mice significantly restored leptin-induced signaling pathway, thus suggesting that BP3 can trigger a downstream molecular response that mimicks that of leptin. Neutralization of endogenous FGF15 drastically reduced the induction of the aforementioned pathways and metabolic functions upon BP3 treatment, thus indicating that BP3 can sensitize and modulate FGF15/19-driven downstream biochemical signaling. The integration of BP3/FGF19/FGFR4-induced transcriptional regulation with activated pathways analyzed in silico is depicted in the schematic model in
FIG. 3C . - The impact of BP3 treatment on ad libitum fed normoglycemic wild type C57BL mice was examined using a similar protocol as above. Similar to the results seen in the ob/ob mice, a single intraperitoneal dose of BP3, but not of a control protein, was sufficient to significantly reduce plasma glucose levels (
FIG. 5A ). Similarly to what observed in ob/ob mouse livers, BP3 treatment of wild type mice induced a marked downregulation of hepatic CYP7A1, CYP8B1, G6PC, and PPARGC1B, and an upregulation of IL6 and SOCS3 (FIG. 5B ) and a prominent increase of endogenous AKT and STAT3 phosphorylation levels, without affecting basal expression (FIG. 5B ). Conversely, CYP8B1 transcription was not suppressed by BP3 treatment in ob/ob mice. Lastly, in this experimental setting, in silico analysis of BP3-activated pathways in C57BL mice (FIG. 5D ) was commensurate with the results obtained from the ob/ob model (seeFIG. 4 ). - FGF19 exhibits a high affinity for FGFR4, and BP3 can enhance FGFR4/FGF19 complex formation. The data herein shows that BP3 binds to FGFR4, but not to other FGFRs (
FIG. 6A ), and contains a high-affinity binding site for FGFR4, as determined in a dose response assay by surface plasmon resonance (SPR) and ELISA (FIG. 6B-C ). A 66 amino acid-long BP3 C-terminal fragment has been previously identified as the FGF2 binding domain. This binding domain was purified, and an MBP-tagged C66 fusion protein (referred as C66) (FIG. 7A ) and used as a tool for binding studies. Whilst C66 retained its ability to bind to FGF2, its binding to FGF19 was even higher. The C66 fragment, however, did not bind to FGFR4, indicating that the FGF-binding domain of BP3 is not sufficient to elicit BP3 binding to FGFR4 (FIG. 7B ). The contribution of C66 to FGFR4/FGF19 complex formation was assessed in vitro. C66 significantly enhanced FGF19 binding to immobilized FGFR4, but immobilized FGFR1 did not display any binding to FGF19 or C66, either alone or in combination, which is similar to the binding characteristics of full length BP3 (FIG. 7C ). Moreover, when administered to ad libitum fed ob/ob diabetic mice for four hours in a single dose, C66 reverted hyperglycemia to normoglycemic levels, indistinguishable from those resulted from the treatment with full-length BP3, whereas mice treated with a control protein remained diabetic (FIG. 7D ). Analogous to full-length BP3 treatment, gene expression analysis of livers from C66-treated ob/ob mice revealed a marked suppression of CYP7A1 and G6PC and upregulation of IL6 (FIG. 7E ). From these experiments, the FGF-binding domain of BP3 is sufficient to increase FGFR4/FGF19 binding affinity and to reduce hyperglycemia in diabetic mice to the same extent as full-length BP3. Moreover, binding of BP3 to FGFR4 is not required for BP3 regulation of glucose homeostasis. - Five week old female ob/ob mice, obtained from Jackson Laboratories, were randomly assigned to treatment groups with FGF19 alone, or FGF19 in combination with BP3. The substances were administered in the morning by single intraperitoneal (i.p.) injection. To assess potential therapeutic effects on the metabolic profile with FGF19 or FGF19+BP3 treatments, glucose tolerance tests were performed and changes in body weights were measured. Two series of experiments were performed to investigate acute and sustained effects with the different treatments.
-
Series I Series II 1. Baseline (non-treated) 1. Baseline 2. FGF19 or FGF19 + BP3 treated 2. Post-treatment (2 days (1 dose/day for 5 days) following a single dose) 3. Post-treatment (9 days after treatment) - For all experiments reported in
FIGS. 8-10 , mice were fasted overnight (14 h) before they were subjected to a standard glucose tolerance test (GTT). The GTT is used to evaluate the ability of an organism to metabolize exogenous glucose. In clinical practice the GTT is used to uncover patients with latent diabetes or patients at risk for diabetes, e.g. during pregnancy and is performed after a fasting period by oral administration of a glucose containing drink. In the animal model oral GTT is performed by gavage of a glucose solution, i.e. orally administration. Intraperitoneal injection of a sterile glucose solution was chosen because this allows for a tighter control of dosing of the glucose. Before starting the experiments, the animals were weighed to determine the amount of glucose to inject. The glucose tolerance test was performed in a quiet room and handling was kept down to a minimum to reduce stress during the procedure. A bolus of glucose (1 g/kg) was injected into the intraperitoneal cavity (30% D-glucose:H2O solution) and blood was sampled from the tail tip at 0, 15, 30, 60, 120 and 180 minutes after glucose injection. Blood glucose levels were determined with a portable glucose meter (Contour®, Bayer HealthCare). - Ob/ob mice exhibit glucose intolerance and are generally used as a leptin-deficient diabetes model and reflect the human disease well. To examine the metabolic capacity of the animals for glucose the intraperitoneal glucose tolerance test (IPGTT) was performed in ob/ob mice and blood glucose levels were read at 0, 15, 30, 60, 120 and 180 minutes post-injection of glucose. The baseline blood glucose levels were much higher than the normal range (70-120 mg/dl) (
FIG. 8A, 8B , Baseline). After 5 days of treatment (one dose/day) with FGF19, ob/ob mice (n=6) showed an improved glucose tolerance as evidenced by reduced blood glucose levels at 180 minutes post-injection of glucose (p<0.05). The glucose tolerance test (IPGTT) was repeated 9 days after treatment and glucose levels returned to the levels seen before the treatment (FIG. 8A ). - For the combination treatment group (FGF19+BP3, n=5), the glucose tolerance test (IPGTT) was seen improved at 60, 120 and 180 minutes post-injection of glucose (p<0.05). The respective blood glucose levels were significantly lower than those of the control group. This effect was sustained even 9 days after the last of 5 doses of FGF19+BP3 (
FIG. 8B ). The results from the glucose tolerance test, shown as the area under the curve of the blood glucose levels after glucose injection (AUC), was reduced by 26% compared with baseline in the FGF19 group and by 33% in the FGF19+BP3 group. Most strikingly, the FGF19+BP3 group displayed an improved AUC compared with baseline even 9 days after completed treatment (p<0.01), whereas no such effect was observed in mice receiving FGF19 alone (FIG. 8C ). In addition, the combination treatment (n=5) improved the AUC even 2 days after a single dose of FGF19+BP3, while FGF19 alone (n=3) did not (p<0.01,FIG. 9 ). These data suggest that BP3 dramatically enhances the FGF19 effect in a standardized IPGTT glucose tolerance test in diabetic mice. - The effects of acute single doses of BP3 or FGF19 alone, and the combination of BP3+FGF19 were tested in ob/ob mice by IPGTT described above (
FIG. 10A , B). Mice were starved overnight (14 hours) and then treated for two hours by i.p. injection of vehicle (filled squares,FIG. 10A ,B) or FGF19 alone or BP3+FGF19 (open squaresFIG. 10A ,B). The IPGTT test was initiated two hours after treatments. The combination of BP3+FGF19 induced a striking effect and normalized the baseline blood glucose and the IPGTT blood glucose curve (FIG. 10A ). This effect was still present 24 hours after the single dose of BP3+FGF19 (FIG. 10C ). FGF19 alone showed no significant effect in the test (FIG. 10B ). Also, treatment of animals with BP3 alone (without FGF19) showed no significant effect, and even 7 days of dosing of BP3 with a single daily dose of BP3 lacked an effect on the baseline blood glucose or on the IPGTT test (FIG. 10D ). - Two days after a single dose treatment with FGF19 (n=3) or FGF19+BP3 (n=5), ob/ob mice were weighed and the changes in body weight were compared between the two groups. No significant changes in body weight were found (
FIG. 11A , 11B, 1 treatment) between the groups. However, 5 days of treatment (one dose/day) with FGF19+BP3 lowered the body weight by 1.0±0.7 g/animal, whereas 5 days of treatment (one dose/day) with FGF19 alone was associated with an increase in body weight (2.2±0.8 g/animal), which was similar to vehicle treated animals. For the 5 days of treatment, the changes in body weight were significantly different, in both grams and in percent body weight (p<0.05,FIG. 11A , 11B, 5 daily treatments). - BP3 enhances and prolongs the effects of FGF19 on glucose homeostasis, i.e., there is an improvement of the glucose tolerance, from either single or multiple doses. The combination of FGF19+BP3 also reduced the body weight per mouse after 1 dose/day for 5 days, while the animals in FGF19 treatment group gained weight. The combination of BP3 and FGF19 has an unexpectedly better therapeutic effect on obesity than FGF19 alone.
- Given the proposed mechanism of action, it was surprising that a FGFBP3 enhanced the effects of FGF19, i.e., a molecule that that will not be retained by the extracellular matrix and that administration of a complex of FGF19 and FGFBP3 would affect metabolism more quickly, (i.e. after a single dose), for a longer period (i.e for up to two days after a single dose) and more profoundly (i.e. better efficacy in normalizing glucose tolerance) than FGF19 alone. These data support the concept that BP3+FGF19 treatment improves glucose metabolism in subjects with diabetes and that a single daily dose may be sufficient to last beyond 24 hours.
- To further explore a possible mechanism of action, HepG2 cells (hepatocellular carcinoma cells) were treated with FGF19 with or without BP3.
FIG. 12 shows that the presence of BP3 enhanced the ability of FGF19 to induce phosphorylation of Erk1/2. - To understand the longterm effect, FGFBP3 was expressed in ob/ob, leptin deficient mice that develop metabolic disease. For this experiment, mouse FGFBP3 was used to avoid immune rejection of a human protein. Exogenously introduced FGFBP3 expression was achieved by injecting mice twice weekly with a liposomally packaged FGFBP3 expression plasmid to achieve uptake and expression of the exogenously introduce FGFBP3 gene. After 2½ weeks of treatments the content of blood in mice was analyzed as shown in
FIG. 13 .
Claims (20)
Priority Applications (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US15/784,730 US20180214514A1 (en) | 2013-03-14 | 2017-10-16 | Compositions and Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
US16/840,046 US20200246425A1 (en) | 2013-03-14 | 2020-04-03 | Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
Applications Claiming Priority (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US201361782347P | 2013-03-14 | 2013-03-14 | |
US201361782382P | 2013-03-14 | 2013-03-14 | |
PCT/US2014/026937 WO2014152089A1 (en) | 2013-03-14 | 2014-03-14 | Compositions and treatments of metabolic disorders using fgf binding protein 3 |
PCT/US2014/026938 WO2014152090A1 (en) | 2013-03-14 | 2014-03-14 | Compositions and treatments of metabolic disorders using fgf binding protein 3 and fgf 19 |
US14/853,482 US9789160B2 (en) | 2013-03-14 | 2015-09-14 | Treatments for lowering glucose levels using FGF binding protein 3 |
US15/784,730 US20180214514A1 (en) | 2013-03-14 | 2017-10-16 | Compositions and Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/853,482 Division US9789160B2 (en) | 2013-03-14 | 2015-09-14 | Treatments for lowering glucose levels using FGF binding protein 3 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/840,046 Continuation US20200246425A1 (en) | 2013-03-14 | 2020-04-03 | Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
Publications (1)
Publication Number | Publication Date |
---|---|
US20180214514A1 true US20180214514A1 (en) | 2018-08-02 |
Family
ID=55016240
Family Applications (3)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/853,482 Active US9789160B2 (en) | 2013-03-14 | 2015-09-14 | Treatments for lowering glucose levels using FGF binding protein 3 |
US15/784,730 Abandoned US20180214514A1 (en) | 2013-03-14 | 2017-10-16 | Compositions and Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
US16/840,046 Abandoned US20200246425A1 (en) | 2013-03-14 | 2020-04-03 | Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US14/853,482 Active US9789160B2 (en) | 2013-03-14 | 2015-09-14 | Treatments for lowering glucose levels using FGF binding protein 3 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US16/840,046 Abandoned US20200246425A1 (en) | 2013-03-14 | 2020-04-03 | Treatments of Metabolic Disorders Using FGF Binding Protein 3 |
Country Status (1)
Country | Link |
---|---|
US (3) | US9789160B2 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
PE20141727A1 (en) | 2011-07-01 | 2014-11-26 | Ngm Biopharmaceuticals Inc | COMPOSITIONS, USES AND METHODS FOR THE TREATMENT OF METABOLIC DISORDERS AND DISEASES |
EP3798228A1 (en) | 2012-11-28 | 2021-03-31 | NGM Biopharmaceuticals, Inc. | Compositions and methods for treatment of metabolic disorders and diseases |
US9290557B2 (en) | 2012-11-28 | 2016-03-22 | Ngm Biopharmaceuticals, Inc. | Compositions comprising variants and fusions of FGF19 polypeptides |
AU2013370404B2 (en) | 2012-12-27 | 2017-11-02 | Ngm Biopharmaceuticals, Inc. | Methods for modulating bile acid homeostasis and treatment of bile acid disorders and diseases |
US9273107B2 (en) | 2012-12-27 | 2016-03-01 | Ngm Biopharmaceuticals, Inc. | Uses and methods for modulating bile acid homeostasis and treatment of bile acid disorders and diseases |
US10456449B2 (en) | 2014-06-16 | 2019-10-29 | Ngm Biopharmaceuticals, Inc. | Methods and uses for modulating bile acid homeostasis and treatment of bile acid disorders and diseases |
US10434144B2 (en) | 2014-11-07 | 2019-10-08 | Ngm Biopharmaceuticals, Inc. | Methods for treatment of bile acid-related disorders and prediction of clinical sensitivity to treatment of bile acid-related disorders |
ES2871036T3 (en) | 2015-11-09 | 2021-10-28 | Ngm Biopharmaceuticals Inc | Method for treating bile acid-related disorders |
US11370841B2 (en) | 2016-08-26 | 2022-06-28 | Ngm Biopharmaceuticals, Inc. | Methods of treating fibroblast growth factor 19-mediated cancers and tumors |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20020155543A1 (en) | 1997-11-25 | 2002-10-24 | Genentech, Inc. | Fibroblast growth factor-19 (FGF-19) nucleic acids and polypeptides and methods of use for the treatment of obesity and related disorders |
WO2003072126A2 (en) | 2002-02-28 | 2003-09-04 | Switch Biotech Ag | Use of a fibroblast growth factor-binding protein for the treatment and diagnosis of diabetic wound healing problems |
EP1877545A2 (en) | 2005-04-20 | 2008-01-16 | Wyeth | Mammalian expression systems |
WO2010065439A1 (en) | 2008-12-05 | 2010-06-10 | Eli Lilly And Company | Variants of fibroblast growth factor 21 |
-
2015
- 2015-09-14 US US14/853,482 patent/US9789160B2/en active Active
-
2017
- 2017-10-16 US US15/784,730 patent/US20180214514A1/en not_active Abandoned
-
2020
- 2020-04-03 US US16/840,046 patent/US20200246425A1/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
US20200246425A1 (en) | 2020-08-06 |
US20160000861A1 (en) | 2016-01-07 |
US9789160B2 (en) | 2017-10-17 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20200246425A1 (en) | Treatments of Metabolic Disorders Using FGF Binding Protein 3 | |
WO2014152089A1 (en) | Compositions and treatments of metabolic disorders using fgf binding protein 3 | |
WO2014152090A1 (en) | Compositions and treatments of metabolic disorders using fgf binding protein 3 and fgf 19 | |
US20190192630A1 (en) | Mutated fibroblast growth factor (fgf) 1 and methods of use | |
EP2152294B1 (en) | A bad bh3 domain peptide for use in treating or delaying the onset of diabetes. | |
AU2013311777B2 (en) | Fusion proteins for treating a metabolic syndrome | |
JP7028775B2 (en) | FGF21 mutant | |
AU2011239386B2 (en) | Methods for treating metabolic disorders using FGF | |
JP2008535797A (en) | Polypeptide derived from human leptin and use thereof | |
KR102442984B1 (en) | Methods for treating type 1 diabetes using glucagon receptor antagonistic antibodies | |
US20190151416A1 (en) | Fibroblast growth factor (fgf) 1 proteins with glucose lowering ability and reduced mitogenicity | |
EP3285793A2 (en) | Fibroblast growth factor (fgf) 1 mutants and methods of use to reduce blood glucose | |
WO2010086867A2 (en) | Peptides, pharmaceutical compositions comprising same and uses thereof | |
US20230097335A1 (en) | Fibroblast growth factor 1 (fgf1) mutant proteins that selectively activate fgfr1b to reduce blood glucose | |
US20070218504A1 (en) | Human leptin-derived polypeptides and uses thereof | |
US20220118052A1 (en) | Materials and methods for modulating glucose uptake | |
US20090197800A1 (en) | Insulin Receptor Binding Peptides with Non-Insulin Gene Activation Profiles and Uses Thereof | |
KR20230034356A (en) | GLP-1R agonist peptides with reduced activity | |
US20190276510A1 (en) | Use of fibroblast growth factor 1 (fgf1)-vagus nerve targeting chimeric proteins to treat hyperglycemia | |
US20040033954A1 (en) | Therapeutic method for combined use of modified CNTF and thiadolidinedione |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
STPP | Information on status: patent application and granting procedure in general |
Free format text: NON FINAL ACTION MAILED |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: RESPONSE TO NON-FINAL OFFICE ACTION ENTERED AND FORWARDED TO EXAMINER |
|
STPP | Information on status: patent application and granting procedure in general |
Free format text: FINAL REJECTION MAILED |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |
|
AS | Assignment |
Owner name: NATIONAL INSTITUTES OF HEALTH (NIH), U.S. DEPT. OF HEALTH AND HUMAN SERVICES (DHHS), U.S. GOVERNMENT, MARYLAND Free format text: CONFIRMATORY LICENSE;ASSIGNOR:GEORGETOWN UNIVERSITY;REEL/FRAME:066207/0957 Effective date: 20230808 |