CN114903905B - Application of glycodeoxycholic acid in preparing medicament for treating intrahepatic cholestasis and pharmaceutical composition thereof - Google Patents
Application of glycodeoxycholic acid in preparing medicament for treating intrahepatic cholestasis and pharmaceutical composition thereof Download PDFInfo
- Publication number
- CN114903905B CN114903905B CN202110175232.0A CN202110175232A CN114903905B CN 114903905 B CN114903905 B CN 114903905B CN 202110175232 A CN202110175232 A CN 202110175232A CN 114903905 B CN114903905 B CN 114903905B
- Authority
- CN
- China
- Prior art keywords
- fxr
- icp
- gdca
- mice
- bile acid
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- WVULKSPCQVQLCU-BUXLTGKBSA-N glycodeoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 WVULKSPCQVQLCU-BUXLTGKBSA-N 0.000 title claims abstract description 65
- 108010035713 Glycodeoxycholic Acid Proteins 0.000 title claims abstract description 62
- WVULKSPCQVQLCU-UHFFFAOYSA-N Glycodeoxycholic acid Natural products C1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)C(O)C2 WVULKSPCQVQLCU-UHFFFAOYSA-N 0.000 title claims abstract description 62
- 239000003814 drug Substances 0.000 title claims abstract description 11
- 208000001024 intrahepatic cholestasis Diseases 0.000 title claims abstract description 11
- 230000007872 intrahepatic cholestasis Effects 0.000 title claims abstract description 11
- 239000008194 pharmaceutical composition Substances 0.000 title abstract description 6
- 241000606125 Bacteroides Species 0.000 claims abstract description 42
- 241000124008 Mammalia Species 0.000 claims abstract description 7
- 210000001035 gastrointestinal tract Anatomy 0.000 claims description 10
- 230000035935 pregnancy Effects 0.000 claims description 7
- 102100038495 Bile acid receptor Human genes 0.000 claims description 5
- 101000603876 Homo sapiens Bile acid receptor Proteins 0.000 claims description 5
- 230000005764 inhibitory process Effects 0.000 claims description 5
- 238000011282 treatment Methods 0.000 claims description 4
- CRDAMVZIKSXKFV-FBXUGWQNSA-N (2-cis,6-cis)-farnesol Chemical compound CC(C)=CCC\C(C)=C/CC\C(C)=C/CO CRDAMVZIKSXKFV-FBXUGWQNSA-N 0.000 claims description 3
- 239000000260 (2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1-ol Substances 0.000 claims description 3
- 229940043259 farnesol Drugs 0.000 claims description 3
- 229930002886 farnesol Natural products 0.000 claims description 3
- CRDAMVZIKSXKFV-UHFFFAOYSA-N trans-Farnesol Natural products CC(C)=CCCC(C)=CCCC(C)=CCO CRDAMVZIKSXKFV-UHFFFAOYSA-N 0.000 claims description 3
- 238000004519 manufacturing process Methods 0.000 claims description 2
- 230000000968 intestinal effect Effects 0.000 abstract description 26
- 238000002474 experimental method Methods 0.000 abstract description 9
- 238000011160 research Methods 0.000 abstract description 7
- 239000000556 agonist Substances 0.000 abstract description 6
- 238000012404 In vitro experiment Methods 0.000 abstract description 5
- 229940121360 farnesoid X receptor (fxr) agonists Drugs 0.000 abstract description 4
- 201000002161 intrahepatic cholestasis of pregnancy Diseases 0.000 abstract description 4
- 230000009471 action Effects 0.000 abstract description 3
- 229940079593 drug Drugs 0.000 abstract description 3
- 230000007246 mechanism Effects 0.000 abstract description 3
- 241001465754 Metazoa Species 0.000 abstract description 2
- 230000009286 beneficial effect Effects 0.000 abstract description 2
- 102100039215 Guanine nucleotide-binding protein G(t) subunit alpha-3 Human genes 0.000 abstract 2
- 101000888145 Homo sapiens Guanine nucleotide-binding protein G(t) subunit alpha-3 Proteins 0.000 abstract 2
- 241000699670 Mus sp. Species 0.000 description 54
- 239000003613 bile acid Substances 0.000 description 54
- 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 50
- 241000699666 Mus <mouse, genus> Species 0.000 description 29
- 230000014509 gene expression Effects 0.000 description 26
- 238000004458 analytical method Methods 0.000 description 24
- 108090000623 proteins and genes Proteins 0.000 description 19
- 108010003814 member 2 group B nuclear receptor subfamily 0 Proteins 0.000 description 17
- 238000000034 method Methods 0.000 description 17
- 210000002966 serum Anatomy 0.000 description 16
- 230000002550 fecal effect Effects 0.000 description 14
- 102100023172 Nuclear receptor subfamily 0 group B member 2 Human genes 0.000 description 13
- 210000003608 fece Anatomy 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 210000004185 liver Anatomy 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- 230000000694 effects Effects 0.000 description 10
- RUDATBOHQWOJDD-UHFFFAOYSA-N (3beta,5beta,7alpha)-3,7-Dihydroxycholan-24-oic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(O)=O)C)C1(C)CC2 RUDATBOHQWOJDD-UHFFFAOYSA-N 0.000 description 9
- 241000894006 Bacteria Species 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 230000001605 fetal effect Effects 0.000 description 9
- 239000000463 material Substances 0.000 description 9
- 230000004060 metabolic process Effects 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000003556 assay Methods 0.000 description 8
- 238000003753 real-time PCR Methods 0.000 description 8
- 230000027455 binding Effects 0.000 description 7
- 210000005228 liver tissue Anatomy 0.000 description 7
- GHCZAUBVMUEKKP-UHFFFAOYSA-N ursodeoxycholic acid glycine-conjugate Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)CC2 GHCZAUBVMUEKKP-UHFFFAOYSA-N 0.000 description 7
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 102000003929 Transaminases Human genes 0.000 description 6
- 108090000340 Transaminases Proteins 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000002440 hepatic effect Effects 0.000 description 6
- 201000011461 pre-eclampsia Diseases 0.000 description 6
- 238000012163 sequencing technique Methods 0.000 description 6
- 101000846394 Homo sapiens Fibroblast growth factor 19 Proteins 0.000 description 5
- 101100390675 Mus musculus Fgf15 gene Proteins 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- RUDATBOHQWOJDD-BSWAIDMHSA-N chenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-BSWAIDMHSA-N 0.000 description 5
- 229960001091 chenodeoxycholic acid Drugs 0.000 description 5
- GHCZAUBVMUEKKP-XROMFQGDSA-N glycoursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)CC1 GHCZAUBVMUEKKP-XROMFQGDSA-N 0.000 description 5
- 239000001963 growth medium Substances 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 208000024891 symptom Diseases 0.000 description 5
- 238000002877 time resolved fluorescence resonance energy transfer Methods 0.000 description 5
- 238000002054 transplantation Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 4
- 210000003754 fetus Anatomy 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 238000001746 injection moulding Methods 0.000 description 4
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 4
- 210000002826 placenta Anatomy 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 229920002477 rna polymer Polymers 0.000 description 4
- 239000004017 serum-free culture medium Substances 0.000 description 4
- 210000000813 small intestine Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- RUDATBOHQWOJDD-UZVSRGJWSA-N ursodeoxycholic acid Chemical compound C([C@H]1C[C@@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)CC1 RUDATBOHQWOJDD-UZVSRGJWSA-N 0.000 description 4
- 229960001661 ursodiol Drugs 0.000 description 4
- 239000006216 vaginal suppository Substances 0.000 description 4
- 241000606124 Bacteroides fragilis Species 0.000 description 3
- 206010008635 Cholestasis Diseases 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108010093662 Member 11 Subfamily B ATP Binding Cassette Transporter Proteins 0.000 description 3
- 102000001479 Member 11 Subfamily B ATP Binding Cassette Transporter Human genes 0.000 description 3
- 108091006611 SLC10A1 Proteins 0.000 description 3
- 108010058254 Steroid 12-alpha-Hydroxylase Proteins 0.000 description 3
- 239000005557 antagonist Substances 0.000 description 3
- 210000000941 bile Anatomy 0.000 description 3
- 238000007664 blowing Methods 0.000 description 3
- 230000007870 cholestasis Effects 0.000 description 3
- 231100000359 cholestasis Toxicity 0.000 description 3
- 238000004140 cleaning Methods 0.000 description 3
- 230000006690 co-activation Effects 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 208000035475 disorder Diseases 0.000 description 3
- 230000002496 gastric effect Effects 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 210000000936 intestine Anatomy 0.000 description 3
- 210000005229 liver cell Anatomy 0.000 description 3
- 230000002503 metabolic effect Effects 0.000 description 3
- 230000037353 metabolic pathway Effects 0.000 description 3
- 201000010065 polycystic ovary syndrome Diseases 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 108020004465 16S ribosomal RNA Proteins 0.000 description 2
- 241001202853 Blautia Species 0.000 description 2
- 101150075266 CYP7A1 gene Proteins 0.000 description 2
- 102000029816 Collagenase Human genes 0.000 description 2
- 108060005980 Collagenase Proteins 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102100031734 Fibroblast growth factor 19 Human genes 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- 108010015031 Glycochenodeoxycholic Acid Proteins 0.000 description 2
- 108010007979 Glycocholic Acid Proteins 0.000 description 2
- 239000005089 Luciferase Substances 0.000 description 2
- RFDAIACWWDREDC-UHFFFAOYSA-N Na salt-Glycocholic acid Natural products OC1CC2CC(O)CCC2(C)C2C1C1CCC(C(CCC(=O)NCC(O)=O)C)C1(C)C(O)C2 RFDAIACWWDREDC-UHFFFAOYSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 208000003251 Pruritus Diseases 0.000 description 2
- 102100021988 Sodium/bile acid cotransporter Human genes 0.000 description 2
- 238000010811 Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry Methods 0.000 description 2
- 230000002411 adverse Effects 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 239000008280 blood Substances 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 229960002424 collagenase Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000010219 correlation analysis Methods 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000012470 diluted sample Substances 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- 238000010790 dilution Methods 0.000 description 2
- 239000012895 dilution Substances 0.000 description 2
- 230000007783 downstream signaling Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000029142 excretion Effects 0.000 description 2
- 238000003304 gavage Methods 0.000 description 2
- GHCZAUBVMUEKKP-GYPHWSFCSA-N glycochenodeoxycholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)CC1 GHCZAUBVMUEKKP-GYPHWSFCSA-N 0.000 description 2
- RFDAIACWWDREDC-FRVQLJSFSA-N glycocholic acid Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 RFDAIACWWDREDC-FRVQLJSFSA-N 0.000 description 2
- 229940099347 glycocholic acid Drugs 0.000 description 2
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000007803 itching Effects 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 108020004999 messenger RNA Proteins 0.000 description 2
- 239000012046 mixed solvent Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000009984 peri-natal effect Effects 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000001195 ultra high performance liquid chromatography Methods 0.000 description 2
- 239000012224 working solution Substances 0.000 description 2
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 1
- 102100032645 7-alpha-hydroxycholest-4-en-3-one 12-alpha-hydroxylase Human genes 0.000 description 1
- 101000577181 Aspergillus oryzae (strain ATCC 42149 / RIB 40) Extracellular metalloproteinase NpI Proteins 0.000 description 1
- 108090000145 Bacillolysin Proteins 0.000 description 1
- 101150051438 CYP gene Proteins 0.000 description 1
- 101150079919 Cyp27a1 gene Proteins 0.000 description 1
- 238000007400 DNA extraction Methods 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- -1 FXR Proteins 0.000 description 1
- 208000001951 Fetal Death Diseases 0.000 description 1
- 208000032943 Fetal Distress Diseases 0.000 description 1
- 108090000331 Firefly luciferases Proteins 0.000 description 1
- 206010016852 Foetal damage Diseases 0.000 description 1
- 206010055690 Foetal death Diseases 0.000 description 1
- 206010016855 Foetal distress syndrome Diseases 0.000 description 1
- 206010070538 Gestational hypertension Diseases 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 208000001145 Metabolic Syndrome Diseases 0.000 description 1
- 101000878182 Mus musculus Fibroblast growth factor 15 Proteins 0.000 description 1
- 102000035092 Neutral proteases Human genes 0.000 description 1
- 108091005507 Neutral proteases Proteins 0.000 description 1
- 241000192656 Nostoc Species 0.000 description 1
- 206010036049 Polycystic ovaries Diseases 0.000 description 1
- 208000005107 Premature Birth Diseases 0.000 description 1
- 206010036590 Premature baby Diseases 0.000 description 1
- 108010052090 Renilla Luciferases Proteins 0.000 description 1
- 108700008625 Reporter Genes Proteins 0.000 description 1
- 241000605947 Roseburia Species 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- 241001148134 Veillonella Species 0.000 description 1
- 201000000690 abdominal obesity-metabolic syndrome Diseases 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000008484 agonism Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 238000003149 assay kit Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000010001 cellular homeostasis Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 230000003081 coactivator Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 230000000875 corresponding effect Effects 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 230000002124 endocrine Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 229940011871 estrogen Drugs 0.000 description 1
- 239000000262 estrogen Substances 0.000 description 1
- 239000013613 expression plasmid Substances 0.000 description 1
- 208000011404 female reproductive system disease Diseases 0.000 description 1
- 231100000479 fetal death Toxicity 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
- 239000012634 fragment Substances 0.000 description 1
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 1
- 210000003736 gastrointestinal content Anatomy 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 208000004104 gestational diabetes Diseases 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000047000 human FGF19 Human genes 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000036737 immune function Effects 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 238000002513 implantation Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 210000005027 intestinal barrier Anatomy 0.000 description 1
- 230000007358 intestinal barrier function Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 208000019423 liver disease Diseases 0.000 description 1
- 230000005976 liver dysfunction Effects 0.000 description 1
- 238000003670 luciferase enzyme activity assay Methods 0.000 description 1
- 239000012139 lysis buffer Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000007102 metabolic function Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000003032 molecular docking Methods 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 208000015124 ovarian disease Diseases 0.000 description 1
- 201000004535 ovarian dysfunction Diseases 0.000 description 1
- 231100000543 ovarian dysfunction Toxicity 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000012188 paraffin wax Substances 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 230000008807 pathological lesion Effects 0.000 description 1
- 238000003068 pathway analysis Methods 0.000 description 1
- 239000000546 pharmaceutical excipient Substances 0.000 description 1
- 210000005059 placental tissue Anatomy 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000013062 quality control Sample Substances 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000009933 reproductive health Effects 0.000 description 1
- 101150096065 shp gene Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000009469 supplementation Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000004885 tandem mass spectrometry Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 229940126585 therapeutic drug Drugs 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 238000001890 transfection Methods 0.000 description 1
- 239000012096 transfection reagent Substances 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 229940120293 vaginal suppository Drugs 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
- A61K31/575—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of three or more carbon atoms, e.g. cholane, cholestane, ergosterol, sitosterol
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
Landscapes
- Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Epidemiology (AREA)
- Gastroenterology & Hepatology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
Abstract
The invention discloses application of glycodeoxycholic acid or stereoisomer or derivative thereof in preparing a medicament for preventing and/or treating intrahepatic cholestasis of human or mammals. The invention also discloses a pharmaceutical composition serving as an FXR agonist. The invention has the beneficial effects that: the research finds that the action mechanism of intrahepatic cholestasis caused by the increase of the intestinal bacteroides abundance of ICP patients, and proves that GDCA is used as a novel agonist of FXR through in vitro experiments and animal experiments, and can play a role in preventing and/or relieving ICP by exciting FXR signals, so that the GDCA has the potential of being used for preparing medicines for treating ICP.
Description
Technical Field
The invention belongs to the technical field of medical application of compounds, and particularly relates to application of glycodeoxycholic acid in the field of cholestasis and a composition thereof.
Background
Intrahepatic cholestasis (Intrahepatic cholestasis of pregnancy, ICP) during gestation is a complication specific to the middle and late stages of pregnancy, is clinically characterized by skin itching and bile acid elevation, and can significantly increase the risk of perinatal adverse events such as fetal distress, premature birth, sudden fetal death and the like, and causes great harm to reproductive health of females. At present, the etiology and mechanism of ICP occurrence is still unclear. Because of this, more of the treatments for ICP are symptomatic and empirical. At present, the first-line clinical therapeutic drug is ursodeoxycholic acid (UDCA), which is produced by metabolism of intestinal flora, is the most commonly used drug for ICP treatment at present, and can effectively relieve itching of pregnant women and protect liver dysfunction, but large-sample crowd researches show that the protection effect of the UDCA on reducing serum bile acid and reducing adverse events in the perinatal period of fetuses is not obvious, so that the application of the UDCA in treating ICP is also in great disputes.
Intestinal flora has become a big hot spot in recent years of research and plays an important role in the regulation of various physiological and pathological conditions such as inflammation, metabolic syndrome, endocrine and reproduction. Research of domestic scholars shows that intestinal flora of a female polycystic ovary syndrome patient is obviously disturbed, the implantation of fecal bacteria of the polycystic ovary patient into mice can cause the ovarian dysfunction of the mice, and further the intestinal flora can promote the occurrence of the female polycystic ovary syndrome by regulating and controlling the disorder of bile acid metabolism and immune function; preeclampsia pregnant women have dysenteric flora, which can promote the occurrence of preeclampsia by increasing the translocation of bacteria to the placenta by disturbing the T cell homeostasis and destroying the intestinal barrier, and the preeclampsia pregnant women have the phenotype that preeclampsia can occur in mice due to the transplanted mice with faecal bacteria, which suggests that the regulation of the enteric flora in female reproductive system diseases is more and more interesting and important.
The diet, genetic background of the host, and immune system are closely related to the structural remodeling and alteration of the intestinal flora. In gestation period, the organism can be subjected to serial changes such as hormone, metabolism, immunoregulation and the like, and the stable state of intestinal flora is greatly influenced. The composition of intestinal flora of a normal pregnant woman is obviously changed at different stages of pregnancy, and the composition and the structure of the intestinal flora of the normal pregnant woman are obviously different from those of a common woman. It has been demonstrated that disorders of gestational intestinal flora can lead to gestational diabetes, gestational hypertension, preeclampsia and other pregnancy related disorders. The inventor of the application earlier stage selects normal pregnant women matched with ages, gestational weeks and the like and ICP patient groups, and respectively carries out 16S rRNA sequencing analysis on excrement, so that the result shows that the alpha diversity of intestinal flora of ICP patients is obviously reduced, the composition of the intestinal flora of the two groups of people is obviously different, and Bacteroides (Bacteroides) in the intestinal tract of ICP patients is obviously increased. Further intensive researches show that intestinal flora can participate in regulating and controlling the metabolism of specific bile acid so as to influence the function of a farnesol X receptor (Farnesoid X Receptor, FXR) to promote the occurrence of ICP, and researches show that glycodeoxycholic acid (GDCA) is a novel FXR agonist, can target and activate FXR signals to effectively prevent and treat ICP, and provides a novel intervention means for preventing and treating clinical ICP.
Disclosure of Invention
In view of the above, it is an object of the present invention to provide a use of glycodeoxycholic acid.
The technical scheme is as follows:
use of glycodeoxycholic acid or a stereoisomer or a derivative thereof for the manufacture of a medicament for the prevention and/or treatment of intrahepatic cholestasis in a human or mammal.
Preferably, the derivative of glycodeoxycholic acid is either a pharmaceutically acceptable salt thereof or a pharmaceutically acceptable ester thereof.
Preferably, the intrahepatic cholestasis is caused at least in part by inhibition of Farnesol X Receptor (FXR) downstream signaling in a human or mammalian body.
Preferably, the inhibition of FXR downstream signal is caused by an increase in the abundance of Bacteroides in the human or mammalian intestinal tract.
Preferably, the medicament is used for preventing and/or treating intrahepatic cholestasis during gestation in humans or mammals.
It is a further object of the present invention to provide another use of glycodeoxycholic acid. The technical proposal is as follows:
use of glycodeoxycholic acid or a stereoisomer or a derivative thereof for the preparation of a medicament for use as an agonist of FXR in a human or mammal.
The present invention also provides a pharmaceutical composition. The technical proposal is as follows:
a pharmaceutical composition comprising glycodeoxycholic acid, or a pharmaceutically acceptable salt thereof, or a pharmaceutically acceptable ester thereof, or a stereoisomer thereof.
Preferably, the above pharmaceutical composition further comprises pharmaceutically acceptable carriers, additives or excipients, and unavoidable impurities.
Preferably, the medicine is tablets, granules, capsules or oral liquid.
Drawings
Fig. 1 shows the diversity and composition of fecal flora in ICP patients (ICP) and normal pregnant women (Control), wherein: (a) alpha diversity; (B) beta diversity analysis; (C) flora; (D) LefSe analysis.
FIG. 2 shows the damage of serum bile acid, hepatic transaminase and fetus to pregnant mice transplanted with fecal bacteria from ICP patients and normal pregnant women, wherein: (a) serum bile acids; biochemical indexes such as (B) to (D) hepatic transaminase AST, ALT, ALP; (E) fetal expression; (F) fetal mouse body weight; the two groups are respectively an ICP patient faecal fungus transplanting pregnant rat group (I-FMT) and a normal pregnant woman faecal fungus transplanting pregnant rat group (H-FMT).
FIG. 3 is a metabolic pathway of KEGG analysis of faecal bacteria differences in ICP patients and normal pregnant women;
fig. 4 is a targeted metabonomic analysis of ICP patient (ICP) versus normal Control pregnant woman (Control) bile acid composition differences, wherein: (a) fecal bile acid level; (B) serum bile acid levels; ** P<0.01。
FIG. 5 is an analysis of the correlation of the abundance of Bacteroides in the intestinal tract with the level of bile acid in ICP patients.
FIG. 6 shows the change in the mouse bile juice accumulation and FXR signal after Bacteroides transplantation by GDCA, wherein: (a) serum bile acid levels; (B, C) levels of biochemical indicators such as hepatic aminotransferase ALT, AST, etc.; (D) fetal mouse performance and weight of the fetal mouse; (E-F) qPCR detecting FXR and SHP expression; the groups are respectively an inactivated Bacteroides transplanted pregnant mouse group (Control), a Bacteroides transplanted pregnant mouse group (B.fragilis), a Bacteroides transplanted and GDCA stomach-perfused pregnant mouse group (B.fragilis+GDCA); * P<0.05； ** P<0.01；ns,no significance。
FIG. 7 is a study of the modulation of GDCA and FXR signals, wherein: (A, B) a Docking assay predicts the structural relationship and binding of GDCA to FXR; (C) After the GDCA is incubated with Caco2 cells, qPCR (quantitative polymerase chain reaction) is used for detecting the expression condition result of a downstream gene SHP, DMSO is used as a blank control, and chenodeoxycholic acid (CDCA) is used as a positive control agonist; ** P<0.01。
FIG. 8 shows the effect of TR-FRET FXR coactivation assay to detect FXR agonism by several bile acids such as CDCA, GDCA, GUDCA, with fluorescence emission intensity ratio of 520nm to 495nm as the detection index.
FIG. 9 shows the effect of GDCA on mRNA expression of FXR downstream genes FGF19 and SHP in a cell assay, while studying the effect of FXR antagonists GUDCA and T-. Beta.MCA on the above effects.
FIG. 10 shows the expression of FXR downstream genes FGF15 and SHP in intestinal tissues of a bacteroides transplanted pregnant mouse and a GDCA gastric lavage pregnant mouse; each group is an inactivated Bacteroides transplanted pregnant mouse group (Control), a Bacteroides transplanted pregnant mouse group (B.fragilis), a Bacteroides transplanted and GDCA stomach-perfused pregnant mouse group (BF+GDCA).
Fig. 11 is a comparison of cholestasis symptoms in EE2 injection molded ICP mice and in co-administered GDCA lavage mice, wherein: (a) schematic of the experimental procedure; (b) Bile acid, ALT, AST, ALP, GGT, liver index level; (c) liver tissue section HE staining photographs; each group was a normal mouse group (Control), an EE2 injection molding ICP mouse group (EE 2), an EE2 injection molding and a GDCA gavage mouse group (EE 2+gdca) were given.
FIG. 12 shows the expression of genes related to bile acid metabolism in EE2 injection molded ICP mice and GDCA-infused mice simultaneously, wherein: (a) The expression condition of FXR downstream gene FGF15 and SHP genes in intestinal tracts; (b) The expression of FXR downstream gene SHP and NTCP genes in liver; (c) Bile acid synthesis genes such as Cyp7a1, cyp8b1, cyp27a1, and the like, and expression of bile acid excretion genes such as BSEP, MRP2, and the like; each group was a normal mouse group (Control), an EE2 injection molding ICP mouse group (EE 2), an EE2 injection molding and a GDCA gavage mouse group (EE 2+gdca) were given.
Detailed Description
The invention is further described below with reference to examples and figures.
Example 1
ICP patient and normal pregnant woman intestinal flora characteristic analysis
1.1 metagenomic analysis of intestinal flora differences in ICP patients and control populations
Experimental materials and methods
(1) Collecting faeces and serum samples of 100 ICP patients and 100 normal control pregnant women, expanding the sample volume, establishing a perfect sample library and a follow-up database, respectively taking faeces of two groups of people, dissolving in glycerol solution, and preserving at-80 ℃;
(2) The feces were extracted using the feces DNA extraction kit (MinkaGene Stool DNA Kit) according to the kit procedure: taking about 0.2g of excrement to a 2ml centrifuge tube, adding a lysis buffer solution for high-temperature lysis, centrifuging the suspension, taking the supernatant to a new centrifuge tube, adding proteinase K to digest for 10 minutes at 70 ℃, adding an ethanol solvent, passing through a DNA binding column, repeatedly washing twice, adding an EB buffer solution into DNA precipitate for dissolution, and preserving at-20 ℃ for later use;
(3) DNA is sent to Beijing Nostoc induced biotechnology company for sequencing analysis based on an Illumina HiSeq 2500 platform;
(4) After the original sequencing data is obtained, firstly removing pollution and a joint sequence, and removing host genes according to a human genome reference sequence; high quality data from which contamination and host genes were removed were further assembled by SPAdes; after assembly, predicting orf with MetaGene; clustering the predicted orf sequences by using CD-HI software, wherein each class takes the longest orf sequence as a representative sequence, and constructing a non-redundant gene sequence set; after the non-redundant gene set is obtained, clear reads can be compared to the non-redundant gene set, and the abundance of each gene in each sample is calculated; after obtaining the gene abundance table, performing functional comparison annotation on the genes by using software such as KEGG, GO, COG; species abundance analysis using metaphlan; respectively carrying out alpha and beta diversity analysis according to the gene abundance;
(5) According to the sequencing analysis result, the score of the abundance of each strain in each sample is further analyzed by using least squares discriminant analysis (PLS-DA), and the difference of the two groups of people is most obvious by using variable importance analysis.
Experimental results
The stool DNA of ICP patients and normal pregnant women were subjected to 16S rRNA sequencing analysis, respectively, and as a result, the intestinal flora alpha diversity of ICP patients was found to be significantly lower than that of the control group (FIG. 1A). The beta diversity analysis suggested that there was a significant difference in intestinal flora structure in ICP patients from the normal control group, and the structural composition of the two groups was significant (fig. 1B). The flora composition (fig. 1C) and the LefSe analysis showed that the two groups of populations had a significantly different genus (fig. 1D), and it was found that Bacteroides (bacterioides), lachnoclostrichum, veillonella (bacterioides), ross (Roseburia), escherichia coli-shigella (escherichia_shigella) were significantly enriched in the ICP patient group, while Blautia (Blautia) was significantly reduced. Among them, bacteroides (bacterioides) are significantly enriched in the intestinal tract of ICP patients.
1.2 bacterial group transplantation experiments
Experimental materials and methods
And (3) respectively transplanting the fecal bacteria from ICP patients and normal pregnant women into sterile pregnant mice, and detecting biochemical indexes such as bile acid, hepatic transaminase and the like and fetal indexes. The method comprises the following specific steps:
(1) Preparing a fecal bacterial suspension: taking fresh feces from ICP patients and normal pregnant women respectively, immediately re-suspending in PBS solution containing glycerol, and packaging and storing at-80deg.C for use;
(2) Centrifuging the fecal suspension at a low speed, removing excessive fecal residues, respectively transplanting to sterile mice, and performing gastric lavage on each mouse for 200 μl (0.15 g/ml) of fecal suspension twice a week for 4 weeks;
(3) Mice were small pressed after the gastric lavage was completed: female mice are matched in a ratio of 1:2, the weight change of the female mice is observed and recorded, and vaginal suppositories are observed and recorded as E0.5 d;
(4) Female mice are pregnant for E18d, the feces of the mice are collected, the mice are anesthetized and sacrificed, the blood, placenta and other tissues of the mice are taken, and the number and the weight of fetuses are observed and recorded;
(5) Detecting ALT, AST, ALP and total bile acid level in serum by using a full-automatic biochemical analyzer;
(6) After the liver and placenta are respectively formalin fixed, paraffin embedding and slicing are carried out, and pathological changes of the liver and placenta tissues are detected by HE staining;
experimental results
As shown in figures 2 (A-D), the index of bile acid, hepatic transaminase (AST, ALT, ALP) and the like of the fecal bacteria transplanted mice (I-FMT) derived from ICP patients is obviously higher than that of the fecal bacteria transplanted mice (H-FMT) of normal pregnant women. As shown in FIG. 2 (E, F), fetal performance was observed and fetal mouse body weight was compared, and it was found that the fetal damage of the ICP patient-derived fecal graft mice (I-FMT) was also significantly higher than that of the normal pregnant woman fecal graft mice (H-FMT).
This study showed that the structural changes in the intestinal flora did lead to the occurrence of ICP-related symptoms in pregnant mice.
1.3 metabolic differences in ICP patients and normal pregnant women
1.3.1KEGG analysis of differential Metabolic pathways
Experimental materials and methods
Intestinal flora composition of normal pregnant women and ICP patients was analyzed by metagenomic sequencing, and KEGG metabolic pathway analysis was performed by bioinformatics software.
Experimental results
As shown in fig. 3, in the signal paths of the differential enriched flora participating in regulation in the intestinal tracts of ICP patients and normal pregnant women, the most obvious difference is the bile acid metabolic path, which provides basis for subsequent study of bile acid composition and regulation.
1.3.2 targeted metabonomics detection analysis of bile acid composition and levels in feces and serum of ICP patients and normal pregnant women
Experimental materials and methods
(1) Respectively taking faeces and serum samples of ICP patients and normal pregnant women, and storing in an ultralow temperature refrigerator for standby;
(2) Bile acid metabolism detection is entrusted to the completion of Shanghai Mai painted spectrum biotechnology company;
(3) Preparing a test detection article: bile acid standards were purchased from Steraloids Inc. (Newport, RI, USA) and TRC Chemicals (Toronto, ON, canada), and 10 isotopically labeled bile acid internal standards were purchased from C/D/N Isotopes Inc. (Quebec, canada) and Steraloids Inc. (Newport, RI, USA). The bile acid and the isotope internal standard are precisely weighed and dissolved in methanol to obtain a storage mother solution with the concentration of 5 mM. At the time of use, bile acid stock solutions were mixed and serially diluted in a bile acid-free serum matrix to 2500, 500, 250, 50, 10, 2.5 and 1nM concentration points. 1500, 150 and 5nM bile acid standard solution is prepared from the bile acid-free serum matrix as quality control samples with three concentrations of high, medium and low. The concentration of the internal standard in the concentration point standard solution is the same as that of the internal standard in the quality control sample, namely (GCA-d 4, TCA-d4, TCDCA-d9, CA-d4, GCDCA-d4, CDCA-d4, LCA-d4 and aMCA-d5 are 150 nM);
(4) Sample pretreatment: fecal or serum samples were taken into centrifuge tubes, about 25mg of pre-chilled beads were added, and 200 μl of acetonitrile/methanol (v/v=8:2) mixed solvent containing 10 μl of internal standard was added. After homogenization, the mixture was centrifuged at 13,500rpm for 20min at 4 ℃. Taking 10 mu L of supernatant, diluting with 90 mu L of a mixed solvent of acetonitrile/methanol (80/20) and ultrapure water in a ratio of 1:1, oscillating and centrifuging, and waiting for sample injection analysis, wherein the sample injection volume is 5 mu L;
(5) Ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS, ACQUITY UPLC-Xevo TQ-S, waters Corp., milford, mass., USA) was used for quantitative bile acid detection.
Experimental results
The ICP patient had significantly reduced average of three bile acids in faeces (fig. 4A) and serum (fig. 4B), glycocholic acid (GCA), glycochenodeoxycholic acid (GCDCA) and glycodeoxycholic acid (GDCA), respectively, compared to normal pregnant women.
1.4 analysis of the correlation of Bacteroides with bile acid levels
Experimental materials and methods
(1) Obtaining the relative abundance value of the bacteroides in each ICP patient faeces sample through macrogenomic analysis;
(2) And (3) determining the content of bile acid in the feces of the ICP patient by using the targeted metabonomics, and analyzing the correlation of the bacteroides abundance of the ICP patient and the content of the bile acid by using the correlation analysis.
Experimental results
Correlation analysis of the abundance of Bacteroides in ICP patients with bile acid levels shows that the abundance of Bacteroides is obviously inversely correlated with the GDCA level, as shown in FIG. 5, suggesting that Bacteroides may be involved in the metabolic regulation of GDCA.
Example two
Role of GDCA in Bacteroides regulating ICP Generation
FXR is distributed in the intestine, liver, and is involved in bile acid metabolism. Studies have found that FXR in the liver inhibits bile acid synthesis by inducing a Small Heterodimer Partner (SHP) with downstream signaling. Human fibroblast growth factor 19 (FGF 19) is a bile acid metabolism regulator homologous to mouse fibroblast growth factor 15 (FGF 15), and is a FXR downstream signal. In the intestinal tract, FXR inhibits bile acid synthesis by inducing the signal pathway of FGF19/FGF 15.
This section investigated the effect of GDCA on mouse ICP and mechanism of action.
2.1 Effect of GDCA on mouse ICP symptoms caused by Bacteroides transplantation
Experimental materials and methods
(1) Preparing strains: uniformly coating an anaerobic culture medium plate by a bacteriostasis plate streaking method, culturing in an anaerobic glove box at 37 ℃, picking a single colony to perform anaerobic culture in a liquid culture medium after colony appears for 2-3 days, and measuring the OD value of the culture medium to calculate the cfu value of bacteria;
(2) Bacteroides (2X 10) 8 cfu) is dissolved in 200 mu l of sterile aqueous solution, and the sterile female mice are respectively irrigated, the inactivated bacteroides strains with the same concentration are controlled to be irrigated, and GDCA (30 mg/Kg/d) is respectively added to the inactivated bacteroides strains for the same time, and the inactivated bacteroides strains are irrigated twice a week, and the inactivated bacteroides strains are continuously irrigated for 4 weeks, and the male mice and the female mice are caged in a ratio of 1:2, so that the weight of the mice and the appearance time of vaginal suppositories are observed and recorded;
(3) Female mice are pregnant for E18d, the feces of the mice are collected before the mice are sacrificed, the mice are anesthetized and sacrificed, the blood, intestinal tracts, livers, placenta and other tissues of the mice are taken, and the number and the weight of the fetuses are observed and recorded;
(4) Detecting ALT, AST and total bile acid level in serum by using a full-automatic biochemical analyzer;
(5) The feces, serum, liver and intestinal tissues are respectively detected and analyzed by an ultra-high performance liquid chromatography tandem mass spectrometer (UPLC-MS/MS), and the steps of sample processing and detection and analysis are 1.3.2;
(6) Taking small intestine and liver tissues of a mouse, and separating intestinal epithelial cells and liver cells, wherein the method comprises the following specific steps of: the small intestine is moved to a culture dish, DPBS is repeatedly cleaned, penicillin-containing serum-free culture medium is used for cleaning for a plurality of times, intestinal tissues are sheared into fragments, the serum-free culture medium is cleaned, and the supernatant is centrifuged to obtain a precipitate for later use. Adding collagenase XI and neutral protease I into the precipitate, digesting for 20min at 37deg.C, repeatedly blowing by a pipette, centrifuging, repeatedly cleaning, and re-suspending. Fresh liver tissue is taken, the liver tissue is sheared into small tissue blocks, repeatedly washed and blown by a serum-free culture medium, collagenase is added for digestion, and a hepatocyte suspension is obtained for standby. And (3) respectively extracting RNA (ribonucleic acid) by using a Trizol kit after taking the cell suspension, and detecting the expression of FXR, SHP and other molecules by qPCR.
Experimental results
As shown in 6 (A-D), the bacteroides transplantation can obviously cause the rise of the levels of two hepatic aminotransferases, namely the bile acid level, ALT and AST of the mice, and the appearance of the fetal mice is damaged, and the weight is reduced, namely cholestasis and the fetal mice are damaged. And the expression caused by the bacteroides can be obviously relieved after GDCA is supplemented.
As shown in FIG. 6 (E, F), in vivo experiments show that the Bacteroides transplantation does not affect the expression of FXR molecules, but can obviously inhibit the expression of FXR downstream signal SHP genes, and can recover FXR signals after GDCA is supplemented, which suggests that Bacteroides may regulate and control the change of FXR signals through the change of bile acids such as GDCA and the like.
2.2 in vitro experiments to study the relationship between GDCA and FXR Signal
Based on the above study, further from the viewpoint of the signal path of bile acid metabolism regulation, how GDCA affects body bile acid metabolism and alleviates ICP-related symptoms is studied.
Experimental materials and methods
(1) The binding analysis predicts the binding conformational relationship of each bile acid to FXR as described above: downloading the crystal structure of FXR receptor at RCSB Protein Data Bank (PDB ID:3dct, https:// www.rcsb.org /), performing a Dock assay of GDCA binding to FXR using Surflex-Dock GeomX (SFXC) of SYBYL-X2.0, bile acid binding to FXR using PyMOL and ligplot;
(2) The effect of each bile acid on FXR signal activation was detected by a TR-FRET FXR coactivation assay, which uses LanthaScreen TM TR-FRET Farnesoid X Receptor Coactivator Assay assay kit (Thermo Fisher, cat#A15140) was performed as follows: 1) Preparation Coregulator buffer G was followed by addition of DTT at a final concentration of 10 mM; 2) Diluting a bile acid sample to be detected by using a dilution ratio of 100X sample; 3) Taking 10 mu L of diluted sample diluent, adding the diluted sample diluent into a measuring pore plate, and setting a measuring compound pore; 4) Preparing 4 XFXR-LBD dilution with Coregulator buffer G buffer, adding 5 μl to each corresponding well plate; 5) Preparing a final concentration working solution containing 2.0 mu M fluororescein-SRC 2-2 (4X) and 20nM Tb anti-GST anti-body (4X) by using a Coregulator buffer G buffer solution, and taking 5 mu L of the working solution into the reaction system; 6) Gently shaking and uniformly mixing the reaction mixture, incubating at room temperature in a dark place, and reading plates at the wavelengths of 520nm and 495nm respectively; 7) The TR-FRET ratio (520 nm/495 nm) was calculated, binding curves were drawn and EC50 was calculated and each bile acid was analyzed for binding to FXR and functional effects in comparison to the standard agonist CDCA and the antagonist GUDCA.
(3) The action of each bile acid on FXR transcriptional activity was detected by a dual luciferase reporter: 1) HEK293 cells are cultivated conventionally, after the HEK293 cells grow and meet to 85%, 6 pore plates are inoculated, and the HEK293 cells are cultivated at 37 ℃ until the HEK293 cells meet to 85%; 2) The powder Ji Kai gene company constructs human FXR expression plasmid, PGL4-Shp-TK firefly luciferase reporter vector and Renilla luciferase control vector (pRL-luciferase; promega, madison, wis.) HEK293 cells were co-transfected with the transfection reagent Lipofectamine 3000, and the cells were cultured for an additional 24 hours after transfection; 3) Adding the bile acid with the final concentration of 50-200 mu M into the cell culture solution, and taking CDCA as a positive control; 4) The activity of the fluorescein reporter gene was detected separately using a double luciferase assay kit (Promega), and the specific procedures were performed according to the kit instructions.
(4) After incubation of each bile acid with primary intestinal epithelial cells or Caco2 cells, FXR signal and downstream gene expression were detected: 1) The small intestine of the mouse is taken out from the ultra clean bench, the intestinal content is repeatedly washed by precooled PBS until the content is clear, and the small intestine is washed for a plurality of times in a serum-free culture medium containing penicillin and streptomycin, and the intestinal tissue is sheared into the size smaller than 1mm 2 Transferring to centrifuge tube, repeatedly cleaning without serum culture medium, blowing, centrifuging to remove supernatant, and adding gelatinDigestion of tissue blocks by primordial enzyme and neutral protease, repeated blowing and digestion, and complete culture medium resuspension and culture after centrifugation; 2) Culturing Caco2 cells routinely; 3) After the cells grow and merge to 85%, starving and culturing for 4 hours, adding GDCA with the final concentration of 50-200 mu M respectively, and incubating for 16 hours; the GUDCA or T-beta MCA with the same final concentration is respectively added into the control group; 3) After the incubation is finished, RNA and protein are respectively extracted, and qPCR is used for detecting the expression of SHP, FGF19 and other molecules.
Experimental results
As shown in fig. 7 (A, B), the dock assay predicts that GDCA can bind to FXR, and in vitro experiments have initially found that GDCA can directly promote FXR downstream SHP gene expression, as shown in fig. 7 (C), suggesting that GDCA may exert a regulatory effect as an FXR agonist.
As shown in FIG. 8, the TR-FRET FXR coactivation assay found that GDCA can act as an agonist of FXR to agonize FXR.
As shown in FIG. 9, the results of cell experiments revealed that GDCA can significantly promote mRNA expression of FXR downstream genes FGF19 and SHP, and that the promotion can be inhibited by FXR antagonists GUDCA and T- βMCA.
2.3 in vivo experiments to study the relationship of GDCA and FXR Signal
On the basis of in vitro experiments, the relationship between GDCA and FXR signals was further studied by in vivo experiments.
Experimental materials and methods
(1) Preparing a bacteroides species, in the same manner as part 2.1;
(2) Bacteroides (2X 10) 8 cfu) is dissolved in 200 mu l of sterile aqueous solution, the sterile female mice are irrigated, the inactivated bacteroides strains with the same concentration are controlled to be irrigated, GDCA (30 mg/Kg/d) is respectively added to the inactivated bacteroides strains, the inactivated bacteroides strains are irrigated twice a week, the stomach is continuously irrigated for 4 weeks, the male mice and the female mice are caged according to the proportion of 1:2, and the weight and the appearance time of the vaginal suppositories of the mice are observed and recorded;
(3) Female mice are pregnant with E18d, anesthetized and sacrificed, intestinal and liver tissues of the mice are taken, intestinal epithelial cells and liver cells are separated according to the method described in section 2.1, RNA is extracted, and qPCR is used for detecting the expression of downstream genes such as FXR, SHP and the like of the intestines and the livers respectively.
Experimental results
As shown in FIG. 10, after the Bacteroides transplanted mice, the expression of FXR downstream genes of intestinal tissues is detected, and it is found that the Bacteroides transplanted mice can obviously inhibit the expression of FXR downstream genes FGF15 and SHP, and GDCA can obviously restore FXR inhibition caused by the Bacteroides.
In vitro and in vivo studies in this section indicate that GDCA does act as an FXR agonist and is able to promote FXR downstream gene expression.
Example III
Therapeutic effects of GDCA on mouse ICP
Experimental materials and methods
(1) Modeling a standard ICP mouse, namely, constructing an ICP mouse model after subcutaneous injection of EE2 estrogen;
(2) GDCA (30 mg/Kg/d) was infused twice a week for 4 weeks, followed by continuous infusion of stomach, before and after EE2 administration, respectively, in male mice: the female mice are put into cages according to the proportion of 1:2, and the weight and the appearance time of the vaginal suppository of the mice are observed and recorded;
(3) When 18d is pregnant, taking intestinal and liver tissues of the mice, detecting the phenotype change of the ICP mice, separating intestinal epithelial cells according to the method described in 2.2 part, detecting the expression of FGF15, SHP and the like by qPCR, separating liver cells, and detecting the expression of liver SHP, NTCP, CYP A1, CYP8B1, BSEP, MRP2 and the like by qPCR.
Experimental results
Standard EE 2-model ICP mice were obtained using the procedure illustrated in fig. 11a, and biochemical indexes such as bile acid level, ALT, AST, ALP, GGT, liver Index (lever Index) and the like associated with ICP were found to be significantly increased in the model ICP mice group (EE 2) compared to the normal mice group (Control), indicating successful model formation, while the above biochemical indexes of the GDCA intragastric ICP mice group (EE 2+ GDCA) were significantly inhibited (fig. 11b, 11 c), and Liver pathological lesions caused by EE2 were alleviated (fig. 11 d).
As shown in FIG. 12, the FXR downstream genes FGF15, SHP and NTCP of the EE2 model ICP mouse group (EE 2) were significantly inhibited compared with that of the normal mouse group (Control), and GDCA was able to restore the FXR signal of the ICP mouse (FIGS. 12a and 12 b), and at the same time GDCA was able to significantly inhibit the expression of bile acid synthesis genes such as Cyp7a1, cyp8b1 and Cyp27a1 and promote the expression of bile acid excretion genes such as BSEP and MRP2 (FIG. 12 c).
As a result of studying the relationship between GDCA and FXR signal in combination with the in vitro and in vivo experiments, it is known that supplementation of ICP mice with GDCA can alleviate ICP-related indexes and symptoms, and GDCA plays a role in preventing and treating ICP by enhancing the expression of FXR downstream signal genes in the liver and intestines of the mice.
Compared with the prior art, the invention has the beneficial effects that: the research finds that the action mechanism of intrahepatic cholestasis caused by the increase of the abundance of the bacteroides intestinal tract of an ICP patient, and proves that the GDCA serving as a novel agonist of FXR can play a role in preventing or/and relieving ICP by exciting FXR signals through in vitro experiments and animal experiments, so that the GDCA has the potential of being used for preparing medicines for treating ICP.
Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (3)
1. Use of glycodeoxycholic acid for the manufacture of a medicament for the treatment of intrahepatic cholestasis in gestation in a mammal.
2. The use according to claim 1, characterized in that: the intrahepatic cholestasis is caused at least in part by inhibition of the signal downstream of the farnesol X receptor FXR in the mammal.
3. The use according to claim 2, characterized in that: the inhibition of FXR downstream signal of the farnesoid X receptor is caused by the increase of the abundance of bacteroides in the intestinal tract of mammals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110175232.0A CN114903905B (en) | 2021-02-09 | 2021-02-09 | Application of glycodeoxycholic acid in preparing medicament for treating intrahepatic cholestasis and pharmaceutical composition thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110175232.0A CN114903905B (en) | 2021-02-09 | 2021-02-09 | Application of glycodeoxycholic acid in preparing medicament for treating intrahepatic cholestasis and pharmaceutical composition thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114903905A CN114903905A (en) | 2022-08-16 |
| CN114903905B true CN114903905B (en) | 2024-04-02 |
Family
ID=82761020
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110175232.0A Active CN114903905B (en) | 2021-02-09 | 2021-02-09 | Application of glycodeoxycholic acid in preparing medicament for treating intrahepatic cholestasis and pharmaceutical composition thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114903905B (en) |
Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004076657A2 (en) * | 2003-02-28 | 2004-09-10 | Mcgill University | Cell and enzyme compositions for modulating bile acids, cholesterol and triglycerides |
| KR20090059231A (en) * | 2007-12-06 | 2009-06-11 | 원광대학교산학협력단 | Composition comprising oroxylin a for treating and preventing liver disease |
| RU2008130338A (en) * | 2008-07-22 | 2010-01-27 | Леонид Борисович Лазебник (RU) | METHOD FOR DIFFERENTIAL DIAGNOSTICS OF INTRAHEPENOUS CHOLESTASIS IN CHRONIC LIVER DISEASES |
| CN101822659A (en) * | 2010-05-11 | 2010-09-08 | 中国药科大学 | Application of colchicin in preparing cholestatic liver disease drug |
| CN104962521A (en) * | 2015-01-07 | 2015-10-07 | 中山大学 | High-throughput screening model for human farnesoid X receptor (hFXR) agonist used for prevention and treatment of cholestasis |
| CN108697703A (en) * | 2016-02-22 | 2018-10-23 | 诺华股份有限公司 | Use the method for FXR agonists |
| CN110368393A (en) * | 2019-08-30 | 2019-10-25 | 上海交通大学医学院附属瑞金医院 | A kind of bile acid is preparing the application in metabolic disease drug |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2014521699A (en) * | 2011-08-04 | 2014-08-28 | ルメナ ファーマシューティカルズ，インク． | Bile acid recycling inhibitors for the treatment of pancreatitis |
-
2021
- 2021-02-09 CN CN202110175232.0A patent/CN114903905B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004076657A2 (en) * | 2003-02-28 | 2004-09-10 | Mcgill University | Cell and enzyme compositions for modulating bile acids, cholesterol and triglycerides |
| KR20090059231A (en) * | 2007-12-06 | 2009-06-11 | 원광대학교산학협력단 | Composition comprising oroxylin a for treating and preventing liver disease |
| RU2008130338A (en) * | 2008-07-22 | 2010-01-27 | Леонид Борисович Лазебник (RU) | METHOD FOR DIFFERENTIAL DIAGNOSTICS OF INTRAHEPENOUS CHOLESTASIS IN CHRONIC LIVER DISEASES |
| CN101822659A (en) * | 2010-05-11 | 2010-09-08 | 中国药科大学 | Application of colchicin in preparing cholestatic liver disease drug |
| CN104962521A (en) * | 2015-01-07 | 2015-10-07 | 中山大学 | High-throughput screening model for human farnesoid X receptor (hFXR) agonist used for prevention and treatment of cholestasis |
| CN108697703A (en) * | 2016-02-22 | 2018-10-23 | 诺华股份有限公司 | Use the method for FXR agonists |
| CN110368393A (en) * | 2019-08-30 | 2019-10-25 | 上海交通大学医学院附属瑞金医院 | A kind of bile acid is preparing the application in metabolic disease drug |
Non-Patent Citations (2)
| Title |
|---|
| Gut microbiota alters host bile acid metabolism to contribute to intrahepatic cholestasis of pregnancy;Bo Tang et al.;nature communications;1-17 * |
| 胆汁淤积性肝病患者肠道菌群特征及其临床相关性研究;伍灵怡等;第三军医大学学报;第42卷;1-8 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114903905A (en) | 2022-08-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| IL258881B1 (en) | Faecalibacterium prausnitzii ו– desulfovibrio piger לשימוש בטיפול או מניעת סכרת ומחלות מעיים | |
| KR101726488B1 (en) | Composition for the treatment of pregnancy-related diseases comprising the extracellular vesicles derived from Bacillus spp. | |
| US20190153446A1 (en) | Mir-149-3p and method for treating metabolic disease using the same | |
| He et al. | Lactiplantibacillus plantarum CCFM1019 attenuate polycystic ovary syndrome through butyrate dependent gut–brain mechanism | |
| Nian et al. | Gut microbiota metabolite TMAO promoted lipid deposition and fibrosis process via KRT17 in fatty liver cells in vitro | |
| CN114903905B (en) | Application of glycodeoxycholic acid in preparing medicament for treating intrahepatic cholestasis and pharmaceutical composition thereof | |
| Caron et al. | Effects of sustained hyperprolactinemia in late gestation on mammary development of gilts | |
| CN109868313A (en) | Application of the HMGA2 gene in Stein-Leventhal syndrome disease | |
| CN115645407A (en) | Application of pyrroloquinoline quinone in product for improving obesity male sterility disorder | |
| Yildiz et al. | Hepatic lipid overload triggers biliary epithelial cell activation via E2Fs | |
| CN115518077B (en) | Cell medicine for treating inflammatory bowel disease | |
| Gao et al. | DNA Methylation Pattern of CALCA and CALCB in Extremely Premature Infants with Monochorionic Triplets after Single‐Embryo Transfer | |
| CN115804843B (en) | Use of SKOR gene as target in treatment of type 2 diabetes and/or inflammation | |
| CN117205225B (en) | Application of geniposide in preparation of medicament for treating myeloproliferative neoplasm | |
| NL2035207B1 (en) | Application of lactobacillus paracasei in preparation for reducing serum cholesterol levels | |
| Zou et al. | Human gestational diabetes mellitus–derived exosomes impair glucose homeostasis in pregnant mice and stimulate functional maturation of offspring-islets | |
| CN114990004B (en) | Secretory immunoglobulin A-packaged lactobacillus reuteri and application thereof in preventing and treating gestational diabetes | |
| Huang et al. | Bile acids metabolism in the gut-liver axis mediates liver injury during lactation | |
| Li et al. | Sodium oligomannate’s amelioration of reproductive and metabolic phenotypes in a letrozole-induced PCOS-like mouse model depends on the gut microbiome | |
| Hejie et al. | ��� յ��±����� ̥� ּ��� ֲ������ Ӧ�� | |
| CN116814539A (en) | Construction method of fetal osteoarthritis model caused by prednisone exposure during pregnancy, intervention target and application of intervention target | |
| Wu et al. | A study of protective effects of Mus81 in preeclampsia | |
| Moss | Investigating Progesterone Resistance in Endometriosis Using Menstrual Effluent-Derived Endometrial Stromal Cells | |
| Liu et al. | GPR120 agonist ameliorated insulin resistance and improved ovarian function | |
| Wu et al. | Integrative omics analysis reveals gene regulatory mechanisms distinguishing organoid-derived hepatocytes from primary human hepatocytes |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |