MXPA00009863A - Fatty acid derivatives of bile acids and bile acid derivatives - Google Patents
Fatty acid derivatives of bile acids and bile acid derivativesInfo
- Publication number
- MXPA00009863A MXPA00009863A MXPA/A/2000/009863A MXPA00009863A MXPA00009863A MX PA00009863 A MXPA00009863 A MX PA00009863A MX PA00009863 A MXPA00009863 A MX PA00009863A MX PA00009863 A MXPA00009863 A MX PA00009863A
- Authority
- MX
- Mexico
- Prior art keywords
- bile
- acid
- fatty acid
- conjugates
- fatty
- Prior art date
Links
- 239000003613 bile acid Substances 0.000 title claims abstract description 53
- 229940053200 antiepileptics Fatty acid derivatives Drugs 0.000 title 1
- 210000000941 Bile Anatomy 0.000 claims abstract description 92
- 235000014113 dietary fatty acids Nutrition 0.000 claims abstract description 53
- 239000000194 fatty acid Substances 0.000 claims abstract description 53
- 150000004665 fatty acids Chemical class 0.000 claims abstract description 42
- 239000003833 bile salt Substances 0.000 claims abstract description 40
- 201000001883 cholelithiasis Diseases 0.000 claims abstract description 23
- 230000021615 conjugation Effects 0.000 claims abstract description 11
- 206010003210 Arteriosclerosis Diseases 0.000 claims abstract description 10
- 230000001603 reducing Effects 0.000 claims abstract description 10
- 150000004671 saturated fatty acids Chemical class 0.000 claims abstract description 6
- 210000004940 Nucleus Anatomy 0.000 claims abstract description 5
- 125000004432 carbon atoms Chemical group C* 0.000 claims abstract description 5
- 235000003441 saturated fatty acids Nutrition 0.000 claims abstract description 5
- HVYWMOMLDIMFJA-DPAQBDIFSA-N (3β)-Cholest-5-en-3-ol 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 claims description 124
- 229940107161 Cholesterol Drugs 0.000 claims description 62
- 235000012000 cholesterol Nutrition 0.000 claims description 62
- 239000002253 acid Substances 0.000 claims description 56
- 150000001875 compounds Chemical class 0.000 claims description 18
- QIQXTHQIDYTFRH-UHFFFAOYSA-N Stearic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 claims description 15
- 230000015572 biosynthetic process Effects 0.000 claims description 15
- 230000002265 prevention Effects 0.000 claims description 15
- IPCSVZSSVZVIGE-UHFFFAOYSA-N Palmitic acid Chemical compound CCCCCCCCCCCCCCCC(O)=O IPCSVZSSVZVIGE-UHFFFAOYSA-N 0.000 claims description 13
- 239000008194 pharmaceutical composition Substances 0.000 claims description 13
- 238000005755 formation reaction Methods 0.000 claims description 12
- 239000011780 sodium chloride Substances 0.000 claims description 10
- 238000004090 dissolution Methods 0.000 claims description 9
- 238000006722 reduction reaction Methods 0.000 claims description 9
- 235000021355 Stearic acid Nutrition 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 239000008117 stearic acid Substances 0.000 claims description 8
- 239000002904 solvent Substances 0.000 claims description 7
- 235000021314 Palmitic acid Nutrition 0.000 claims description 6
- XOAAWQZATWQOTB-UHFFFAOYSA-N Taurine Chemical compound NCCS(O)(=O)=O XOAAWQZATWQOTB-UHFFFAOYSA-N 0.000 claims description 6
- 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 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 239000004471 Glycine Substances 0.000 claims description 4
- 229960001661 Ursodiol Drugs 0.000 claims description 4
- 238000010521 absorption reaction Methods 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
- 230000028327 secretion Effects 0.000 claims description 4
- 229940115889 ursodeoxycholic acid Drugs 0.000 claims description 4
- 229960003080 Taurine Drugs 0.000 claims description 3
- 238000003786 synthesis reaction Methods 0.000 claims description 3
- 230000002194 synthesizing Effects 0.000 claims description 3
- KXGVEGMKQFWNSR-LLQZFEROSA-N Deoxycholic 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)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims description 2
- 239000002775 capsule Substances 0.000 claims description 2
- 239000000969 carrier Substances 0.000 claims description 2
- 229960003964 deoxycholic acid Drugs 0.000 claims description 2
- 239000003995 emulsifying agent Substances 0.000 claims description 2
- 239000000839 emulsion Substances 0.000 claims description 2
- 239000003623 enhancer Substances 0.000 claims description 2
- 230000002708 enhancing Effects 0.000 claims description 2
- 239000003112 inhibitor Substances 0.000 claims description 2
- OVBPIULPVIDEAO-LBPRGKRZSA-N folic acid Chemical compound C=1N=C2NC(N)=NC(=O)C2=NC=1CNC1=CC=C(C(=O)N[C@@H](CCC(O)=O)C(O)=O)C=C1 OVBPIULPVIDEAO-LBPRGKRZSA-N 0.000 claims 2
- 229960000304 Folic Acid Drugs 0.000 claims 1
- 125000001204 arachidyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims 1
- 235000019152 folic acid Nutrition 0.000 claims 1
- 239000011724 folic acid Substances 0.000 claims 1
- 125000001421 myristyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 claims 1
- 150000003839 salts Chemical class 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 2
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 90
- 239000000243 solution Substances 0.000 description 58
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 48
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 37
- BHQCQFFYRZLCQQ-OELDTZBJSA-N Cholic 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)[C@@H](O)C1 BHQCQFFYRZLCQQ-OELDTZBJSA-N 0.000 description 33
- 150000004702 methyl esters Chemical class 0.000 description 32
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 30
- 229940099352 cholate Drugs 0.000 description 27
- 239000000203 mixture Substances 0.000 description 24
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 22
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 22
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 22
- ZMANZCXQSJIPKH-UHFFFAOYSA-N triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 22
- 239000011541 reaction mixture Substances 0.000 description 20
- 150000003904 phospholipids Chemical class 0.000 description 19
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 18
- 238000002425 crystallisation Methods 0.000 description 17
- 230000005712 crystallization Effects 0.000 description 17
- 150000002632 lipids Chemical class 0.000 description 17
- 229940067631 Phospholipids Drugs 0.000 description 15
- 238000003756 stirring Methods 0.000 description 15
- 238000010899 nucleation Methods 0.000 description 14
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 14
- BHQCQFFYRZLCQQ-UMZBRFQRSA-N 4-[(3R,5S,7R,12S)-3,7,12-trihydroxy-10,13-dimethyl-2,3,4,5,6,7,8,9,11,12,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-17-yl]pentanoic acid Chemical class C([C@H]1C[C@H]2O)[C@H](O)CCC1(C)C1C2C2CCC(C(CCC(O)=O)C)C2(C)[C@@H](O)C1 BHQCQFFYRZLCQQ-UMZBRFQRSA-N 0.000 description 13
- 229940093761 Bile Salts Drugs 0.000 description 13
- 230000000694 effects Effects 0.000 description 13
- 239000004380 Cholic acid Substances 0.000 description 12
- 229960002471 cholic acid Drugs 0.000 description 12
- 235000019416 cholic acid Nutrition 0.000 description 12
- 239000002244 precipitate Substances 0.000 description 12
- 239000000741 silica gel Substances 0.000 description 12
- 229910002027 silica gel Inorganic materials 0.000 description 12
- 239000008079 hexane Substances 0.000 description 11
- 229940086542 triethylamine Drugs 0.000 description 11
- 241000699800 Cricetinae Species 0.000 description 10
- 241001465754 Metazoa Species 0.000 description 10
- 101700067048 CDC13 Proteins 0.000 description 9
- 210000004185 Liver Anatomy 0.000 description 9
- 239000000727 fraction Substances 0.000 description 9
- 210000000232 Gallbladder Anatomy 0.000 description 8
- UIIMBOGNXHQVGW-UHFFFAOYSA-M NaHCO3 Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 8
- 239000012223 aqueous fraction Substances 0.000 description 8
- IXCSERBJSXMMFS-UHFFFAOYSA-N hydrogen chloride Substances Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 8
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 8
- 210000004369 Blood Anatomy 0.000 description 7
- 208000001130 Gallstone Diseases 0.000 description 7
- 239000008280 blood Substances 0.000 description 7
- PMZURENOXWZQFD-UHFFFAOYSA-L na2so4 Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 7
- -1 palmitoyl cholate Chemical compound 0.000 description 7
- 229910052938 sodium sulfate Inorganic materials 0.000 description 7
- 235000011152 sodium sulphate Nutrition 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 125000001160 methoxycarbonyl group Chemical group [H]C([H])([H])OC(*)=O 0.000 description 6
- 125000003696 stearoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 6
- 210000000936 Intestines Anatomy 0.000 description 5
- 125000000118 dimethyl group Chemical group [H]C([H])([H])* 0.000 description 5
- 238000004809 thin layer chromatography Methods 0.000 description 5
- 230000002792 vascular Effects 0.000 description 5
- 210000002751 Lymph Anatomy 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 4
- 150000007513 acids Chemical class 0.000 description 4
- 125000001124 arachidoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 4
- XKRFYHLGVUSROY-UHFFFAOYSA-N argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 230000004087 circulation Effects 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 4
- SJRJJKPEHAURKC-UHFFFAOYSA-N n-methylmorpholine Chemical compound CN1CCOCC1 SJRJJKPEHAURKC-UHFFFAOYSA-N 0.000 description 4
- KDLHZDBZIXYQEI-UHFFFAOYSA-N palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 235000017557 sodium bicarbonate Nutrition 0.000 description 4
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 4
- 238000005063 solubilization Methods 0.000 description 4
- 241000894007 species Species 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- 229960002449 Glycine Drugs 0.000 description 3
- TUNFSRHWOTWDNC-UHFFFAOYSA-N Myristic acid Chemical compound CCCCCCCCCCCCCC(O)=O TUNFSRHWOTWDNC-UHFFFAOYSA-N 0.000 description 3
- 210000003240 Portal Vein Anatomy 0.000 description 3
- WBWWGRHZICKQGZ-HZAMXZRMSA-N Taurocholic 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)NCCS(O)(=O)=O)C)[C@@]2(C)[C@@H](O)C1 WBWWGRHZICKQGZ-HZAMXZRMSA-N 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 239000003858 bile acid conjugate Substances 0.000 description 3
- 230000000747 cardiac effect Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000001839 systemic circulation Effects 0.000 description 3
- 239000012085 test solution Substances 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- 230000032258 transport Effects 0.000 description 3
- 208000004998 Abdominal Pain Diseases 0.000 description 2
- VKOBVWXKNCXXDE-UHFFFAOYSA-N Arachidic acid Chemical compound CCCCCCCCCCCCCCCCCCCC(O)=O VKOBVWXKNCXXDE-UHFFFAOYSA-N 0.000 description 2
- 208000002881 Colic Diseases 0.000 description 2
- FUZZWVXGSFPDMH-UHFFFAOYSA-N Hexanoic acid Chemical compound CCCCCC(O)=O FUZZWVXGSFPDMH-UHFFFAOYSA-N 0.000 description 2
- POULHZVOKOAJMA-UHFFFAOYSA-N Lauric acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 description 2
- HOGDNTQCSIKEEV-UHFFFAOYSA-N N'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 description 2
- 229940014499 Ursodeoxycholate Drugs 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 125000002511 behenyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 238000001704 evaporation Methods 0.000 description 2
- 239000005457 ice water Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000000051 modifying Effects 0.000 description 2
- WTBAHSZERDXKKZ-UHFFFAOYSA-N octadecanoyl chloride Chemical compound CCCCCCCCCCCCCCCCCC(Cl)=O WTBAHSZERDXKKZ-UHFFFAOYSA-N 0.000 description 2
- 230000003287 optical Effects 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 238000001907 polarising light microscopy Methods 0.000 description 2
- 230000002035 prolonged Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 239000010414 supernatant solution Substances 0.000 description 2
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 2
- GTRMJEVFVTWDIU-KPNWGBFJSA-N (3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-[(2R)-6-methylheptan-2-yl]-2,3,4,7,8,9,11,12,14,15,16,17-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol;hydrate Chemical compound O.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 GTRMJEVFVTWDIU-KPNWGBFJSA-N 0.000 description 1
- NTBLHFFUSJRJQO-DOFZRALJSA-N (5Z,8Z,11Z,14Z)-icosa-5,8,11,14-tetraenoyl chloride Chemical compound CCCCC\C=C/C\C=C/C\C=C/C\C=C/CCCC(Cl)=O NTBLHFFUSJRJQO-DOFZRALJSA-N 0.000 description 1
- IFABLCIRROMTAN-KTKRTIGZSA-N (Z)-1-chlorooctadec-9-ene Chemical compound CCCCCCCC\C=C/CCCCCCCCCl IFABLCIRROMTAN-KTKRTIGZSA-N 0.000 description 1
- ZAKBPRIDLSBYQQ-VHSXEESVSA-N 1,2-diacyl-sn-glycero-3-phospho-(1'-sn-glycerol) Chemical compound CC(=O)OC[C@@H](OC(C)=O)COP(O)(=O)OC[C@@H](O)CO ZAKBPRIDLSBYQQ-VHSXEESVSA-N 0.000 description 1
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- YAYNEUUHHLGGAH-UHFFFAOYSA-N 1-chlorododecane Chemical compound CCCCCCCCCCCCCl YAYNEUUHHLGGAH-UHFFFAOYSA-N 0.000 description 1
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- QEYMMOKECZBKAC-UHFFFAOYSA-M 3-chloropropanoate Chemical compound [O-]C(=O)CCCl QEYMMOKECZBKAC-UHFFFAOYSA-M 0.000 description 1
- 210000001015 Abdomen Anatomy 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
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- MTCFGRXMJLQNBG-UWTATZPHSA-N D-serine Chemical compound OC[C@@H](N)C(O)=O MTCFGRXMJLQNBG-UWTATZPHSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- 210000002969 Egg Yolk Anatomy 0.000 description 1
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- 229960001269 Glycine Hydrochloride Drugs 0.000 description 1
- 241000282412 Homo Species 0.000 description 1
- 239000005639 Lauric acid Substances 0.000 description 1
- 229940067606 Lecithin Drugs 0.000 description 1
- RSHAOIXHUHAZPM-UHFFFAOYSA-N Magnesium hydride Chemical compound [MgH2] RSHAOIXHUHAZPM-UHFFFAOYSA-N 0.000 description 1
- 230000035633 Metabolized Effects 0.000 description 1
- QARBMVPHQWIHKH-UHFFFAOYSA-N Methanesulfonyl chloride Chemical compound CS(Cl)(=O)=O QARBMVPHQWIHKH-UHFFFAOYSA-N 0.000 description 1
- 239000012359 Methanesulfonyl chloride Substances 0.000 description 1
- 235000021360 Myristic acid Nutrition 0.000 description 1
- PSHKMPUSSFXUIA-UHFFFAOYSA-N N,N-dimethylpyridin-2-amine Chemical compound CN(C)C1=CC=CC=N1 PSHKMPUSSFXUIA-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000002378 acidificating Effects 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000000540 analysis of variance Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
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- 238000002192 cholecystectomy Methods 0.000 description 1
- CRBHXDCYXIISFC-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CC[O-] CRBHXDCYXIISFC-UHFFFAOYSA-N 0.000 description 1
- 230000000875 corresponding Effects 0.000 description 1
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- 235000005911 diet Nutrition 0.000 description 1
- 230000037213 diet Effects 0.000 description 1
- 230000001079 digestive Effects 0.000 description 1
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- ZGTMUACCHSMWAC-UHFFFAOYSA-L disodium;2-[2-[carboxylatomethyl(carboxymethyl)amino]ethyl-(carboxymethyl)amino]acetate Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- QTHQYNCAWSGBCE-UHFFFAOYSA-N docosanoyl chloride Chemical compound CCCCCCCCCCCCCCCCCCCCCC(Cl)=O QTHQYNCAWSGBCE-UHFFFAOYSA-N 0.000 description 1
- 125000003438 dodecyl group Chemical group [H]C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])* 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013345 egg yolk Nutrition 0.000 description 1
- 239000008344 egg yolk phospholipid Substances 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000037406 food intake Effects 0.000 description 1
- 244000144993 groups of animals Species 0.000 description 1
- ARBOVOVUTSQWSS-UHFFFAOYSA-N hexadecanoyl chloride Chemical compound CCCCCCCCCCCCCCCC(Cl)=O ARBOVOVUTSQWSS-UHFFFAOYSA-N 0.000 description 1
- YWGHUJQYGPDNKT-UHFFFAOYSA-N hexanoyl chloride Chemical compound CCCCCC(Cl)=O YWGHUJQYGPDNKT-UHFFFAOYSA-N 0.000 description 1
- 238000005984 hydrogenation reaction Methods 0.000 description 1
- 230000003382 ingestive Effects 0.000 description 1
- 230000000968 intestinal Effects 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 229910012375 magnesium hydride Inorganic materials 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- WVJVHUWVQNLPCR-UHFFFAOYSA-N octadecanoyl octadecanoate Chemical compound CCCCCCCCCCCCCCCCCC(=O)OC(=O)CCCCCCCCCCCCCCCCC WVJVHUWVQNLPCR-UHFFFAOYSA-N 0.000 description 1
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid group Chemical group C(CCCCCCC\C=C/CCCCCCCC)(=O)O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 description 1
- 125000002811 oleoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])/C([H])=C([H])\C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 125000001312 palmitoyl group Chemical group O=C([*])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000000611 regression analysis Methods 0.000 description 1
- 230000001105 regulatory Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- VMHLLURERBWHNL-UHFFFAOYSA-M sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 description 1
- 239000001632 sodium acetate Substances 0.000 description 1
- 235000017281 sodium acetate Nutrition 0.000 description 1
- 239000011343 solid material Substances 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 230000000707 stereoselective Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 125000001424 substituent group Chemical group 0.000 description 1
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- LPWCRLGKYWVLHQ-UHFFFAOYSA-N tetradecanoyl chloride Chemical compound CCCCCCCCCCCCCC(Cl)=O LPWCRLGKYWVLHQ-UHFFFAOYSA-N 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- QWCKQJZIFLGMSD-UHFFFAOYSA-N α-Aminobutyric acid Chemical compound CCC(N)C(O)=O QWCKQJZIFLGMSD-UHFFFAOYSA-N 0.000 description 1
Abstract
The present invention relates to bile acid or bile salt fatty acid conjugates (hereinafter called"BAFAC"), to their use in dissolving cholesterol gallstones in bile, preventing their occurrence or recurrence, to their use in reducing or preventing arteriosclerosis and to methods for the treatment of said diseases. The conjugates are of the formula W - X - G in which G is a bile acid or bile salt radical, W stands for one or two saturated fatty acid radicals and X is either a direct bond or a bonding member between said bile acid or bile salt and the fatty acid(s). The conjugation is advantageously performed at a position selected among the 3, 6, 7, 12 and 24 positions of the bile acid or bile salt nucleus. The fatty acids are preferably saturated fatty acids having (14 - 22) carbon atoms.
Description
DERIVATIVES OF FATTY ACIDS OF BILIARY ACIDS AND DERIVATIVES OF BILIARY ACIDS
FIELD OF THE INVENTION The present invention relates to conjugates of fatty acids, bile acids or bile salts (hereinafter later called "BAFAC"), with their use in the solution of ca.; cholesterol bile in the bile, preventing its or urgency or recurrence, with its use in the reduction or prevention of arteriosclerosis and with the mécodes for the treatment of diseases.
BACKGROUND OF THE INVENTION It should be noted that the terms bile acids and bile salts are similar and used interchangeably. Gallstones are found in approximately 15% of people in most industrialized countries. The majority of gallstones are cholesterol gallstones, meaning that cholesterol is your cor, main speaker. In this way, cholesterol gallstones represent a major health problem. Bile is often supersaturated with cholesterol, which tends to crystallize. The prevention of crystallization of cholesterol in the bile will prevent the formation of cholesterol gallstones or recurrence after procedures such as lithotripsy, dissolution, or extraction of the calculus. The residence time of newly secreted bile in the gallbladder is short-less than 12-24 hours. The prevention of cholesterol crystallization in bile during such a period could prevent the formation of gallstones. It has been proven that gallstones of cholesterol can be released medically and their recurrence prevented by utilizing certain bile salts such as acidic or ursodeoxycholic acid. The therapy with bile salts is, however, of low efficiency, it consumes a lot of time and has been abandoned to a great extent. In this way, more effective therapies are required. Recent works have shown the main role played by i .s phospholipids in the solubilization of cholesterol in the bile. (T. Gilat et al., Biochimica et Biophysic-i Acta 1286, (1996), 95-115, Y. Ringel et al., Biochimicu et Biophysica Acta 1390, (1998), 293-300), and J. Heo ^ tology, 28, (1998), 1008-1014). The phospholipids sor: a main or unique component of the lipids that solubilize the cholesterol added in the bile. It has been shown that the gradual addition of phospholipids to the bile will progressively prolong the nucleation time of cholesterol in the bile. (Z. Halpern et al., Gut 34 (1993) 110-115). The main differences between certain molecular species of phospholipids in their potency of inhibiting the crystallization of cholesterol in human bile or model have been demonstrated. The phospholipids differ from each other mainly in the fatty acids present in the positions of the stereospecific number sn-1 and / or sn-2 and in their main groups. It has been demonstrated that the largest prolongations in nucleation time and the greatest reductions in the growth rate of cholesterol crystals and in the total mass of cholesterol crystals are achieved with changes in phospholipid molecular species without changing the absolute amounts or relative of phospholipids. The crystallization of cholesterol was markedly retarded when the sn-2 fatty acid was saturated, when the head group was serine instead of choline, etc. (Y. Ringer et al., Above). It has also been shown that several phospholipid components by themselves (without the entire phospholipid molecule), for example, saturated fatty acids such as palmitic acid or stearic acid; or phosphatidyl glycerol have a strong inhibitory activity of cholesterol crystallization. Thus, by enriching human bile with phospholipids in general, or specific phospholipids or their components, such fatty acids would significantly retard the crystallization of cholesterol in the bile and the desired results would be achieved. The problem was how to enrich human bile in vivo with phospholipids or their components. When bile salts are fed to humans, they are absorbed very efficiently, being absorbed by the liver and excreted into the bile. This also applies to analogues of synthetic bile salts. There are specific and very efficient transport mechanisms in the body for these purposes. Thus, when ursodeoxycholic acid (which is normally present in human bile in minimal amounts) is fed regularly it is absorbed and secreted in the bile and eventually constitutes 32-50% of the bile acids in the bile. However, as indicated above, bile salt therapy for the dissolution of cholesterol gallstones is not satisfactory. The phospholipids and their components are well absorbed and taken up by the liver. The secretion of phospholipids into bile is, however, closely regulated by the liver and only limited amounts and species of phospholipids are secreted in the bile and in association with the secretion of bile salts and cholesterol. Currently there is no efficient method to modulate, quantitatively and qualitatively, the human biliary phospholipid components to any considerable degree. When the phospholipids of the diet reach the liver they can be metabolized, secreted into the blood or stored in the liver. Only small amounts and predetermined species are secreted into the bile with minimal possibilities of modulation. Therefore, it has been desirable to find a satisfactory method for the transport of phospholipids or one of their components to the bile, which would improve the solubilization of bile cholesterol and prevent the formation of cholesterol gallstones or dissolve existing gallstones. In the Israeli Patent Specification No. 95668 and the corresponding US Specifications, bile acid derivatives of the general formula I are known:
W X G in which G is a bile acid radical, W is a portion of active compound of a medicament and X is a direct bond or a linking member between the bile acid radical and the active compound. A large list of substituents is given in the specifications but W is not specifically mentioned as being important for a fatty acid radical, neither for a saturated one nor for an unsaturated one, ie the specification does not mention anything about the BAFAC. Furthermore, among all the objects of the compounds, there has not yet been found a clue that any of the compounds can be used to increase the solubilization of biliary cholesterol, to prevent the formation of cholesterol gallstones, to dissolve the gallstones of existing cholesterol, and to reduce or prevent arteriosclerosis. It has now been found that acids or conjugates of bile salts with fatty acids (saturated or unsaturated) via an X linkage (here subsequently BAFAC) can serve as vehicles for transporting fatty acids into bile using the enterohepatic circulation in a very efficient of bile salts and acids. An ester link between fatty acids and bile acids is inadequate since it would be broken by digestive juices and intestinal bacteria, resulting in the breakdown of BAFACs and the separate absorption of their components. It has also been shown that BAFACs are absorbed from the intestine, taken up by the liver and secreted in the bile. BAFACs improve the solubilization of cholesterol in the bile and markedly retard its crystallization. BAFACs are, therefore, useful agents for the prevention of the formation or recurrence of cholesterol gallstones and for the dissolution of cholesterol gallstones. The administration of BAFAC also has an inhibitory effect on the crystallization of cholesterol in the vascular tree. In the physiological situation the ingested acids or bile salts are absorbed in the intestine, transported via the portal vein to the liver and excreted via the bile into the intestine. They, in this way, recirculate in the enterohepatic circulation, with only minimal amounts reaching the systemic circulation (the vascular tree). BAFACs behave more like lipids, which after intestinal absorption are transported via the lymph to the systemic circulation. The BAFACs were transported both via the lymph and via the portal vein. By both routes they are captured by the liver and secreted in the bile. In each enterohepatic circulation they are excreted into the intestine, are again partially absorbed via the lymph and recirculated to the vascular tree before the uptake by the liver. Since there are 10-12 cycles of enterohepatic circulation daily, the net effect will be the recirculation of BAFACs in the vascular tree. Administering BAFAC orally in divided doses during the course of the day will increase this effect. The inhibitory effect of BAFAC on cholesterol crystallization and its ability to solubilize existing cholesterol crystals has been tested. In this way, also its value in the reduction and / or prevention of crystallization of cholesterol in the vascular tree, that is to say arteriosclerosis.
BRIEF DESCRIPTION OF THE INVENTION The present invention thus consists of bile acid grade acid conjugates or bile salts of the general formula II:
W-X-G in which G has the same meaning as in formula I, means one or two fatty acid radicals and X is a linking member between the bile acid radical or bile salt and the fatty acid radicals. Suitable bile acids can be mentioned, for example, cholic acid, cenodeoxycholic acid, ursodeoxycholic acid and deoxycholic acid. The bile acids used may be conjugated or, as in bile, conjugated at position 24 with glycine, taurine or a suitable amino acid. These possibilities are within the definition of a bile acid and thus within the scope of the present invention. The conjugation with the fatty acid radicals is very often preferred at the 3-position of the nucleus depending on the bile acid being used. It is also possible to carry out the conjugation with the fatty acid radical in different positions, for example 6, 7, 12 and 24. When the bile acid is conjugated with glycine or taurine, the conjugation with the fatty acid radical can not be carried out in the position 24. The conjugation between the fatty acid radical and the bile acid can be in the a or β configuration. Binding member X is, advantageously, a group -NH- or a group -O-.
Preferred fatty acids are unsaturated, which suitably have 6-26 carbon atoms, advantageously those having 14 to 22 carbon atoms. Preferred saturated acids are behenyl acid, arachidyl acid, stearic acid, palmitic acid and meristinic acid. When it means two fatty acids, they are conjugated in a suitable manner in positions 3 and 7. The present invention also consists of a pharmaceutical composition that allows the dissolution of cholesterol gallstones in the bile and prevents the formation thereof; and which allows the prevention and / or reduction of arteriosclerosis, which comprises as an active ingredient a fatty acid derivative of bile acid of the general formula II. The composition may take the form of a tablet, a capsule, a solution, an emulsion, etc. The composition may comprise additional compounds such as carriers, solvents, emulsifiers, absorption enhancers, inhibitors of the synthesis or secretion of cholesterol in the bile, etc. The composition should advantageously comprise 0.1-1.5 g of the active ingredient. The composition is ingested properly once a day, preferably at bedtime.
It can also be ingested in divided doses during the day. The present invention also consists of the use of a fatty acid derivative of bile acid of general formula II or of a pharmaceutical composition comprising the same, for the dissolution of cholesterol gallstones in the bile and for the prevention of the formation of cholesterol. same. The present invention also consists of the use of a fatty acid derivative of bile acid of the general formula II or of a pharmaceutical composition comprising the same for the prevention and / or reduction of arteriosclerosis. The present invention also consists of a method for the dissolution of cholesterol gallstones in the bile and for the prevention of the formation thereof by the administration of a fatty acid derivative of bile acid in the general formula II or a pharmaceutical composition which comprises the same. The present invention also consists of a method for the prevention and / or reduction of arteriosclerosis by the administration of a fatty acid derivative of bile acid of the general formula II or a pharmaceutical composition comprising the same.
BRIEF DESCRIPTION OF THE DRAWINGS The present invention will now be illustrated with reference to the accompanying Examples and drawings without being limited by them. In the drawings: Figure 1 shows the observation time of crystals. Biliary solution model. Effects of palmitoyl cholate (PalC). A-Control solution, B, C-replacement of 10% and 20% Na taurocholate (NaTC) by equimolar amounts of
PalC, respectively. D-replacement of 20% phospholipids by PalC. E, F addition of 10 mM and 20 mM of
PalC to the solution, respectively; Figure 2 shows the mass of cholesterol crystals. Biliary solution model. Effects of PalC, A, B, C, D, E and F - as in Figure 1; Figure 2 shows the growth rate of the crystals. Biliary solution model. Effects of PalC, A, B, C, D, E and F - as in Figure 1; Figure 4 shows thin layer chromatography. A-standards of PalC in pure solution (left) and in bile of hamsters. B-bile from control animal hamster (left) and hamster bile fed with PalC; Figure 5A shows the steps in the conjugation of cholic acid (at C-3) with: behenyl acid (C-22), arachidic acid (C-20), stearic acid (C-18), palmitic acid (C-16) ), myristic acid (C-14), lauric acid (C-12) and caproic acid (C-6); Figure 5B shows the steps in the synthesis of stearoilcolate conjugated with glycine; Figure 5C shows the conjugation of oleoyl cholate; Figure 5D shows the conjugation of cholic acid with two molecules of stearic acid at the C-3 and C-7 positions of the bile acid core; Figure 6 shows the mass of the cholesterol crystals. Biliary solution model. Effects of myristic (C-14), palmitic (C-16), stearic (C-18) and arachidic (C-20) acids conjugated with cholic acid (C-3). The test compounds replaced 20 mol% of NaTC in the control solution. Figure 7 shows the nucleation time. Biliary solution model. Effects of the compounds used in Figure 6. Figure 8 shows the mass of cholesterol crystals of human bile enriched after 22 days of incubation. Effects of 5 mM palmitoyl (C-16) cholate, stearoyl (C-18) cholate and arachidyl (C-20) cholate added to the bile compared to bile control and bile with 5 mM of added cholic acid.
Figure 9 shows the nucleation time, bile model. Effect of 20 mole% replacement of NaTC with equimolar amounts of caproyl (C-6) cholate, lauryl (C-12) cholate, stearoyl (C-18) cholate, arachidyl (C-20) cholate and disterearoyl ursodeoxycholate compared to bile model and without 20% replacement of NaTC with cholic acid; and Figure 10 shows the levels of stearoyl (C-18) cholate in hamster 1, 2 and 3 hours after ingestion of 30 mg. Concentrations in cardiac blood, portal blood and bile in the gallbladder.
EXAMPLE I 3β-Behenylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid (Figures 5A-3) (a) 1.15 g of methyl ester 3β-behenylamido-7a, 12a-dihydroxy-5β-colanol were dissolved. 24-oico (Figures 5A-1) [French Patent 1017756 December 18, 1952, Chem. Abstr. 52: 1293c] in 30 ml of dry dimethyl fomamide and treated with 15 ml of triethylamine under stirring. 1.13 g of behenoyl chloride in 10 ml of dimethyl formamide were added dropwise to the resulting solution, and stirring was continued overnight. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane ( 6: 4 and 8: 2), to give 0.8 g of the methyl ester 3β-behenylamido-7a, 12a-dihydroxy-5β-colan-24-oico (Figure 5A-2). XH-NMR (CDC13) d, ppm: 0.69 (s, CH3-I8), 0.88
(t, J = lHz, CH3-23), 0.95 (s, CH3-19), 0.99 (d, J = 3Hz, CH3-21), 1.25, 1.14 [s, CH2) 2o- / 2.14 (t, J = 5Hz, CH3-behenyl), 3.67 (s-COOCH3), 3.91 (d, J = 1.5 Hz, CH-7), 3.96 (s, J = 4Hz, CH-12), 3.99 (m, CH-3) , 5.60 (d, J = 4.5
Hz, -CHCO-). (b) The above methyl ester, 0.45 g, was dissolved in 20 ml of methanol, treated with 2 ml of IN sodium hydroxide and left for 24 h at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate which was washed with water, to give 0.41 g of the 3ß-behenylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid (Figure 5A-3).
Example II 3ß-arachidyl amido-7, 12a-dihydroxy-5β-colan-24-oic acid (Figures 5A-5)
(a) 1.0 g of 3β-amino-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figures 5A-1) [see Example I] were dissolved in 30 ml of dry dimethyl fomamide and treated with 15 ml of triethylamine under stirring. 1.0 g of arachidonoyl chloride in 10 ml of dimethyl formamide was added dropwise to the resulting solution, and stirring was continued overnight. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane ( 6: 4 and 8: 2), to give 0.6 g of the 3β-arachidyl amido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-4). XH-NMR (CDC13) d, ppm: 0.70 (s, CH3-I8), 0.88 (t, J = 6Hz, CH3-23), 0.95 (s, CH3-19), 0.99 (d, J = 3Hz, CH3 -21), 1.25, 1.14 [s, CH2) 18], 2.14 (t, J = 5Hz, CH3-arachidyl), 3.67 (s-COOCH3), 3.91 (d, J = 1.5, CH-7) , 3.96 (s, J = 4Hz, CH-12), 4.4 (m, CH-3), 5.60 (d, J = 4.5 Hz, -CH2CONH-).
(b) 0.5 g of 3β-arachidyl amido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-4) was dissolved in 20 ml of methanol, treated with 2 ml of sodium hydroxide IN and they were left for 24 hours at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate, which was washed with water, to give 0.7 g of the 3β-arachidyl-7-a, 12a-dihydroxy-5β-colan-24-oic acid pure (Figure 5A-5).
Example III 3β-Stearyl-amide-7a, 12a-dihydroxy-5β-colan-24-oic acid (Figures 5A-7)Method 1 (a) 1.15 g of 3β-amino-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester were dissolved (Figures 5A-1)
[see Example I] in 30 ml of dry dimethyl fomamide and treated with 15 ml of triethylamine under stirring. 1.13 g of stearoyl chloride in 10 ml of dimethyl formamide was added dropwise to the resulting solution, and the
He continued shaking during the night. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane ( 6: 4 and 8: 2), to give 0.68 g of the 3β-stearylamido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-6). : H-NMR (CDC13) d, ppm: 0.69 (s, CH3-I8), 0.88 (t, J = lHz, CH3-23), • 0.95 (s, CH3-19), 0.99 (d, J = 3Hz , CH3- 21), 1.25, 1.14 [s, CH2) i6-, 2.14 (t, J = 5Hz, CH3-c L ui _l_ _Lo), 3.67 (s-COOCH, 3.91 (d, J = 1.5 Hz, CH -7),
3. 99 (m, CH-3), 4.4 (m, CH-3), 5.60 (d, J = 4.5 Hz, -CH2CONH). (b) 0.45 g of 3β-stearylamido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-6) were dissolved in 20 ml of methanol, treated with 2 ml of sodium hydroxide IN and they were left for 24 hours at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate which was washed with water, to give 0.41 g of the 3β-stearylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid (Figure 5A-7, mp 63-65 ° C.
Method 2 2.5 g of 3ß-amino-7a, 12a-dihydroxy-5β-colanoic-24-oic acid (prepared according to Kramer et al., J. of Lipid Research 24, 910, 1983) were dissolved in acetonitrile and they added to a stirring solution of 1.2 g of stearic acid and 3.6 g of N-hydroxy succinamide in the same solvent. 8 hours later the precipitate was filtered, washed with the solvent and evaporated to dryness. The residue was added to a solution of 1.2 g of stearic acid in 10 ml of N-methyl morpholine and N, N'-dimethyl formamide (1: 3). After being kept at room temperature overnight, the solution was diluted with water, extracted with ethyl acetate, to give 0.6 g of the acid (Figure 5A-7), in a manner identical to Method 1.
Method 3 A solution of 6 g of stearoyl chloride was added dropwise to a stirred solution of 1.6 g of the amine (Figure 5B-18) in toluene at 0 °, and left at the same temperature for 1 hour. The resulting solution was heated at 50 ° for another hour, acidified with 3N hydrochloric acid, and then filtered. The solid material was washed with water and dried at 45 °, to give the acid (Figure 5A-7), in a manner identical to that described above.
Example IV 3β-palmitylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid (Figures 5A-9)
Method 1 (a) 1.0 g of 3β-amino-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester were dissolved (Figures 5A-1)
[see Example I] in 30 ml of dry dimethyl fomamide and treated with 15 ml of triethylamine under stirring. 0.8 g of palmitoyl chloride in 10 ml of dimethyl formamide was added dropwise to the resulting solution, and stirring was continued overnight. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and subjected to chromatography on silica gel with a mixture of ethyl acetate. ethyl and hexane (6: 4 and 8: 2), to give 0.5 g of the 3β-palmitoylamido-7a, 12a-dihydroxy-5β-cholan-i-oic methyl ester (Figure 5A-8).
XH-NMR (CDC13) d, ppm: 0.66 (s, CH3-H), 0.88 (t, J = lHz, CH3-23), 0.91 (s, CHj-19), 0.96 (d, J = 3.5Hz, CH3-2I), 1.22, 1.14 [s, CH2)? 4], 2.13 (t, J = 5Hz, CH3-palmitoyl), 3.67 (s-COOCH3), 3.82 (d, J = 1.5 Hz, CH7), 3.96 (s, J = 3.9 Hz, CH-12), 4.09 (m, CH-3), 5.63 (d, J = 4.5 Hz, -CH2CONH). (b) 0.45 g of the above methyl ester was dissolved in 20 ml of methanol, treated with 2 ml of IN sodium hydroxide and left for 24 hours at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate which was washed with water, to give 0.4 g of the 3β-palmitylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid Figure 5A -9).
Method 2 2.5 g of 3ß-amino-7a, 12a-dihydroxy-5β-colanoic-24-oic acid (Figure 5B-18) (prepared according to Kramer et al., J. of Lipid Research 24, 910, was dissolved. 1983) in acetonitrile and added to a stirring solution of 1.2 g of palmitic acid and 3.6 g of N-hydroxy succinamide in the same solvent. 8 hours later the precipitate was filtered, washed with the solvent and evaporated to dryness. The residue was added to a solution of 1.2 g of palmitic acid in 10 ml of N-methyl morpholine and N, N '-dimethyl formamide (1: 3). After standing at room temperature overnight, the solution was diluted with water, extracted with ethyl acetate, to give 0.6 g of the acid (Figure 5A-9), in a manner identical to Method 1.
EXAMPLE V 3ß-myristylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid (Figures 5A-11)
(a) 0.5 g of 3-amino-7a, 12a-dihydroxy-5β-colan-24-oico (1) methylester [see Example] was dissolved in 30 ml of dry dimethyl-ammonide and treated with 15 ml of low triethylamine agitation. 0.4 g of myristoyl chloride in 10 ml of dimethylformamide was added dropwise to the resulting solution, and stirring was continued overnight. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane ( 6: 4 and 8: 2), to give 0.4 g of the methyl ester 3β-myristoylamido-7a, 12a-dihydroxy-5β-colan-24-oico (Figure 5A-10). XH-NMR (CDC13) d, ppm: 0.69 (s, CH3-I8), 0.88 (t, J = lHz, CH3-23), 0.95 (s, CH3-19), 0.99 (d, J = 3.Hz , CH3-2I), 1.25 [s, CH2)? 2], 2.14 (t, J = 5Hz, CH3-iristoyl), 3.67 (s-COOCH3), 3.91 (d, J = 1.5 Hz, CH-7), 3.99 (s, J = 4Hz, CH-12), 4.4 (m, CH-3), 5.60 (d, J = .5 Hz, -CHCONH). (b) 0.45 g of 3β-myristylamido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-19) was dissolved in 20 ml of methanol, treated with 2 ml of sodium hydroxide IN and they were left for 24 hours at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate which was washed with water, to give 0.26 g of the pure acid (Figure 5A-11).
Example VI 3ß-lauryl amide-7a, 12a-dihydroxy-5β-colan-24-oic acid
(Figures 5A-13) _ (a) 0.6 g of 3-amino-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-1) was dissolved in 30 ml of dry dimethyl formamide and treated with 15 ml of triethyl amine under stirring. 0.6 g of lauryl chloride in 10 ml of dimethyl formamide were added dropwise to the resulting solution, and stirring was continued overnight. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane ( 6: 4 and 8: 2), to give 0.5 g of the methyl ester 3β-laurylamido-7a, 12a-dihydroxy-5β-colan.-24-oico (Figure 5A-12). XH-NMR (CDC13) d, ppm: 0.67 (s, CH3-I8), 0.89
(t, J = lHz, CH3-23), 0.94 (s, CH3-19), 0.99 (d, J = 3.Hz,
CH3-2I), 1.25 [s, CH2)? 0], 2.14 (t, J = 5Hz, CH3-lauryl), 3.67 (S-COOCH3), 3.91 (d, J = 1.5 Hz, CH-7), 3.99 (s,
J = 3.9Hz, CH-12), 4.4 (m, CH-3), 5.60 (d, J = 4.5 Hz, -CH2CONH). (b) 0.45 g of 3β-laurylamido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-12) were dissolved in 20 ml of methanol, treated with 2 ml of sodium hydroxide IN and they were left for 24 hours at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate which was washed with water, to give 0.41 g of the pure acid (Figure 5A-13). pf. 82-88.
Example VII 3ß-caprylamido-7a / 12a-dihydroxy-5β-colan-24-oic acid
(Figures 5A-15)
• (a) 1.0 g of 3-amino-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-1) [see Example 1] was dissolved in 30 ml of dry methylene chloride and treated with 15 ml of triethyl amine under stirring. 1.2 g of caproyl chloride in 10 ml of methylene chloride were added dropwise to the resulting solution, and stirring was continued overnight. The reaction mixture was poured into water, extracted with methylene chloride, the organic fraction was then dried over sodium sulfate, evaporated to dryness and chromatographed on silica gel with a mixture of ethyl acetate and hexane ( 6: 4 and 8: 2), to give 0.7 g of 3-bis-caprylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid methyl ester (Figure 5A-14). XH-NMR (CDC13) d, ppm: 0.69 (s, CH3-I8), 0.88 (t, J = lHz, CH3-23), 0.95 (s, CH3-19), 0.99 (d, J = 3.Hz , CH3-2I), 1.25 [s, CH2) 4], 2.14 (t, J = 5Hz, CH3-capryl), 3.67 (S-COOCH3), 3.91 (d, J = 1.5 Hz, CH-7), 3.99 (s, J = 4Hz, CH-12), 4.4 (m, CH-3), 5.60 (d, J = .5 Hz, -CH2CONH). (b) 0.5 g of 3β-caprylamido-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-14) was dissolved in 20 ml of methanol, treated with 2 ml of sodium hydroxide IN and they were left for 24 hours at room temperature. The methanol was then distilled, 10 ml of water was added and the reaction mixture was extracted with ethyl acetate. The aqueous fraction was then acidified with dilute hydrogen chloride, resulting in a white precipitate which was washed with water, to give 0.4 g of the pure acid (Figure 5A-15).
Example VIII N- (-carboxymethyl) -3β-stearylamido-7a, 12a-dihydroxy-5β-colan-24 amide (Figures 5A-17)
(a) 0.5 g of 3β-stearylamido-7a, 12a-dihydroxy-5β-colanoic acid (Figure 5A-7) was dissolved in 25 ml of dry 1,4-dioxane and cooled to -10 °. The stirred solution was treated with 0.15 ml of triethylamine, then 0.085 ml of chloroethyl formate was added and stirred at the same temperature during
minutes. The solution was then left at room temperature, treated with 0.1 ml of triethylamine and with 14 g of ethyl glycine hydrochloride, and left overnight. The reaction mixture was poured into water, extracted with ethyl acetate and washed-with water.
The extract was evaporated to dryness and subjected to chromatography on silica gel, using a mixture of ethyl acetate: hexane 60:40, pure ethyl acetate and then ethyl acetate: methanol 9: 1, to give 0.27 g of the product (Figure 5B-16). (b) 0.27 g of the above compound was dissolved in 20 ml of methanol and treated with 2 ml of IN sodium hydroxide. 24 hours later the methanol was evaporated to dryness, dissolved in water and extracted with ethyl acetate. The aqueous fraction was acidified with 1N HCl. The precipitate obtained was washed with water and dried, to give 0.24 g of the dried material (Figure 5A-17).
EXAMPLE IX 3ß-Oleylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid
(Figures 5A-20)
(a) 1.6 g of 3-amino-7a, 12a-dihydroxy-5β-colan-24-oic methyl ester (Figure 5A-1) was dissolved in 30 ml of dry dimethyl fomamide and treated with 3 ml of triethyl amine under stirring . A solution of 1.38 g of oleyl chloride in 10 ml of dry DMF was added dropwise, and the resulting solution was kept at room temperature overnight. The reaction mixture was poured into water, extracted with ethyl acetate, the organic fraction was purified by washing with dilute hydrochloric acid, sodium bicarbonate and then with water. Evaporation to dryness in vacuo resulted in 3.1 g, which were subjected to chromatography on silica gel, using a mixture of ethyl acetate / hexane (4: 6 and 10: 8), to give
1. 8 g of the methyl ester (Figure 5A-19). (b) A solution of 1.2 g of the methyl ester was dissolved in 20 ml of methanol, treated at room temperature with a solution of 5 ml of IN sodium hydroxide and left at room temperature for 48 hours and then evaporated to dryness . The residue was dissolved in 20 ml of water and extracted with 25 ml of ethyl acetate 3 times. The extracted water was acidified with a hydrochloric solution to give. a precipitate, which leaked. This residue was chromatographed on silica gel with a mixture of ethyl acetate: hexxane: acetic acid 1 - (10: 4: 0.3), to give 0.3 g of 3β-oleylamido-7-otic acid, 12a-dihydroxy-5β-colan -24-oico (Figure 5C-20).
EXAMPLE X 3β, 7α-Distearylamido-5β-ursodeoxycolan-24-oic acid
(Figures 5D-26)
(a) 20 g of ursodesoci-colan-24-oic acid were dissolved in 200 ml of absolute methanol, treated with 1 ml of concentrated sulfuric acid and stirred for 24 hours. The majority of the solvent was distilled and the residue was poured into water and extracted with methylene chloride. The organic extract was washed with a solution of sodium bicarbonate and sodium chloride, and evaporated to dryness resulting in 19.5 g of 3,7-dihydroxy-5β-ursodeoxycolan-24-oic acid methyl ester (Figure 5D- twenty-one) .
^ -RN (CDCI3) d, ppm: 0.66 (s, CH3-I8), 0.90 (t, J = lHz, CH3-23), 0.93 (s, CH3-19), 0.94 (d, J = 3Hz, CH3 - 21), 3.58 (m, CH-3, CH-7), 3.65 (s, COOCH3). (b) 4.06 g of methyl ester 5 (Figure 5D-21) were dissolved in 30 ml of dry pyridine and cooled to
0 ° C. The reaction mixture was stirred and treated by dropping for 15 minutes with a solution of 1.49 g of methanesulfonyl chloride in 5 ml of pyridine. After allowing to stand for 3 hours at the same temperature, the
The reaction mixture was poured onto ice and water, and then extracted with ethyl acetate. The organic phase was washed with hydrochloric acid, sodium bicarbonate and sodium chloride solution, filtered and evaporated in vacuo. The residue consisting of four compounds
was subjected to chromatography on a silica column using a mixture of ethyl acetate and hexane as eluent. The less polar compound, 5.3 g, was the methyl ester of 7-dimesyl-5β-ursodeoxylanoic acid 24-oico (Figure 5D-22). Í:: H-NMR (CDClj) d, ppm: 0.65 (s, CHj-18), 0.90
(t, J = lHz, CH3-23), 0.97 (s, CH3-19), 1.2 (d, J = 3Hz, CH3-21), 2.97 (m, CH3S02) 2.98 (s, CH3S02), 3.64 (s) , CH3S02), 4.09 (c, J = 12Hz, H-7), 4.62 (m, H-7). (c) 5.65 g of the dimesyl derivative was dissolved in 50 ml of dry DMF, treated with dry sodium azide and heated at 130 ° for 2 hours. The reaction mixture was cooled, poured into ice water and extracted with ethyl acetate. The extract was then washed with a solution of sodium acetate and sodium chloride, filtered and evaporated to dryness, resulting in 4.6 g of the 3β, 7a-diazido-5β-ursodeoxycolan-24-oic acid methyl ester (Figure 5D). -2. 3). (d) 4.5 g of the diazido compound (Figure 5D-23) was dissolved in 120 ml of methanol to which 150 mg of 5% palladium on carbon was added and hydrogenated at atmospheric pressure for 4 days. The hydrogenation was repeated with an additional 150 mg of 5% palladium on carbon. The hydrogenated mixture was filtered and evaporated in vacuo to give 3 g of 3β, 7α-diamino-5β-ursodeoxycolan-24-oic acid methyl ester (Figure 5D-24.). XH-NMR (CDC13) d, ppm: 0.65 (s, CH3-I8), 0.92 (t, J = 4Hz, CH3-23), 0.96 (s, CH3-19), 1.2 (t, J = 3Hz, CH3 -21), 3.68 (s, COOCH3), 3.72, 3.95 (m, 2H-7.3). (e) 1.47 g of 3β, 7a-diamino-5β-ursodeoxycolan-24-oic acid methyl ester (FIG. 5D-24) was dissolved in 50 ml of a dry 1: 1 mixture of DMSO and DMF, treated with 2% of the mixture. ml of triethylamine and 30 mg of dimethylamino pyridine and 5.1 g of stearic anhydride. The reaction mixture was heated to 50 °, stirred for 18 hours, poured into ice water and extracted 3 times with ethyl acetate. The organic phase was washed with hydrochloric acid, sodium bicarbonate and sodium chloride solution. After evaporation of the organic solvent, 2.05 g of an oily residue were obtained. Separation on silica gel using ethyl acetate: hexane as eluent (1: 4) resulted in a number of fractions, one of which was 80 mg, was methyl ester 3β, 7a-distearylamido-5β-ursodeoxycolan- 4-oico (Figure 5D-25), according to its MS and: H-NMR EM FAB: MgH2 + 937 (MW) 936). ^ -RM (CDC13) d, ppm: 0.66 (s, CH3-I8), 0.86 (d, J = 4Hz, CH3-23), 0.96 (s, CH-19), 1.2 (t, J = 3Hz, CH3 -21), 1.26 [s, CH2) 16], 3.64 (s, COOHCH3), 3.05 (d, J = 7.Hz, H-7), 5.75 (m, CH-3). (f) 78 mg of methyl ester (Figure 5D-25) was dissolved in 20 ml of methanol, treated with 2 ml of IN sodium hydroxide and left for 48 hours at room temperature. The methanol was evaporated in vacuo, the residue was dissolved in 25 ml of water, filtered and then acidified with dilute hydrochloric acid to give a precipitate which consisted of 3β, 7α-distearylamido-5β-ursodeoxycolan-24-oic acid ( Figure 5D-26y).
Example XI Materials and Methods Cholesterol (Sigma, St. Louis, Mo.) Was recrystallized twice from hot ethanol; Na-taurocholate (Na-TC; Sigma, St Louis, Mo.) was recrystallized twice from ethanol and ether (J. L Pope, J. Lipid Res. 8, (1967) 146-147); Egg yolk lecithin (EYL) (Avanti Polar Lipíds, Alabaster, Al.) was used without further purification. All the lipids used in this study were purified by TLC (Thin Layer Chromatography) standard. The synthetic bile acid conjugate used in Examples XII to XIV was palmitylamido-7a, 12a-dihydroxy-5β-colan-24-oic acid (PalC) (prepared as described in Example IV).
1. Preparation of Bilis A. Bilis Model Mixtures of EYL, cholesterol and Na-TC were dissolved in CHCH3 / CH3OH (2: 1 v / v) dried at room temperature under N2, lyophilized overnight and stored at room temperature. ° C under argon until they were used. The model bile solutions were prepared by suspending the dry lipids in 150mM NaCl, 1.5mM disodium EDTA, 50mM Tris-HCl at pH 8.0 and incubating the suspensions at 55 ° C for 1 hour. The solubilized model bile was incubated, in sealed bottles under argon, at 37 ° C for the duration of the experiment. The aliquots of the models were examined daily. All models were prepared in triplicate and stored at the same conditions throughout the experimental period. The bile model compositions were: 15 mM cholesterol, 30 mM EYL, 150 mM Na-TC. One hundred percent EYL was used for the preparation of the control solution. The other model bile solutions investigated were prepared by adding or substituting (10-20%) of the EYL or Na-TC for the synthetic bile acid conjugate (PalC).
B ^ Native human gall bladder bile Native human gall bladder bile was obtained from patients with gallstones of cholesterol in cholecystectomy. The bile collected from several patients was enriched in cholesterol by incubation with dry cholesterol or by mixing with a concentrated bile model before being used in experiments to facilitate crystallization.
2. Evaluation of the formation and growth of cholesterol crystals 2.1 Test of the observation time of the crystals (TOC) The TOC (also called "Nucleation Time") was determined according to what was described by Holán et al. in Gastroenterology 77, (1979) 611-617. The aliquots of each model bile were examined daily by polarized light microscopy. The TOC was defined as the initial time of detection of at least three crystals of cholesterol monohydrate per microscopic field at a magnification of 100 times.
2. 2 Crystal growth rate assay (CGR) The growth of the crystal was verified spectrophotometrically using a microplate reader (SPECTRA-STL, Austria) (G.J. Somjen, et al.,
J. Lipid Re. 38, (1977) 1048-1052). The aliquots (50 μl) of lipid solutions were mixed and vigorously shaken with equivalent volumes of Na-taurodeoxycholate (200mM), in microplate wells.
After 60 minutes at room temperature, the microplates were shaken again and the absorbance at 405 nm was measured in each well. Each model was prepared by triplicate and sampled in triplicate for measurement.
Data were collected and analyzed by an IBM compatible personal computer, and optical density (OD), averaged for triplicate preparations, was calculated. A graph is used that describes the average DO changes for each solution. The slope in the inclined region of the curve was determined by linear regression adjustment for at least three measurements and was defined as the CGR. The different CGR and DO were calculated between day 0 and day 14 for each model.
2. 3 Measurement of crystal mass Chemical analyzes of cholesterol were performed on each sample on the last day of the experiment (day 14), as described previously (G.J. Somjen, see above). Samples were collected from the microwells, centrifuged in an Airfuge (Beckman) at 70,000 rpm for 5 minutes. Separate determinations were made on the total sample (before centrifugation), as well as on the supernatant solution. The amount of cholesterol in the precipitated sediment was calculated by subtracting the amounts in the total supernatant solutions. The crystalline character of the sediment was confirmed by polarized light microscopy. The crystalline mass was also measured spectrophotometrically as the difference of OD between day 0 and day 14 of the incubation.
3. Data analysis Each lipid dispersion was prepared in triplicate and aliquots were measured in triplicate from all samples. The mean values of the DO and standard errors were calculated: The growth rates of the crystals were calculated from the linear regression analysis of the crystal growth curves as explained above. The comparisons between the different model solutions were carried out by means of an analysis of variance.
Example XII The effect of the bile salt fatty acid conjugate prepared in Example IV (PalC hereinafter "test compound") on the crystallization kinetics of cholesterol in model and human bile. Replacement and addition experiments were carried out. The following results were obtained.
A. Bilis Model a. In a model ft ^ bile solution (composition: 150 M taurocholate, 15 mM cholesterol, 30 mM egg lecithin, 10.3 gm / dl total lipids) 5 when 20% Na taurocholate was replaced by the test compound ( PalC) the nucleation time was extended by 167%; the growth rate of the cholesterol crystals was reduced by 67% and the mass of total cholesterol crystals after 14 days of 10 incubation was reduced by 53%. b. When the test compound was added to the complete model bile solution (at a concentration of 20% bile salts) the effects were as follows: The nucleation time was prolonged by 200%; the growth rate of the cholesterol crystals was reduced by 59% and the total mass of the crystals after 14 days of incubation was reduced by 51%.
B. Native human bile, When the test compound (at a concentration of 10 mM) was incubated with the native human gallbladder bile collected from patients with cholesterol gallstones, the results were
the following:
In the native human bile, from which the cholesterol crystals were removed by previous ultracentrifugation for 2 hours, new cholesterol crystals were observed from day 2 of incubation (at 37 ° C). The crystalline members increased progressively reaching a peak of more than 150 crystals per microscopic field on day 14. In the same bile incubated with '10 mM of the test compound no cholesterol crystals were observed through the 21 day incubation period.
EXAMPLE XIII Cholesterol crystal nucleation studies were performed in bile model as follows: PalC was added to the model bile in the following proportions (mole%) NaTC replacement in 10%, 20% ("B", "C" ); Replacement PC by 20% ("D"); and Adding 10%, 20% of the total NaTC ("E", "F") The results of the experiments of the
Examples XII and XIII are summarized in the accompanying Figures 1 to 3 below (in all Figures 1 to 3"A" represents the bile control model without PalC): Figure 1 illustrates the observation time of the crystal = nucleation time. The results are given as means of triplicates. The crystal observation time was extended by 167% in C and 200% in F. Figure 2 illustrates the mass of cholesterol crystals. The cholesterol mass on day 14 was reduced by 17% in B and by 53% in C. This was reduced by 51% in F. Figure 3 illustrates the growth rate of the crystals. The growth rate of the crystals was reduced by 70% in and by 59% in F. These experimental data confirmed that conjugates of bile salts and fatty acids prevent or retard the crystallization of cholesterol in model and human bile.
Example XIV A. Animals 6-7 week old male hamsters weighing 79-83 g were kept in an animal housing with access to water and feed. The test hamsters were given by means of a special syringe 10-15 mg / animal / day of PalC dissolved in 1 ml of saline solution. The control animals were given an equivalent volume of saline alone. Both groups behaved normally during the course of treatment. The second day 4 hours after the application of PalC in saline to the respective animals, were sacrificed by means of an overdose of chloroform vapors. The abdomens were opened, the bile ducts were cut, the biliary vesicles were removed and rinsed twice in saline. They were then placed on top of conical plastic tubes and cut. The bile is collected at the bottom of the tubes. 2 series of animals were examined; I. 5 control animals and 5 test animals each receiving 10 mg of PalC / day; II. 3 control animals and 3 test animals each receiving 15 mg of PalC / day.
B. Biochemical procedures Bile samples were centrifuged (Eppendorf centrifuge) for 5 minutes at 2000 rpm. The remains were discarded and the supernatant was extracted according to the Folch procedure (chloroform: methanol 2: 1). After partitioning with water, the lipid phase was analyzed by thin-layer chromatography on 60 thin plates of silica gel (Merck). The eluent was dichloromethane: methanol: acetic acid (100: 5: 1, v: v: v). The samples were compared to a true standard, Reference source (Rf) approximately 0.2 after development with I2 vapors.
C. Results In series I, 140 μl of control bile and 100 μl of test bile were obtained. In series II, 95 μl of control bile and 240 μl of test bile were obtained. The control bile and test bile from each series were collected separately. The analysis by TLC (shown in Figure 4) of the extracted lipids demonstrated the presence of PalC in the bile of the test animals. This proves that the ingestive PalC is absorbed and excreted in the bile. Figure 4 illustrates comparisons of TLC from the Experiments performed in Example XIV: A. illustrates PalC standards: in pure solution (left and in bile (right): B. illustrates hamster bile: from control hamsters (left); of hamster fed with PalC (right) A PalC band is observed in this column Figures 5A, 5B, Figures 5C and 5D illustrate the formulas of the compounds described in Examples I to X, respectively.
Example XV Methods The model bile solution had the following composition: Cholesterol 15m; EYL 30ml, NaTCl 150 mL. It was prepared as described in Example XI. In the test solutions, 20 mol% of NaTC was replaced by an equimolar amount of each specific fatty acid / bile acid conjugate tested. The results obtained with the conjugates of saturated fatty acids with a chain length of C? 4, Ci6, m and C2o respectively, were conjugated with cholic acid at the C3 position as shown in Figures 6 and 7. Figure 6 shows the effect of those conjugates on the mass of cholesterol crystals after 14 days of incubation of cholesterol solutions and test. All of the above conjugates reduced the final crystalline mass compared to the control solution. The C? 8 conjugate reduced this by 14% of the control; the C2o conjugate reduced it by 38%. A C22 conjugate tested in a different experiment showed activity similar to that of C2o- Figure 7 shows the nucleation time (crystal observation time) of the different test solutions compared to the control solution. The replacement of 20% of the bile salt (NaTC) by the specific conjugates resulted in a prolongation of the nucleation time with the conjugates of C? 6, Cig and C2o- The Ci4 did not prolong the nucleation time. The C2o conjugate prolonged the nucleation time by more than 360%.
Example XVI Bile of human gall bladder put together
• 10 obtained in the operations was enriched with a concentrated lipid solution to improve cholesterol crystallization. The final concentration in bile of added lipids was 60mM NaTC, 18.4mM EYL and 9.2mM cholesterol. Enriched bile was
ultracentrifuged at 50,000 rpm for 1 hour to remove cholesterol crystals and was distributed
• then in jars. The first bottle had only enriched bile (control). To the other 4 bottles were added the following solutions (at 5 mM): acid
< - -J colic, cholate of (palmitolyl) C-16, cholate
(stearoyl) C-18 and C20 (arachidyl) cholate. After 22 days of incubation at 32 ° C all the bile were centrifuged in an Airfugue at 70,000 rpm. The sediment was removed and its cholesterol content
chemically measured. The results are shown in Figure 8, as μmol of cholesterol in the sedimented crystalline mass. It is obvious that the three bile salt / fatty acid conjugates markedly reduced cholesterol crystallization compared to control bile with or without cholic acid.
Example XVII A model bile solution was prepared as described in Example XI, with the same lipid composition, and served as a control. In all the other samples, 20 mol% of NaTC were replaced by equimolar amounts of: cholic acid, C6 cholate, C? 2 cholate, Cis cholate, Co cholate (all those fatty acids were conjugated at the C3 position of cholate) ) and di-stearoyl ursodeoxylate (with conjugated stearic acid radicals in equal proportions at the C3 and C positions of bile acid). All samples were incubated at 37 ° C in the same manner as described in Example XI and the nucleation time was determined by periodic simple microscopy observations. The results are shown in Figure 9. The results proved that: 1) all the conjugates (BAFAC) tested retarded the cholesterol crystallization compared to the bile control model and with equimolar amounts of cholic acid. 2) That the BAFAC with longer fatty acid chains were more effective than those with shorter chains. 3) That the conjugate with two fatty acids (distearoyl ursodeoxycholate) was particularly effective.
Example XVIII Absorption and Transport of Stearoyl Collate (C-18 cholate) ^ Female hamsters with a weight of 80-100 gm
gave them an overdose of 30 mg of C-18 cholate by intragastric administration. Animals were sacrificed alone at 1, 2 and 3 hours after administration. The cardiac blood, portal blood and bile of the gallbladder were sampled. Ib studied two groups of animals (A and B) in parallel. Stearoyl cholate levels were measured with a CLAP instrument (Kontron Switzerland) using a UV detector at 206 nm. The results are shown in Figure 10. 20 In group A: The levels in cardiac blood after 1, 2 and 3 hours were 99, 7, 2 μm, while the levels in the portal blood were 68, 99 and 133 μm, respectively. The cholate levels of C-18 in gallbladder bile were 540 and 270 μm at 2 and 3 hours, respectively. The results in group B were similar. The data show: 1) That stearoyl cholate of C-18 is absorbed from the intestine. 2) That it is transported both in the systemic circulation (via the lymph) and in the portal vein. 3) That it is actively secreted in the bile and concentrated in it.
Example XIX A model bile solution was prepared in the same manner as described in Example XI. This had the same lipid composition and served as a control. In colic acid from the test solutions, cholate (C-18: 0) stearoyl and cholate (C -18: 1) oleoyl were added in concentrations of 20 mM: All samples were incubated at 37 ° C for 100 hours. The difference in optical density between 100 hours and 0 hours (as described in Example XI) was used to measure the total crystalline mass at 100 hours. In comparison with the control solution (100%), the crystalline mass with cholic acid was 114%, with 62% stearoyl cholate and 55% oleoyl cholate. These results prove that BAFACs with a saturated acid as well as an unsaturated (oleic) acid both decreased cholesterol crystallization compared to control bile and equimolar amounts of cholic acid. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.
•
Claims (25)
1. The conjugates of the fatty acid of bile acid or bile salt of general formula II W-X-G characterized in that G is a radical of bile acid or bile salt, W means one or more fatty acid radicals and X is a linking member between radical of bile acid and bile salt and fatty acid radicals.
2. The conjugates of the fatty acid of bile acid or bile salt according to the claim 1, characterized in that the bile acids are selected from folic acid, cenodeoxycholic acid, ursodeoxycholic acid and deoxycholic acid.
3. The conjugates of the fatty acid of bile acid or bile salt according to claim 1 or 2, characterized in that the bile acid is conjugated in position 24 with glycine or taurine.
4. The conjugates of the fatty acid of bile acid or bile salt according to any of claims 1 to 3, characterized in that the conjugation with the fatty acid radical is carried out in the 3-position of the bile acid nucleus.
5. The conjugates of the fatty acid of bile acid or bile salt according to any of claims 1 to 3, characterized in that the conjugation with the fatty acid radical is carried out in a position selected from position 6, 7, 12 and 24 of the bile acid nucleus.
6. The conjugates of the fatty acid of bile acid or bile salt according to any of claims 1 to 5, characterized in that the conjugation between the fatty acid radical and the bile acid is selected from the configuration a or ß.
7. The conjugates of the fatty acid of bile acid or bile salt according to any of claims 1 to 6, characterized in that the binding member is selected from a group -NH- and a group -0-.
8. The conjugates of the fatty acid of bile acid or bile salt according to any of claims 1 to 8, characterized in that the fatty acids are saturated fatty acids having 6-26 carbon atoms.
9. The conjugates of the fatty acid of bile acid or bile salt according to claim 8, characterized in that the saturated fatty acids have 14-22 carbon atoms. 10. The conjugates of the fatty acid of bile acid or bile salt according to any of claims 8 or 9, characterized in that the saturated fatty acid is selected from behenylic acid, arachidyl acid, stearic acid, palmitic acid and myristyl acid. 11. 3-β-Behenylamido-7a, 12a-dihydroxy-5-colan-24-oic acid. 12. 3-β-arachidyl amido-7a, 12a-dihydroxy-5-colan-24-oic acid. 13. The 3-β-sterilamido-7, 12a-dihydroxy-5-colan-24-oic acid. 14. 3-β-palmitylamido-7a, 12a-dihydroxy-5-cholan-2 -oic acid. 15. The 3-ß-myristylamido-7a, 12a-dihydroxy-5-cholan-2 -oic acid. 16. N- (-carboxymethyl) -3β-esterylamido-7a, 12-dihydroxy-5β-colan-24-amide. 17. The fatty acid conjugates of bile acid or bile salt according to any of claims 1 to 4 and 6 to 10, characterized in that W means two fatty acids, which are conjugated in positions 3 and 7 of the biliary nucleus. 18. A pharmaceutical composition that allows the dissolution of cholesterol gallstones in the bile and to prevent the formation thereof and allow the prevention and / or reduction of arteriosclerosis, characterized in that it comprises as an active ingredient a fatty acid derivative of acid biliary salt or bile salt of the general formula II in accordance with any of
10. Claims 1 to 17. The pharmaceutical composition according to claim 18, characterized in that it is selected from among a tablet, a capsule, a solution and an emulsion. i 20. The pharmaceutical composition according to claim 18 or 19, characterized in that it comprises an additional compound selected from a carrier., a solvent, an emulsifier, an absorption enhancer and an inhibitor of the synthesis or secretion of 0 cholesterol in the bile. 21. The pharmaceutical composition according to any of claims 18 to 20, characterized in that it comprises 0.1-1.5 g of the active ingredient. 22. The use of a bile acid fatty acid conjugate or bile salt according to any one of claims 1 to 17 or of a pharmaceutical composition according to any of claims 18 to 21 for the dissolution of cholesterol gallstones in the bile and for the prevention of their formation. 23. The use of a bile acid fatty acid conjugate or bile salt according to any one of claims 1 to 17 or of a pharmaceutical composition according to any of claims 18 to 21 for the prevention and / or reduction of the arteriosclerosis. 24. A method for the dissolution of cholesterol gallstones in the bile and for the prevention of the formation thereof, characterized in that a bile acid fatty acid conjugate or bile salt is administered according to any of claims 1 to 17 or a pharmaceutical composition according to any of claims 18 to 21. 25. A method for the prevention and / or reduction of arteriosclerosis, characterized in that a conjugate of bile acid or bile salt is administered according to any of the claims 1 to 17 or a pharmaceutical composition according to any of claims 18 to 21.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| IL123998 | 1998-04-08 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| MXPA00009863A true MXPA00009863A (en) | 2002-05-09 |
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