AU2003300161B2 - Heparin-derived polysaccharide mixtures, preparation thereof and pharmaceutical compositions containing same - Google Patents
Heparin-derived polysaccharide mixtures, preparation thereof and pharmaceutical compositions containing same Download PDFInfo
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- AU2003300161B2 AU2003300161B2 AU2003300161A AU2003300161A AU2003300161B2 AU 2003300161 B2 AU2003300161 B2 AU 2003300161B2 AU 2003300161 A AU2003300161 A AU 2003300161A AU 2003300161 A AU2003300161 A AU 2003300161A AU 2003300161 B2 AU2003300161 B2 AU 2003300161B2
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- methanol
- heparin
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- 239000000203 mixture Substances 0.000 title claims abstract description 144
- 229920000669 heparin Polymers 0.000 title claims abstract description 90
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 title claims abstract description 56
- 229960002897 heparin Drugs 0.000 title claims abstract description 50
- 150000004676 glycans Chemical class 0.000 title claims abstract description 11
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 11
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 11
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 9
- 229920001542 oligosaccharide Polymers 0.000 claims abstract description 17
- 150000002482 oligosaccharides Chemical class 0.000 claims abstract description 17
- 150000003839 salts Chemical group 0.000 claims abstract description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 485
- 239000000243 solution Substances 0.000 claims description 95
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 90
- 239000003055 low molecular weight heparin Substances 0.000 claims description 75
- 159000000000 sodium salts Chemical class 0.000 claims description 71
- -1 benzyl ester Chemical class 0.000 claims description 57
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 45
- 239000012429 reaction media Substances 0.000 claims description 36
- 238000000034 method Methods 0.000 claims description 35
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 31
- 229960003872 benzethonium Drugs 0.000 claims description 22
- 238000006243 chemical reaction Methods 0.000 claims description 22
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 20
- 230000032050 esterification Effects 0.000 claims description 20
- 238000005886 esterification reaction Methods 0.000 claims description 20
- 239000002585 base Substances 0.000 claims description 19
- 238000007127 saponification reaction Methods 0.000 claims description 19
- 239000001632 sodium acetate Substances 0.000 claims description 19
- 235000017281 sodium acetate Nutrition 0.000 claims description 19
- 238000000746 purification Methods 0.000 claims description 17
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 16
- SIYLLGKDQZGJHK-UHFFFAOYSA-N dimethyl-(phenylmethyl)-[2-[2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethoxy]ethyl]ammonium Chemical compound C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 SIYLLGKDQZGJHK-UHFFFAOYSA-N 0.000 claims description 15
- 239000011734 sodium Substances 0.000 claims description 13
- 229960001950 benzethonium chloride Drugs 0.000 claims description 10
- UREZNYTWGJKWBI-UHFFFAOYSA-M benzethonium chloride Chemical compound [Cl-].C1=CC(C(C)(C)CC(C)(C)C)=CC=C1OCCOCC[N+](C)(C)CC1=CC=CC=C1 UREZNYTWGJKWBI-UHFFFAOYSA-M 0.000 claims description 10
- 150000002148 esters Chemical class 0.000 claims description 8
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical compound ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 claims description 7
- 229940073608 benzyl chloride Drugs 0.000 claims description 7
- WMFOQBRAJBCJND-UHFFFAOYSA-M Lithium hydroxide Chemical compound [Li+].[OH-] WMFOQBRAJBCJND-UHFFFAOYSA-M 0.000 claims description 6
- KWYUFKZDYYNOTN-UHFFFAOYSA-M Potassium hydroxide Chemical compound [OH-].[K+] KWYUFKZDYYNOTN-UHFFFAOYSA-M 0.000 claims description 6
- 239000003814 drug Substances 0.000 claims description 6
- ZFGMDIBRIDKWMY-PASTXAENSA-N heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims description 6
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims description 5
- 150000008044 alkali metal hydroxides Chemical class 0.000 claims description 5
- 239000012736 aqueous medium Substances 0.000 claims description 5
- 230000002265 prevention Effects 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 claims description 4
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 4
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 claims description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- QZUPHAGRBBOLTB-UHFFFAOYSA-N NSC 244302 Chemical compound C=1C=CC=CC=1P(C(C)(C)C)C1=CC=CC=C1 QZUPHAGRBBOLTB-UHFFFAOYSA-N 0.000 claims description 4
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 4
- FGUUSXIOTUKUDN-IBGZPJMESA-N C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 Chemical compound C1(=CC=CC=C1)N1C2=C(NC([C@H](C1)NC=1OC(=NN=1)C1=CC=CC=C1)=O)C=CC=C2 FGUUSXIOTUKUDN-IBGZPJMESA-N 0.000 claims description 3
- 230000001476 alcoholic effect Effects 0.000 claims description 3
- 230000002785 anti-thrombosis Effects 0.000 claims description 3
- 206010012601 diabetes mellitus Diseases 0.000 claims description 3
- 230000035484 reaction time Effects 0.000 claims description 3
- 238000007920 subcutaneous administration Methods 0.000 claims description 3
- VSCBATMPTLKTOV-UHFFFAOYSA-N 2-tert-butylimino-n,n-diethyl-1,3-dimethyl-1,3,2$l^{5}-diazaphosphinan-2-amine Chemical compound CCN(CC)P1(=NC(C)(C)C)N(C)CCCN1C VSCBATMPTLKTOV-UHFFFAOYSA-N 0.000 claims description 2
- GNFTZDOKVXKIBK-UHFFFAOYSA-N 3-(2-methoxyethoxy)benzohydrazide Chemical compound COCCOC1=CC=CC(C(=O)NN)=C1 GNFTZDOKVXKIBK-UHFFFAOYSA-N 0.000 claims description 2
- 206010002388 Angina unstable Diseases 0.000 claims description 2
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 2
- 206010003658 Atrial Fibrillation Diseases 0.000 claims description 2
- 206010051055 Deep vein thrombosis Diseases 0.000 claims description 2
- 206010028980 Neoplasm Diseases 0.000 claims description 2
- 208000010378 Pulmonary Embolism Diseases 0.000 claims description 2
- 208000007536 Thrombosis Diseases 0.000 claims description 2
- 208000007814 Unstable Angina Diseases 0.000 claims description 2
- 206010047249 Venous thrombosis Diseases 0.000 claims description 2
- 230000033115 angiogenesis Effects 0.000 claims description 2
- 239000002870 angiogenesis inducing agent Substances 0.000 claims description 2
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 2
- 210000001367 artery Anatomy 0.000 claims description 2
- 201000011510 cancer Diseases 0.000 claims description 2
- 229960001927 cetylpyridinium chloride Drugs 0.000 claims description 2
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 claims description 2
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 claims description 2
- 201000010099 disease Diseases 0.000 claims description 2
- 208000035475 disorder Diseases 0.000 claims description 2
- 239000003102 growth factor Substances 0.000 claims description 2
- 201000004332 intermediate coronary syndrome Diseases 0.000 claims description 2
- 238000001990 intravenous administration Methods 0.000 claims description 2
- 208000010125 myocardial infarction Diseases 0.000 claims description 2
- 208000031225 myocardial ischemia Diseases 0.000 claims description 2
- 239000003960 organic solvent Substances 0.000 claims description 2
- 239000007800 oxidant agent Substances 0.000 claims description 2
- 230000002093 peripheral effect Effects 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims description 2
- 230000035755 proliferation Effects 0.000 claims description 2
- 230000002685 pulmonary effect Effects 0.000 claims description 2
- 238000009472 formulation Methods 0.000 claims 2
- 208000007342 Diabetic Nephropathies Diseases 0.000 claims 1
- 206010012689 Diabetic retinopathy Diseases 0.000 claims 1
- 239000000654 additive Substances 0.000 claims 1
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
- 210000000663 muscle cell Anatomy 0.000 claims 1
- 239000003981 vehicle Substances 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 84
- 230000001858 anti-Xa Effects 0.000 abstract description 40
- 239000003513 alkali Substances 0.000 abstract description 5
- 150000001720 carbohydrates Chemical group 0.000 abstract description 5
- 239000002253 acid Substances 0.000 abstract description 4
- 239000002184 metal Substances 0.000 abstract 2
- 239000002244 precipitate Substances 0.000 description 127
- 238000003756 stirring Methods 0.000 description 100
- 239000000725 suspension Substances 0.000 description 81
- 239000006228 supernatant Substances 0.000 description 73
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 71
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 48
- 239000011521 glass Substances 0.000 description 48
- 238000001035 drying Methods 0.000 description 37
- 239000007787 solid Substances 0.000 description 37
- 238000003760 magnetic stirring Methods 0.000 description 36
- 235000011121 sodium hydroxide Nutrition 0.000 description 27
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 25
- 239000012528 membrane Substances 0.000 description 24
- 239000013049 sediment Substances 0.000 description 24
- 239000011780 sodium chloride Substances 0.000 description 24
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 23
- 238000004090 dissolution Methods 0.000 description 23
- 239000012153 distilled water Substances 0.000 description 16
- 239000011541 reaction mixture Substances 0.000 description 15
- 229940040526 anhydrous sodium acetate Drugs 0.000 description 11
- 239000000706 filtrate Substances 0.000 description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 8
- CQUCJWQSILDSQR-UHFFFAOYSA-N 1,2-dimethyldiazaphosphinane Chemical compound CN1N(CCCP1)C CQUCJWQSILDSQR-UHFFFAOYSA-N 0.000 description 6
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 6
- 229910052784 alkaline earth metal Inorganic materials 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- RVIIEUUMDMVPLO-UHFFFAOYSA-N 4-butylimino-n,n-diethyl-1,3-dimethyldiazaphosphinan-2-amine Chemical compound CCCCN=C1CCN(C)N(N(CC)CC)P1C RVIIEUUMDMVPLO-UHFFFAOYSA-N 0.000 description 5
- 229940127215 low-molecular weight heparin Drugs 0.000 description 5
- GKTNLYAAZKKMTQ-UHFFFAOYSA-N n-[bis(dimethylamino)phosphinimyl]-n-methylmethanamine Chemical compound CN(C)P(=N)(N(C)C)N(C)C GKTNLYAAZKKMTQ-UHFFFAOYSA-N 0.000 description 5
- 150000003254 radicals Chemical class 0.000 description 5
- 238000001816 cooling Methods 0.000 description 4
- 229910052757 nitrogen Inorganic materials 0.000 description 4
- 239000002002 slurry Substances 0.000 description 3
- 235000017557 sodium bicarbonate Nutrition 0.000 description 3
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 3
- 230000019635 sulfation Effects 0.000 description 3
- 238000005670 sulfation reaction Methods 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 229920000936 Agarose Polymers 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- NEUSVAOJNUQRTM-UHFFFAOYSA-N cetylpyridinium Chemical compound CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 NEUSVAOJNUQRTM-UHFFFAOYSA-N 0.000 description 2
- 229960004830 cetylpyridinium Drugs 0.000 description 2
- RLGQACBPNDBWTB-UHFFFAOYSA-N cetyltrimethylammonium ion Chemical class CCCCCCCCCCCCCCCC[N+](C)(C)C RLGQACBPNDBWTB-UHFFFAOYSA-N 0.000 description 2
- 238000004587 chromatography analysis Methods 0.000 description 2
- 239000000470 constituent Substances 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- IIPYXGDZVMZOAP-UHFFFAOYSA-N lithium nitrate Chemical compound [Li+].[O-][N+]([O-])=O IIPYXGDZVMZOAP-UHFFFAOYSA-N 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- SLRMQYXOBQWXCR-UHFFFAOYSA-N 2154-56-5 Chemical class [CH2]C1=CC=CC=C1 SLRMQYXOBQWXCR-UHFFFAOYSA-N 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
- 108090000935 Antithrombin III Proteins 0.000 description 1
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 229920000045 Dermatan sulfate Polymers 0.000 description 1
- 108010029144 Factor IIa Proteins 0.000 description 1
- 108010014173 Factor X Proteins 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- 102100021194 Glypican-6 Human genes 0.000 description 1
- OBIAROUDANLMOS-UHFFFAOYSA-N N1N(P=CC=C1)N Chemical compound N1N(P=CC=C1)N OBIAROUDANLMOS-UHFFFAOYSA-N 0.000 description 1
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 description 1
- 206010038923 Retinopathy Diseases 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 229960000583 acetic acid Drugs 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 125000000217 alkyl group Chemical group 0.000 description 1
- 230000003024 amidolytic effect Effects 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 230000002429 anti-coagulating effect Effects 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 229960005348 antithrombin iii Drugs 0.000 description 1
- 229960004676 antithrombotic agent Drugs 0.000 description 1
- 239000000010 aprotic solvent Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 125000001797 benzyl group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])* 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229910052791 calcium Inorganic materials 0.000 description 1
- 239000011575 calcium Substances 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 210000005056 cell body Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 229960001701 chloroform Drugs 0.000 description 1
- 239000003593 chromogenic compound Substances 0.000 description 1
- AGVAZMGAQJOSFJ-WZHZPDAFSA-M cobalt(2+);[(2r,3s,4r,5s)-5-(5,6-dimethylbenzimidazol-1-yl)-4-hydroxy-2-(hydroxymethyl)oxolan-3-yl] [(2r)-1-[3-[(1r,2r,3r,4z,7s,9z,12s,13s,14z,17s,18s,19r)-2,13,18-tris(2-amino-2-oxoethyl)-7,12,17-tris(3-amino-3-oxopropyl)-3,5,8,8,13,15,18,19-octamethyl-2 Chemical class [Co+2].N#[C-].[N-]([C@@H]1[C@H](CC(N)=O)[C@@]2(C)CCC(=O)NC[C@@H](C)OP(O)(=O)O[C@H]3[C@H]([C@H](O[C@@H]3CO)N3C4=CC(C)=C(C)C=C4N=C3)O)\C2=C(C)/C([C@H](C\2(C)C)CCC(N)=O)=N/C/2=C\C([C@H]([C@@]/2(CC(N)=O)C)CCC(N)=O)=N\C\2=C(C)/C2=N[C@]1(C)[C@@](C)(CC(N)=O)[C@@H]2CCC(N)=O AGVAZMGAQJOSFJ-WZHZPDAFSA-M 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000012691 depolymerization reaction Methods 0.000 description 1
- 229940051593 dermatan sulfate Drugs 0.000 description 1
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- MUJOVVKDDICVMS-UHFFFAOYSA-N diazaphosphinine Chemical compound C1=CN=NP=C1 MUJOVVKDDICVMS-UHFFFAOYSA-N 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 229960000610 enoxaparin Drugs 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- XEKSTYNIJLDDAZ-JASSWCPGSA-F fondaparinux sodium Chemical compound [Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].[Na+].O[C@@H]1[C@@H](NS([O-])(=O)=O)[C@@H](OC)O[C@H](COS([O-])(=O)=O)[C@H]1O[C@H]1[C@H](OS([O-])(=O)=O)[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](OS([O-])(=O)=O)[C@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@@H]4[C@@H]([C@@H](O)[C@H](O)[C@@H](COS([O-])(=O)=O)O4)NS([O-])(=O)=O)[C@H](O3)C(O)=O)O)[C@@H](COS([O-])(=O)=O)O2)NS([O-])(=O)=O)[C@H](C(O)=O)O1 XEKSTYNIJLDDAZ-JASSWCPGSA-F 0.000 description 1
- 239000012362 glacial acetic acid Substances 0.000 description 1
- 239000002628 heparin derivative Substances 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 208000017169 kidney disease Diseases 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 159000000003 magnesium salts Chemical class 0.000 description 1
- 125000000325 methylidene group Chemical group [H]C([H])=* 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 150000007530 organic bases Chemical class 0.000 description 1
- UNQNIRQQBJCMQR-UHFFFAOYSA-N phosphorine Chemical compound C1=CC=PC=C1 UNQNIRQQBJCMQR-UHFFFAOYSA-N 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 229910052700 potassium Inorganic materials 0.000 description 1
- 239000011591 potassium Substances 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 125000001453 quaternary ammonium group Chemical group 0.000 description 1
- 210000000329 smooth muscle myocyte Anatomy 0.000 description 1
- URGAHOPLAPQHLN-UHFFFAOYSA-N sodium aluminosilicate Chemical compound [Na+].[Al+3].[O-][Si]([O-])=O.[O-][Si]([O-])=O URGAHOPLAPQHLN-UHFFFAOYSA-N 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 229940061706 sulfated mucopolysaccharides Drugs 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P11/00—Drugs for disorders of the respiratory system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/12—Drugs for disorders of the urinary system of the kidneys
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
New mixtures (A) of heparin-derived sulfated polysaccharides have average molecular weight 1500-3000, anti-Xa activity 120-200 IU/mg, anti-IIa activity 0-10 IU/mg and ratio of anti-Xa to anti-IIa activity more than 30; comprise 2-26 saccharide units; have a 4,5- unsaturated uronic acid 2-O-sulfate unit at one terminal; contain a specific hexasaccharide moiety designated DELTAIIa-IIs-Is hexasaccharide; and are in alkali(ne earth) metal salt form. New mixtures (A) of sulfated polysaccharides have the general structure of the constitutive polysaccharides of heparin, an average molecular weight of 1500-3000, an anti-Xa activity of 120-200 IU/mg, an anti-IIa activity of 0-10 IU/mg and a ratio of anti-Xa to anti-IIa activity of more than 30. The constitutive oligosaccharides of (A) comprise 2-26 saccharide units; have a 4,5- unsaturated uronic acid 2-O-sulfate unit at one terminal; contain a hexasaccharide moiety of formula (I) (designated DELTAIIa-IIs-Is hexasaccharide) and are in alkali(ne earth) metal salt form. An Independent claim is included for the preparation of (A).
Description
WO 2004/033503 - 1 - PCT/FR2003/002960 HEPARIN-DERIVED POLYSACCHARIDE MIXTURES, PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS - CONTAINING SAME 5 The present invention relates to mixtures of poly saccharides derived from heparin, their method of preparation and pharmaceutical compositions containing them. 10 Heparin is a mixture of sulfated mucopolysaccharides of animal origin which is used in particular for its anti coagulant and antithrombotic properties. Heparin nevertheless has disadvantages which limit the 15 conditions for its use. In particular, its high anticoagulant activity (anti-IIa activity) can cause hemorrhages. Low-molecular-weight heparins obtained by basic 20 depolymerization of heparin esters have been proposed (EP40144); however, these products still have a high anti-IIa activity. Very-low-molecular-weight heparins have also been 25 described in US 6384021. However, the anti-Xa activity values obtained in the examples described do not exceed 120 IU, and the anti-Xa/anti-IIa ratio obtained is between 15 and 50. 30 In WO-0208295, very-low-molecular-weight heparins are prepared by a method different from US 6384021 and exhibit an activity between 100 and 150 IU with anti Xa/anti-IIa ratios which are also very high for certain examples of application. 35 A constant need however exists in this class of medicament to improve the anti-Xa activities, in particular in obtaining activities greater than - 2 150 IU/mg, and the anti-Xa/anti-IIa ratio, and therefore to develop novel generations of heparin derivatives. 5 One of the objectives of the invention is therefore to improve the anti-Xa activity and the anti-Xa/anti-IIa ratio by modifying the methods described in the prior art, in particular by controlling the percentage of water during the depolymerization step. The heparins 10 thus obtained thus exhibit an excellent antithrombotic activity and possess an aXa activity similar to that of heparin while reducing the risks of hemorrhage with a very low aIIa activity. Likewise, the products of the invention exhibit half-life periods which are markedly 15 greater than that of heparin. The subject of the invention is therefore novel mixtures of polysaccharides derived from heparin possessing a more selective activity toward activated 20 factor X (factor Xa) and toward activated factor II (factor IIa) than heparin. It is understood that the mixtures of polysaccharides having a mean molecular weight of 1500 to 3000 Da can 25 be termed as oligosaccharides. The subject of the present invention is therefore the mixtures of sulfated oligosaccharides having the general structure of the constituent polysaccharides of 30 heparin and having the following characteristics: - they have a mean molecular weight of 1500 to 3000 daltons, an anti-Xa activity of 120 to 200 IU/mg, an anti-IIa activity of less than 10 IU/mg and an anti Xa activity/anti-IIa activity ratio of greater than 30, 35 - the constituent oligosaccharides of the mixtures contain 2 to 26 saccharide units, have a 4,5 unsaturated uronic acid 2-0-sulfate unit at one of their ends, and contain the hexasaccharide AIIa-IIs-Is of formula: -3 Na Na Na Na;'~O 0 Na 0 0 Na 0 0 0- 0- 0-// O 0 0 6H OH. mH 'OH OH OH NH OH 0 . NH 0 NH NaO Na Alla s Is The hexasaccharide Alla-Ils-Is contained in the mixture 5 of oligosaccharides described in the present invention is a sequence which has a high affinity for ATIII and characterized by an aXa activity greater than 740 U/mg. The mixture of oligosaccharides described in the 10 present invention is in the form of an alkali or alkaline-earth metal salt. As alkali or alkaline-earth metal salt, the sodium, potassium, calcium and magnesium salts are preferred. 15 The mean molecular weight is determined by high pressure liquid chromatography using two columns in series, for example those marketed under the name TSK G3000 XL and TSK G2000 XL. The detection is carried out 20 by refractometry. The eluent used is lithium nitrate and the flow rate is 0.6 ml/min. The system is calibrated with standards prepared by fractionation of enoxaparin (AVENTIS) by chromatography on agarose polyacrylamide gel (IBF). This preparation is carried 25 out according to the technique described by Barrowcliffe et al., Thromb. Res., 12, 27-36 (1977-78) or D.A. Lane et al., Thromb. Res., 12, 257-271 (1977-78). The results are calculated with the GPC6 software (Perkin Elmer).
- 4 The anti-Xa activity is measured by the amidolytic method on a chromogenic substrate described by Teien et al.-,- Thromb. Res., 10, 399-410 (1977), with, as 5 standard, the first international standard for low molecular-weight heparins. The anti-IIa activity is measured by the technique described by Anderson L.O. et al., Thromb. Res., 15, 10 531-541 (1979), with, as standard, the first inter national standard for low-molecular-weight heparins. The hexasaccharide fraction preferably represents from 15 to 25% of the mixture of oligosaccharides. 15 Preferably, the mixtures according to the invention contain from 8 to 15% of the hexasaccharide AIIa-IIs-Is in the hexasaccharide fraction of the mixture of oligo saccharides. 20 The percentage of the hexasaccharide fraction may be analytically determined by high-pressure liquid chromatography on TSK G3000 XL and TSK G2000 XL columns or alternatively by preparative separation of the hexa 25 saccharide fraction. The mixture is in this case chromatographed on columns filled with a polyacrylamide agarose type gel such as that marketed under the trademark Ultrogel ACA202R (Biosepra) . The mixture is eluted with a sodium hydrogen carbonate solution. 30 Preferably, the sodium hydrogen carbonate solution is a 0.1 mol/l to 1 mol/l solution. Still more preferably, the separation is carried out at a concentration of 1 mol/l. The determination is carried out by UV spectrometry (254 nm) . After fractionation, the hexa 35 saccharide fraction in solution in sodium hydrogen carbonate is neutralized with glacial acetic acid. The solution is then concentrated under reduced pressure so as to obtain a sodium acetate concentration greater than 30% by weight. The hexasaccharide fraction is - 5 precipitated by adding from 3 to 5 volumes of methanol. The hexasaccharide fraction is recovered by filtration on No. 3 sintered glass. The hexasaccharide mixture obtained may be analyzed by HPLC (High-Performance 5 Liquid Chromatography) in order to determine the content of hexasaccharide AIIa-IIs-Is. Hexasaccharide AIIa-IIs-Is may be isolated by preparative HPLC chromatography or by affinity chromatography on an antithrombin III sepharose column according to the 10 techniques used by persons skilled in the art (M. Hook, I. Bjork, J. Hopwood and U. Lindahl, F.E.B.S letters, vol 656(1) (1976)). Most particularly, the mixtures according to the 15 invention have an anti-Xa activity of between 150 IU/mg and 200 IU/mg. Preferably, the mixtures according to the invention have an anti-IIa activity of less than 5 IU/mg, and 20 most particularly of 0.5 to 3.5 IU/mg. The examples of applications described below demonstrate values of between 1.1 and 1.6 IU/mg when the preferred characteristics of the process are used. 25 Preferably, the mixtures exhibit an anti-Xa activity/anti-IIa activity ratio greater than 50 and most particularly greater than 100. Preferably, the mixtures according to the invention 30 have a mean molecular weight of between 2000 and 3000 Daltons, and most particularly a mean molecular weight of between 2400 and 2650 Da. The subject of the invention is therefore most 35 particularly the mixtures as defined above, having an anti-Xa activity of between 150 and 200 IU/mg, an anti IIa activity of between 0.5 and 3.5 IU/mg and a mean molecular weight of between 2400 and 2650 Da.
- 6 The mixtures of oligosaccharides according to the invention may be prepared by depolymerization of a quaternary ammonium salt of the benzyl ester of heparin in an-organic medium, by means of a strong organic base 5 with a pKa preferably greater than 20 (properties preferably similar to the family of phosphazenes defined for example according to R. Schwesinger et al., Angew. Chem. Int. Ed. Engl. 26, 1167-1169 (1987), R. Schwesinger et al., Angew. Chem. 105, 1420 (1993)), 10 conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt, saponification of the residual esters and optionally purification. The method according to the invention repeats the main steps of the method as 15 described in WO 0208295 while adding an essential characteristic which makes it possible to obtain the mixtures of oligosaccharides according to the invention with the physicochemical characteristics and the activities described above. 20 Indeed, in order to obtain the specific mixtures of oligosaccharides according to the invention, it is necessary to control the selectivity of the base by a - very precise control of the water content of the 25 mixture during the depolymerization step. The method according to the present invention is indeed characterized by a control of the high selectivity of the base during the depolymerization. It makes it 30 possible to depolymerize the heparin while preserving as much as possible the sequences with an affinity for ATIII such as the hexasaccharide AIIa-IIs-Is described in the present invention. This critical step of the method makes it possible to obtain the polysaccharides 35 according to the invention. This characteristic of the method results in unexpected aXa activities in terms of the mean molecular weight of the mixtures of oligosaccharides (150 IU/mg < aXa < - 7 200 IU/mg for a mean molecular weight of between 2000 Da and 3000 Da) . This selectivity is due to the very particular physicochemical characteristics of the phosphazene bases which have a pKa greater than 20, a 5 very high steric hindrance and a weak nucleophilicity. This effect is fully expressed when the reaction medium is anhydrous. On the other hand, when the water content of the reaction medium increases, a drastic reduction 10 in the selectivity of the depolymerization is observed. The preservation of the sequences with affinity for ATIII decreases and the consequence is a large drop in the aXa activity. In the presence of a low quantity of water, the phosphazene base becomes protonated and the 15 reactive species becomes a quaternary ammonium hydroxide. In this case, the very high steric hindrance and weak nucleophilicity properties are lost and greatly influence the quality of the product obtained. When depolymerization trials are carried out with a 20 measured and controlled water content, this effect can be seen to be fully expressed. The following table summarizes the impact of the water content on the selectivity of the depolymerization 25 (only this parameter is variable in the trials: the stoichiometry of the reagents, the dilutions, the temperatures remain constant according to the criteria of persons skilled in the art. The base used is the phosphazene base: 2-tert-butylimino-2-diethylamino-1,3 30 dimethylperhydro-1,2,3-diazaphosphorine).
-8 Water 0.05% 0.1% 0.2% 0.3% 0.4% 0.57% 1.8% 2.5% content aXa~ 192 177 161 132 122 120 105 99 IU/mg aIIa 1.3 1.5 1.4 1.4 1.3 1.4 3.1 13.4 IU/mg aXa/ 148 118 115 94 94 85.7 34 7.4 aIIa For an optimum selectivity and a maximum preservation of the sequences with affinity for ATIII, it is preferable to carry out the procedure at water contents 5 of less than 0.6% and most particularly less than 0.3% when 1 molar equivalent of phosphazene base is used relative to the benzyl ester of heparin, benzethonium salt. 10 The subject of the invention is therefore most particularly the step of depolymerization of the quaternary ammonium salt of the benzyl ester of heparin obtained according to methods known to persons skilled in the art, characterized in that a base of the family 15 of phosphazenes, in particular in dichloromethane solution containing a percentage of water of less than 0.6%, is used. Preferably, this percentage of water should be chosen less than 0.3% and most particularly less than 0.2%. 20 Advantageously, the strong base/ester mol ratio is between 0.2 and 5, preferably between 0.6 and 2 and most particularly between 0.8 and 1.2. The use of the equimolar ratio therefore forms part of the preferred 25 embodiments of the invention. Other aprotic solvents known to persons skilled in the art may be used, such as THF or DMF.
9 The quaternary ammonium salt of the benzyl ester of heparin is preferably the benzethonium, cetylpyridinium or cetyltrimethylammonium salt. 5 The bases of the family of phosphazenes are preferably those of formula: R3 R2-N R4 Ri-N=P-N-R5 N-R6 R7 in which the radicals R1 to R7 are identical or different and represent alkyl 10 radicals. In particular, the subject of the invention is the method as defined above, characterized in that the base used (step d) for depolymerization) is 2-tert butylimino-2-diethylamino-1,3-dimethylperhydro-1,3,2-diazaphosphorine (2-[(1,1 15 dimethylethyl)imino]-N,N-diethyl-1,2,2,2,3,5,6-octahydro-1,3-dimethyl-1,3,2 diazaphosphorine-2-amine): NCH -Et or tert-butyliminotri(pyrrolidino)phosphorane. 20 In the preceding formulae, the alkyl radicals contain 1 to 6 carbon atoms in the form of a straight or branched chain.
9a The subject of the invention is therefore a method for preparing the mixtures of oligosaccharides according to the invention comprising the following steps a) transalification of sodium heparin by the action of benzethonium chloride, b) esterification of benzethonium heparinate by the action of benzyl chloride, 5 c) transalification of the benzyl ester obtained to a quaternary ammonium salt, d) depolymerization of the quaternary ammonium salt of the benzyl ester of heparin by the method as defined above, e) conversion of the quaternary ammonium salt to a sodium salt, f) optionally saponification of the heparin by the action of a base such as 10 sodium hydroxide, g) optionally purification in particular by the action of an oxidizing agent such as hydrogen peroxide. 15 10 The following reaction scheme illustrates the present invention: 0 OP4 O(SO NagX 0-/-,.70 H2 TA O(SOMA)Y H(sN)Z0 Stp Sodium heparinate m Benzethonium chloride Mn-25 ( C) n= X+Y+Z (mean over level of sulfation of the disacchride) X= Degree of sulfation of the site, the remainder is represented by the radical H Y= Degree of sutfaton of the site, the remainder is represented by the radical H Z= Degree of sulfation of the site, the remaMdr is represented by the radical COCH, 6 oions oisopy .F0 OW o" WO "ai S cH oLaoficaion 2' MeOH. AcONa O(SO,NaY NiXONII''LlONl ~SO,Na)Zj n, Stop ba)Y NH(So,Na! ( S b2 degree of esteification b- degree of esterifc action Crude benzyl ester Pure benzyl ester, sodium salt C on)b o(soy ejx o ../)NC 0 Trn-ln o- )o i Pure benzy ester, sodium salt + C* Step Benzethonium chlodde _O(soHY)Y NH(SO,Hy)Z ( Hr Cl ) m =2-- 2 Pure benzyl ester, benzethonlum salt O ONa 0 ONa O(oNal 10 | - Saponification H0 HO O HO _O NaOH 4C Depolymerization Step f 2 / AcNa O(SOVNa)Y Noqso.Ne O(SONa)Y NH(SONa)Z Step d and e m=-3 Crude ULMWH (residual esters) o ONa O(So 0-ONa2 o.S-o-,Nag ONa ( ONe oOsoa O 0-ON 0 OH 0- , H202 0 0 0 O(SONa)Y NM(sONa) t O(SO 3 Na)Y NH(SONaIz O(SO,Na)Y N(SONa)z m m--3 me-3 Crude ULMWH pure ULMWH The conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin to a sodium salt (step e) is generally carried out by treating the reaction medium with an alcoholic solution of sodium acetate and preferably with a 10% solution of sodium acetate in methanol (weight/volume), at a temperature of between 15 and 25 0 C. The equivalent by weight of acetate added is preferably 3 times greater than the mass of quaternary ammonium salt of the benzyl ester of heparin used in the depolymerization reaction.
- 11 The saponification (step f) is generally carried out by means of an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in an -aqiieous medium, at a temperature of between 0 and 5 200C and preferably between 0 and 100C. 1 to 5 molar equivalents of alkali metal hydroxide will be generally used. Preferably, the saponification will be carried out in the presence of 1 to 2 molar equivalents of alkali metal hydroxide. 10 The final product may be optionally purified (step g) by any known method of purifying depolymerized heparins (for example EP 0037319B1). Preferably, the purifi cation is carried out by means of hydrogen peroxide, in 15 an aqueous medium, at a temperature of 10 to 500C. Preferably, this operation is carried out between 20 and 400C. The quaternary ammonium salt of the benzyl ester of 20 heparin may be prepared according to the following reaction scheme: a) conversion of the heparin to the form of a sodium salt by means of benzethonium chloride in order to . obtain benzethonium heparinate (transalification), 25 b) esterification of the benzethonium salt obtained above by means of benzyl chloride and treatment with an alcoholic solution of sodium acetate in order to obtain the sodium salt of the benzyl ester of heparin, c) transalification of the sodium salt of the benzyl 30 ester of heparin to a quaternary ammonium salt and preferably to a benzethonium, cetylpyridinium or cetyltrimethylammonium salt. The reaction of step a) is carried out by the action of 35 benzethonium chloride in excess, on sodium heparin, at a temperature in the region of 15 to 250C. Advan tageously, the salt/sodium heparin molar ratio is between 3 and 4.
- 12 The starting heparin used is preferably a pig heparin. The latter may be purified beforehand in order to reduce its dermatan sulfate level according to the method described in patent FR2663639. 5 The esterification of step b) is preferably carried out in a chlorinated organic solvent (for example chloro form or methylene chloride), at a temperature of between 25 and 45'C and preferably between 30 and 400C. 10 The ester in the form of a benzethonium salt is then recovered in the form of a sodium salt by precipitation by means of sodium acetate at 10% by weight in an alcohol such as methanol. 1 to 1.2 volumes of alcohol are generally used per volume of reaction medium. The 15 quantity of benzyl chloride and the reaction time are adjusted in order to obtain a degree of esterification of between 50 and 100% and preferably between 70 and 90%. Preferably, 0.5 to 1.5 parts by weight of benzyl chloride are used for 1 part by weight of benzethonium 20 salt of heparin. Likewise, preferably the reaction time will be between 10 and 35 hours. The transalification step c) is carried out by means of a quaternary ammonium chloride and preferably by means 25 of benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride, in an aqueous medium, at a temperature of between 10 and 25*C. Advan tageously, the quaternary ammonium chloride/sodium salt of the benzyl ester of heparin mol ratio is between 2 30 and 3. The mixtures according to the invention, in the form of a sodium salt, may be converted to another alkali or alkaline-earth metal salt. The passage from one salt to 35 another is optionally achieved using the method described in patent FR 73 13 580. The mixtures according to the invention are not toxic and may thus be used as medicaments.
- 13 The mixtures of oligosaccharides of the present invention may be used . as antithrombotic agents. In particular, they are useful for the treatment or 5 prevention of venous and arterial thromboses, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation. They are also useful in the prevention 10 and treatment of the proliferation of smooth muscle cells, atheriosclerosis and arteriosclerosis, for the treatment and.prevention of cancer by modulating angio genesis and growth factors, and for the treatment and prevention of diabetic disorders such as diabetic 15 retinopathies and nephropathies. The present invention also relates to the pharma ceutical compositions containing, as active ingredient, a mixture of formula (I), optionally in combination 20 with one or more inert excipients. The pharmaceutical compositions are for example solutions which can be injected by the subcutaneous or intravenous route. They are also useful for 25 administration by the pulmonary route (inhalation) or by the oral route. The dosage may vary according to the age, weight and state of health of the patient. For an adult, it is in 30 general between 20 and 100 mg per day by the intramuscular or subcutaneous route. The following examples illustrate the invention without however limiting it. 35 Example A: PREPARATION OF THE BENZETHONIUM SALT OF BENZYL OF HEPARINATE - 14 Benzethonium heparinate A solution of 25 g of benzethonium chloride in 125 ml of water is added to a solution of 10 g of heparin in the- -form of a sodium salt in 100 ml of water. The 5 product is filtered, washed with water and dried. Benzyl ester of heparin (sodium salt) 16 ml of benzyl chloride are added to a solution of 20 g of benzethonium heparinate in 80 ml of methylene 10 chloride. The solution is heated at a temperature of 30'C for 12 hours. 108 ml of a 10% solution of sodium acetate in methanol are then added, the mixture is filtered, washed with methanol and dried. 7.6 g of benzyl ester of heparin are thus obtained in the form 15 of a sodium salt whose degree of esterification is 77%. Benzyl ester of heparin (benzethonium salt) 36 g (0.0549 mol) of benzyl ester of heparin (sodium salt) and 540 ml of distilled water are introduced into 20 a 2-litre Erlenmeyer flask A. After homogenization at a temperature of about 20 0 C, a pale yellow solution is obtained. A solution of 64.45 g (0.1438 mol) of benzethonium chloride and 450 ml of water is prepared, with magnetic stirring, in a 1-litre Erlenmeyer flask 25 B. The solution in Erlenmeyer B is poured over about 35 minutes into the solution in Erlenmeyer A, with stirring. The formation of an abundant white precipitate is observed. The Erlenmeyer B is rinsed with 200 ml of distilled water and the wash water is 30 introduced into the Erlenmeyer A. The stirring is then stopped and the suspension is allowed to settle for 12 hours. Once this time has elapsed, the clear portion of the supernatant is removed and discarded. 560 ml of water are added to the sedimented precipitate (slurry 35 appearance) and the mixture is stirred for 20 minutes. The precipitate is allowed to resediment for about 30 minutes. The supernatant is removed and discarded (560 ml) . This operation of washing with about 560 ml of distilled water is repeated twice on the sedimented - 15 precipitate. In the last washing operation, the precipitate is left in suspension and filtered through a No. 3 sintered glass. The cake is then washed with 4 times-200 ml of distilled water. The wet white solid is 5 drained and then dried under reduced pressure (2.7 kPa) at a temperature in the region of 60'C. After drying for 12 hours, 87.5 g of benzyl heparinate, benzethonium salt, are obtained. The yield obtained is 94.9%. 10 Example B: DESCRIPTION OF THE HEXASACCHARIDE ATIII (AIIa-IIs-Is) Na Na Na O 0 0 O=S'O O=sO O=S'O Na 0 0 Na 0 0 Na 0 0 O O( 0- 0 o 0o-0 6 H O tt O OH O ' O w O OH O".OH-o0 OH 0"' o .O.. H O., OH OH OH NH OH O. NH O. O O. NH 0-* ' '" 90-s 0 NaO 0 Na O Na Alla l1S Is 15. - Proton spectrum in D 2 0, 500 MHz, T=298 K, 8 in ppm 1.97 (3H, s), 3.18 (lH, dd, 10 and 3Hz), 3.30 (1H, t, 8Hz), 3.37 (lH, dd, 10 and 3Hz), 3.60 (2H, m), between 3.65 and 3.85 (6H, m), 3.87 (2H, m), 3.95 (1H, d, 8Hz), 4.03 ( 1 H, d, 8Hz), between 4.05 and 4.13 (4H, m), 20 between 4.16 and 4.45 (8H, m), 4.52 (lH, d, 8Hz), 4.67 (1H, m), 5.06 (lH, d, 6Hz), 5.10 (1H, d, 3Hz), 5.33 (lH, d, 4Hz), 5.36 (lH, d, 3Hz), 5.46 (1H, d, 3Hz), 5.72 (lH, d, 4Hz) Decasodium salt of 4-deoxy-a-L-threo-hexenepyranosyl 25 uronic acid- (1-4) -2-deoxy-2-acetamido-6-0-sulfo-a-D glucopyranosyl- (1->4) -- D-glucopyranosyluronic acid (1-*4)-2-deoxy-2-sulfamido-3,6-di-O-sulfo-a-D-gluco pyranosyl) - (1->4) -2-0-sul fo-a-L-idopyranosyluronic acid (1->4) -2-deoxy-2-sulfamido-6-0-sulfo-a-D-glucopyranose. 30 - 16 . Examples 1 to 7 and 12 illustrate the influence of the water content on the selectivity of the polymerization reaction and the aXa and aIIa activity of the products obtained. 5 . Examples 8 to 10 illustrate the influence of the number of base equivalents on the aXa and aIIa activity of the product obtained (with a water content of 0.1%) . Example 11 illustrates the use of a phosphazene base other than 2-tert-butylimino-2-diethylamino-1,3 10 dimethylperhydro-1,2,3-diazaphosphorine: Use of tert-butyliminotri(pyrrolidino)phosphorane EXAMPLE 1 Depolymerization and conversion to the sodium salt 15 (0.1% water): 70 ml of dichloromethane are placed in an Erlenmeyer flask A. 10 g (0.006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A are added slowly 20 with stirring. The water content of the reaction medium is adjusted to 0.1%. The solution is heated to 400C under nitrogen. After total dissolution, the solution is cooled to a temperature in the region of 200C, followed by addition of 1.75 ml (0.006 mol) of 2-tert 25 butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3 diazaphosphorine. The resulting mixture is stirred at a temperature in the region of 20 0 C for 24 hours. During this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in an 30 Erlenmeyer flask B. After total dissolution, 5 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic solution of sodium acetate at a temperature in the region of 35 5C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 1 hour 30 minutes. The clear part of the supernatant is separated out and discarded (220 ml). 220 ml of methanol are added to the sedimented precipitate and - 17 the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour 20 minutes. The supernatant is separated out and discarded (250 ml). 250-ml of methanol are added to the sedimented 5 precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 100 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure 10 (6 kPa) at a temperature in the region of 40*C. After drying for 18 hours, 2.51 g of the sodium salt of depolymerized heparin in Celite (5 g) are obtained. The yield obtained is 64%. 15 Saponification: 2.5 g (0.0038 mol) of the crude depolymerized heparin, sodium salt in Celite (5 g) obtained above, and 17 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass 20 and rinsed with twice 5 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.4 ml (0.004 mol) of 30% caustic soda is introduced with magnetic stirring, at a temperature in the region of 5*C. After addition, the mixture is stirred for 25 2 hours. The solution is neutralized by adding 1N HCl and 3 g of sodium chloride are added. After dissolution, 21 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 44 ml of methanol are added, followed by stirring for 1 hour. 30 Stirring is then stopped and the suspension is left to sediment for 45 minutes at a temperature in the region of 50C. The supernatant is then separated out and discarded (90 ml) . 90 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 35 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (80 ml) . 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then - 18 filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40'C. After 5 drying for 18 hours, 1.31 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 66%. Purification: 10 1.3 g of crude depolymerized heparin obtained above and 13 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is 15 filtered through a 0.45 pm membrane and 0.07 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 20*C, the mixture is neutralized by adding 1N HCl, followed by addition of 2 g of sodium chloride. 20 The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in. The solution is then cooled to 100C and stirred for about 15 minutes. 36 ml of methanol are then added, followed . by stirring for 1 hour. Stirring is then stopped and 25 the suspension is left to sediment for about 15 minutes. The supernatant is then separated out and discarded (50 ml) . 50 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for 30 about 25 minutes. The supernatant is separated out and discarded (50 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced 35 pressure (6 kPa) at a temperature in the region of 40'C. After drying for 18 hours, 1.13 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 87%.
- 19 The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2600 Daltons anti-Xa activity: 177 IU/mg 5 anti-IIa activity: 1.5 IU/mg anti-Xa activity/anti-IIa activity ratio: 118 EXAMPLE 2 Depolymerization and conversion to the sodium salt 10 (0.2%) 70 ml of dichloromethane are placed in a Erlenmeyer flask A. 10 . g (0.006 mol) of the benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A are added 15 slowly, with stirring and under nitrogen pressure. The water content of the reaction medium is adjusted to 0.2%. After total dissolution, 1.75 ml (0.006 mol) of 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro 1,2,3-diazaphosphorine are added. The mixture is 20 stirred for 24 hours at a temperature in the region of 20 0 C. During this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 5 g of -- Hyflo supercel Celite are added to the solution. The 25 reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic solution of sodium acetate with magnetic stirring, at a temperature in the region of 50C. After stirring for 5 minutes, the suspension is left to settle for 30 2 hours. The clear part of the supernatant is separated out and discarded (220 ml). 220 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 2 hours. The supernatant is 35 separated out and discarded (230 ml). 230 ml of methanol are added to the sedimented precipitate. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 150 ml of methanol. The pale yellow wet solid is - 20 drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying for 18 hours, 2.63 g of crude depolymerized heparin in Celite (5 g) are obtained. The yield obtained is 67%. 5 Saponification: 2.5 g (0.0038 mol) of the sodium salt of depolymerized heparin in Celite (5 g) obtained above and 18 ml of water are placed in 50 ml Erlenmeyer flask. The 10 suspension is filtered through a No. 3 sintered glass and rinsed with twice 5 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.4 ml (0.004 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 50C. After 15 addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 2 g of sodium chloride are added. 14 ml of methanol are poured into the reaction mixture. After stirring for 15 minutes, 36 ml of methanol are added, followed by 20 stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 45 minutes at a temperature in the region of 5*C. The supernatant is then separated out and discarded (80 ml). 80 ml of - methanol are added to the sedimented precipitate and 25 the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region 30 of 400C. After drying for 48 hours, 2.3 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 65%. Purification: 35 1.4 g of crude depolymerized heparin obtained above and 15 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is - 21 filtered through a 0.45 pm membrane and 0.07 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 40'C, the mixture is cooled to a temperature 5 in the region of 200C and then neutralized by addition of 1N HCl. 2 g of sodium chloride are added to the reaction medium. The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in. The solution is then cooled to 100C and 10 stirred for 15 minutes. 36 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 15 minutes. The supernatant is then separated out and discarded (40 ml). 40 ml of methanol are added to the 15 sedimented precipitate (slurry appearance) and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (50 ml). The precipitate in suspension is then filtered through a 20 No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying for - 18 hours, 1.2 g of pure depolymerized heparin (sodium 25 salt) are obtained. The yield obtained is 86%. The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2650 daltons anti-Xa activity: 161 IU/mg 30 anti-IIa activity: 1.4 IU/mg anti-Xa activity/anti-IIa activity ratio: 115 EXAMPLE 3 Depolymerization and conversion to the sodium salt 35 (0.3% water) 70 ml of dichloromethane are placed in a Erlenmeyer flask A. 10 g (0.006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A are added with - 22 stirring and under nitrogen pressure. The water content of the reaction medium is adjusted to 0.3%. After total dissolution, 1.75 ml (0.006 mol) of 2-tert-butylimino 2-diethylamino-1,3-dimethylperhydro-1,2,3-diaza 5 phosphorine are added. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 5 g of 10 Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in the region of 50C, with magnetic stirring. After stirring for 5 minutes, 15 the suspension is left to settle for 1 hour 10 minutes. The clear part of the supernatant is separated out and discarded (220 ml). 220 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then 20 filtered through a No. 3 sintered glass. The cake obtained is then washed with 100 ml of methanol. The wet solid is drained and, then dried under reduced pressure (6 kPa) at a temperature in the region of 40 0 C. After drying for 18 hours, 2.57 g of crude 25 depolymerized heparin are obtained, as the sodium salt in Celite (5 g). The yield obtained is 66%. Saponification: 2.5 g (0.0038 mol) of the sodium salt of crude 30 depolymerized heparin in Celite (5 g) obtained above and 18 ml of water are placed in 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 5 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer 35 flask. 0.4 ml (0.004 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 50C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 3 g of sodium chloride are added. 15 ml of methanol - 23 are poured into the reaction mixture. After stirring for 15 minutes, 36 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the- -suspension is left to sediment for 1 hour. The 5 supernatant is then separated out and discarded (70 ml) . 70 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then 10 washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40"C. After drying for 48 hours, 1.42 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 62%. 15 Purification: 1.4 g of crude depolymerized heparin obtained above and 14 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 400C with 20 magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.07 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the 25 region of 20 0 C, the mixture is neutralized by adding 1N HCl, followed by addition of 2 g of sodium chloride. After dissolution, the solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in. The filtrate is then cooled to 100C and 30 stirred for 15 minutes. 36 ml of methanol are then added, followed by stirring for about 1 hour. Stirring is then stopped and the suspension is left to sediment for 40 minutes. The supernatant is then separated out and discarded (50 ml) . 50 ml of methanol are added to 35 the sedimented precipitate (slurry appearance) and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 25 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then - 24 washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying for 18 heurs, 1.24 g of pure depolymerized heparin (sodium 5 salt) are obtained. The yield obtained is 89%. The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2400 daltons anti-Xa activity: 132 IU/mg 10 anti-IIa activity: 1.4 IU/mg anti-Xa activity/anti-IIa activity ratio: 94 EXAMPLE 4 Depolymerization and conversion to the sodium salt 15 (0.4% water): 70 ml of dichloromethane are placed in a Erlenmeyer flask A. 10 g (0.006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, 20 with stirring. The water content of the reaction medium is adjusted to 0.4%. The solution is heated to 300C under nitrogen. After total dissolution, the mixture is cooled to a temperature in the region of 200C, followed by addition of 1.75 ml (0.006 mol) of 2-tert 25 butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3 diazaphosphorine. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a 30 Erlenmeyer flask B. After total dissolution, 5 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in the region of 5*C, 35 with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 2 hours. The clear part of the supernatant is separated out and discarded (80 ml) . 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes.
- 25 The precipitate is left to resediment for about 1 hour. The supernatant is separated out and discarded (80 ml). 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. 5 The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 150 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40*C. After 10 drying for 18 hours, 3.25 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (5 g). The yield obtained is 83%. Saponification: 15 3.1 g (0.0018 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above and 21 ml of water are placed in 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 6 ml of water. The 20 filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.7 ml (0.007 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 5 0 C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl 25 and 4 g of sodium chloride are added. 28 ml of methanol are poured into the reaction mixture. After stirring for 15 minutes, 72 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 1 hour. The 30 supernatant is then separated out and discarded (90 ml) . 90 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and 35 discarded (90 ml) . 90 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet - 26 solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying for 48 hours, 1.9 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained 5 is 67%. Purification: 1.9 g of crude depolymerized heparin obtained above and 19 ml of distilled water are placed in a 50 ml 10 Erlenmeyer flask. The mixture is brought to 40'C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.1 ml of aqueous 30% hydrogen peroxide solution is added. After 15 stirring for about 2 hours at a temperature in the region of 200C, the mixture is neutralized by adding 1N HCl, followed by addition of 2 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in, and 20 the mixture is stirred for 15 minutes. 36 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 15 minutes. The supernatant is then separated out and discarded (40 ml). 40 ml of 25 methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (50 ml). 500 ml of methanol are added to the sedimented 30 precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained 35 and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying for 72 hours, 1.56 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 82%.
- 27 The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2350 daltons ant-i Xa activity: 122 IU/mg 5 anti-IIa activity: 1.3 IU/mg anti-Xa activity/anti-IIa activity ratio: 94 EXAMPLE 5 Depolymerization and conversion to the sodium salt 10 (0.57% water): 140 ml of dichloromethane are placed in a Erlenmeyer flask A. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, 15 with stirring. The water content of the reaction medium is adjusted to 0.57%. After total dissolution, 3.5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3 dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 20 200C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to - the solution. The reaction mixture in the Erlenmeyer 25 flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in the region of 40C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 30 minutes. The clear part of the 30 supernatant is separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour. The supernatant is separated out and discarded 35 (420 ml). 420 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is - 28 drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 500C. After drying for 18.hours, 6.66 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (10 g). The 5 yield obtained is 85%. Saponification: 6.66 g (0.0101 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above 10 and 47 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 15 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.1 ml (0.011 mol) of 30% caustic soda are added 15 with magnetic stirring, at a temperature in the region of 5OC. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 9.5 g of sodium chloride are added. 66 ml of methanol are added to the reaction medium. After 20 stirring for 15 minutes, 171 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 1 hour at a temperature in the region of 50C. The . supernatant is then separated out and discarded 25 (160 ml). 160 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (180 ml). 180 ml of methanol are added to the 30 sedimented precipitate and the mixture is stirred for 5 minutes. The suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a 35 temperature in the region of 40*C. After drying for 18 hours, 4.53 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 74%. Purification: - 29 4.53 g of crude depolymerized heparin obtained above and 45 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by 5 addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.25 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 200C, the mixture is neutralized by adding 10 1N HCl, followed by addition of 5.5 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 38 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 200C and stirred for 15 15 minutes. 100 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 20 minutes. The supernatant is then separated out and discarded (90 ml) . 90 ml of methanol are added to the sedimented 20 precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 25 minutes. The supernatant is separated out and discarded (100 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The white 25 cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 500C. After drying for 18 hours, 3.7 g of pure depolymerized heparin (sodium salt) are obtained. The 30 yield obtained is 82%. The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2200 daltons anti-Xa activity: 120 IU/mg 35 anti-IIa activity: 1.4 IU/mg anti-Xa activity/anti-IIa activity ratio: 86 - 30 EXAMPLE 6 Depolymerization and conversion to the sodium salt (1. 8% -water): 5 140 ml of dichloromethane are placed in a Erlenmeyer flask A. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. The water content of the reaction medium 10 is adjusted to 1.8%. After total dissolution, 3.5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3 dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 60 g 15 of anhydrous sodium acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the 20 methanolic sodium acetate solution at a temperature in the region of 40C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 30 minutes. The clear part of the supernatant is separated out and discarded (400 ml). 25 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour. The supernatant is separated out and discarded (420 ml). 420 ml of methanol are added to the 30 sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is drained and then dried under 35 reduced pressure (6 kPa) at a temperature in the region of 500C. After drying for 18 hours, 7.54 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (10 g). The yield obtained is 96%.
- 31 Saponification: 7.54 g (0.0101 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above and- 5-3 ml of water are placed in a 50 ml Erlenmeyer 5 flask. The solution is filtered through a No. 3 sintered glass and rinsed with twice 15 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.25 ml (0.012 mol) of 30% caustic soda are added with magnetic stirring, at a temperature in the 10 region of 40C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 10.5 g of sodium chloride are added. 70 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 190 ml of methanol are added, 15 followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for hi hour at a temperature in the region of 40C. The supernatant is then separated out and discarded (180 ml). 180 ml of methanol are added to the .20 sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (180 ml). 180 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 25 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 30 500C. After drying for 18 hours, 5.53 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 80%. Purification: 35 5.53 g of crude depolymerized heparin obtained above and 55 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is - 32 filtered through a 0.45 pm membrane and 0.31 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours. at a temperature in the region of 200C, the mixture is neutralized by adding 5 1N HCl, followed by addition of 7 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 49 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 200C and stirred for 15 minutes. 126 ml of 10 methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 20 minutes. The supernatant is then separated out and discarded (105 ml) . 105 ml of methanol are added to the sedimented precipitate and 15 the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 25 minutes. The supernatant is separated out and discarded (110 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then 20 washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 550C. After drying for 18 hours, 4.53 g of pure depolymerized heparin (sodium . salt) are obtained. The yield obtained is 82%. 25 The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2600 daltons anti-Xa activity: 105 IU/mg anti-IIa activity: 3.1 IU/mg 30 anti-Xa activity/anti-IIa activity ratio: 34 EXAMPLE 7 Depolymerization and conversion to the sodium salt (2.5% water): 35 140 ml of dichloromethane are placed in a Erlenmeyer flask A. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. The water content of the reaction medium - 33 is adjusted to 2.5%. After total dissolution, 3.5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3 dimethylperhydro-1,2,3-diazaphosphorine are added. The mixture is stirred at a temperature in the region of 5 20 0 C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer 10 flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in the region of 40C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 1 hour. The clear part of the supernatant is 15 separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The clear part of the supernatant is separated out and discarded 20 (400 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region 25 of 50*C. After drying for 18 hours, 7.78 g of crude depolymerized heparin are obtained, as the sodium salt in Celite (10 g). The yield obtained is 99.6%. Saponification: 30 7.78 g (0.0119 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above and 79 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with twice 15 ml of water. 35 The filtrate obtained is placed in a 150 ml Erlenmeyer .flask. 1.3 ml (0.012 mol) of 30% caustic soda are added with magnetic stirring, at a temperature in the region of 40C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl - 34 and 10 g of sodium chloride are added. 60 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 190 ml of methanol are added, followed by stirring for 1 hour. Stirring is then 5 stopped and the suspension is left to sediment for 4 hour at a temperature in the region of 4 0 C. The supernatant is then separated out and discarded (180 ml). 180 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 10 5 minutes. The precipitate is left to resediment for about 20 minutes. The supernatant is separated out and discarded (180 ml). 180 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then 15 filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 50*C. After drying for 18 hours, 5.87 g of crude 20 depolymerized heparin (sodium salt) are obtained. The yield obtained is 82%. Purification: . 5.87 g of crude depolymerized heparin obtained above 25 and 59 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.34 ml of 30 aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 20*C, the mixture is neutralized by adding 1N HCl, followed by addition of 7 g of sodium chloride. The solution is then filtered through a 0.45 pm 35 membrane and 49 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 20 0 C and stirred for 15 minutes. 126 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is - 35 left to sediment for 20 minutes. The supernatant is then separated out and discarded (105 ml) . 105 ml of methanol are added to the sedimented precipitate and the- fixture is stirred for 5 minutes. The precipitate 5 is left to resediment for about 25 minutes. The supernatant is separated out and discarded (110 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained 10 and then dried under reduced pressure (6 kPa) at a temperature in the region of 550C. After drying for 18 hours, 5.21 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 89%. The characteristics of the depolymerized heparin thus 15 obtained are as follows: mean molecular weight: 3550 daltons anti-Xa activity: 99 IU/mg anti-IIa activity: 13.4 IU/mg anti-Xa activity/anti-IIa activity ratio: 7.4 20 EXAMPLE 8 Depolymerization and conversion to the sodium salt (0.5 equivalent of base): - 140 ml of dichloromethane are placed in a Erlenmeyer 25 flask A. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. After total dissolution at a temperature in the region of 30 0 C and cooling to a temperature in 30 the region of 20'C, 1.75 ml (0.006 mol) of 2-tert butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3 diazaphosphorine are added. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 60 g of anhydrous sodium 35 acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. The reaction mixture in the Erlenmeyer flask A is poured into the methanolic sodium acetate solution at a temperature in the region of 40C, with magnetic stirring. After stirring for 1 hour, the - 36 suspension is left to settle for 2 hours. The clear part of the supernatant is separated out and discarded (420 ml). 420 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 30 minutes. The precipitate is left to resediment for about 18 hours. The clear part of the supernatant is separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The supernatant 10 is separated out and discarded (400 ml). The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 100 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure 15 (6 kPa) at a temperature in the region of 600C. After drying, 5.7 g of crude depolymerized heparin are obtained, as the sodium salt. The yield obtained is 73%. 20 Saponification: 5.7 g (0.0086 mol) of the sodium salt of crude depolymerized heparin obtained above and 53 ml of water are placed in a 100 ml Erlenmeyer flask. 0.93 ml (0.009 mol) of 30% caustic soda is added with magnetic 25 stirring, at a temperature in the region of 40C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 6 g of sodium chloride are added. 42 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 30 108 ml of methanol are added, followed by stirring for 30 minutes. Stirring is then stopped and the suspension is left to sediment for 30 minutes at a temperature in the region of 40C. The supernatant is then separated out and discarded (180 ml). 180 ml of methanol are 35 added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and discarded (170 ml). 170 ml of methanol are added to the sedimented precipitate and - 37 the mixture is stirred for 30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 30 mi- of methanol. The wet solid is drained and then S dried under reduced pressure at a temperature in the region of 600C. After drying, 3.5 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 67.4%. 10 Purification: 3.5 g of crude depolymerized heparin obtained above and 35 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.6 ± 0.1 by 15 addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.18 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 200C, the mixture is neutralized by adding 20 1N HC1, followed by addition of 3.6 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 27 ml of methanol are then poured in, at a temperature in the region of 100C. The - solution is then brought to 20'C and stirred for 25 15 minutes. 65 ml of methanol are then added, followed by stirring for 30 minutes. Stirring is then stopped and the suspension is left to sediment for 30 minutes. The supernatant is then separated out and discarded (80 ml) . 80 ml of methanol are added to the sedimented 30 precipitate and the mixture is stirred for 30 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and discarded (70 ml) . 70 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 35 30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with twice 30 ml of methanol. The wet solid is drained and then dried under reduced pressure at a temperature in the region of 600C. After - 38 drying, 2.8 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 80%. The characteristics of the depolymerized heparin thus obtained are as follows: 5 mean molecular weight: 2900 daltons anti-Xa activity: 146.1 IU/mg anti-IIa activity: 5.1 IU/mg anti-Xa activity/anti-IIa activity ratio: 28.6 10 EXAMPLE 9. Depolymerization and conversion to the sodium salt (0.6 equivalent of base): 280 ml of dichloromethane are placed in a three-necked flask A. 40 g (0.024 mol) of benzyl ester of heparin 15 (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. After total dissolution at a temperature in the region of 300C and cooling to a temperature in the region of 200C, 4.2 ml (0.014 mol) of 2-tert 20 butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3 diazaphosphorine are added. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is prepared in a 25 Erlenmeyer flask B. Half of the reaction mixture in the three-necked flask A is poured into the methanolic sodium acetate solution at a temperature in the region of 40C, with magnetic stirring. After stirring for 1 hour, the suspension is left to settle. The clear part 30 of the supernatant is separated out and discarded (310 ml). 310 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 1 hour. The precipitate is left to resediment for about 18 hours. The clear part of the supernatant is 35 separated out and discarded (400 ml). 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 1 hour. The supernatant is separated out and discarded (300 ml). The precipitate in suspension is then filtered through a No. 3 sintered - 39 glass. The cake obtained is then washed with twice 100 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure at a temperature in the region of 60'C. After drying, 6 g of 5 crude depolymerized heparin are obtained, as the sodium salt. The yield obtained is 77%. Saponification: 6 g (0.0091 mol) of the sodium salt of crude 10 depolymerized heparin obtained above and 56 ml of water are placed in a 250 ml Erlenmeyer flask. 1 ml (0.010 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 40C. After addition, the mixture is stirred for 2 hours. The 15 solution is neutralized by adding 1N HCl and 6.4 g of sodium chloride are added. 45 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 115 ml of methanol are added, followed by stirring for 30 minutes. Stirring is then stopped and the suspension 20 is. left to sediment for 30 minutes at a temperature in the region of 40C. The supernatant is then separated out and discarded (170 ml). 170 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate is left to 25 resediment for about 30 minutes. The supernatant is separated out and discarded (140 ml). 140 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered 30 glass. The cake obtained is then washed with twice 30 ml of methanol. The wet solid is drained and then dried under reduced pressure at a temperature in the region of 600C. After drying, 3.6 g of crude depolymerized heparin (sodium salt) are obtained. The 35 yield obtained is 65.3%. Purification: 3.5 g of crude depolymerized heparin obtained above and 35 ml of distilled water are placed in a 100 ml - 40 Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.6 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.18 ml of 5 aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 200C, the mixture is neutralized by adding 1N HCl, followed by addition of 3.5 g of sodium chloride. The solution is then filtered through a 10 0.45 pm membrane and 25 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 200C and stirred for 15 minutes. 63 ml of methanol are then added, followed by stirring for 30 minutes. Stirring is then stopped 15 and the suspension is left to sediment for 30 minutes. The supernatant is then separated out and discarded (70 ml) . 70 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate is left to resediment for a few 20 minutes. The suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with twice 30 ml of methanol. The wet solid is drained and then dried under reduced pressure at a . temperature in the region of 600C. After drying, 2.5 g 25 of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 71.4%. The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2600 daltons 30 anti-Xa activity: 150.5 IU/mg anti-IIa activity: 3.2 IU/mg anti-Xa activity/anti-IIa activity ratio: 47 EXAMPLE 10 35 Depolymerization and conversion to the sodium salt (0.8 equivalent of base): 70 ml of dichloromethane are placed in a Erlenmeyer flask A. 10 g (0.006 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) - 41 obtained as described in example A, are added slowly, with stirring. After total dissolution at a temperature in the region of 300C and cooling to a temperature in the- region of 200C, 1.38 ml (0.004 mol) of 2-tert 5 butylimino-2-diethylamino-1,3-dimethylperhydro-1,2,3 diazaphosphorine are added. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 30 g of anhydrous sodium acetate in 300 ml of methanol is prepared in a 10 Erlenmeyer flask B. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 15 seconds into the methanolic sodium. acetate solution at a temperature in the region of 4*C, with magnetic stirring. After stirring for 5 minutes, the suspension 15 is left to settle for 1 hour. The clear part of the supernatant is separated out and discarded (190 ml). 190 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 20 minutes. The clear part of the supernatant is separated out and discarded (190 ml). 190 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The supernatant is separated out and discarded (190 ml). The precipitate in 25 suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 150 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying for 18 hours, 30 3.05 g of crude depolymerized heparin are obtained, as the sodium salt. The yield obtained is 80%. Saponification: 3.05 g (0.0048 mol) of the sodium salt of crude 35 depolymerized heparin obtained above and 21 ml of water are placed in a 100 ml Erlenmeyer flask. The solution is filtered through a No. 3 sintered glass and rinsed with twice 6 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 0.6 ml (0.006 mol) - 42 of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 40C. After addition, the mixture is stirred for 2 hours. The solution is neutralized by adding 1N HCl and 4 g of sodium chloride 5 are added. 28 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 72 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 30 minutes at a temperature in the region 10 of 40C. The supernatant is then separated out and discarded (80 ml) . 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and 15 discarded (80 ml) . 80 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 30 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 50 ml of methanol. The wet 20 solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying, 1.6 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 57%. 25 Purification: 1.6 g of crude depolymerized heparin obtained above and 16 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.6 ± 0.1 by 30 addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.08 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of .200C, the mixture is neutralized by adding 35 1N HCl, followed by addition of 2 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 14 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 20 0 C and stirred for 15 minutes. 36 ml of - 43 methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 30 minutes. The supernatant is then-separated out and discarded (50 ml). 50 ml of 5 methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is. left to resediment for about 30 minutes. The supernatant is separated out and discarded (50 ml). The precipitate in suspension is then filtered through a 10 No. 3 sintered glass. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 400C. After drying, 1.25 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 78%. 15 The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2400 daltons anti-Xa activity: 154.3 IU/mg anti-IIa activity: 1.6 IU/mg 20 anti-Xa activity/anti-IIa activity ratio: 96.4 EXAMPLE 11 Depolymerization and conversion to the sodium salt: 140 ml of dichloromethane are placed in a Erlenmeyer 25 flask A. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. After total dissolution at a temperature in the region of 400C and cooling to a temperature in 30 the region of 200C, 3.5 ml (0.011 mol) of tert butyliminotri(pyrrolidino)phosphorane are added. The mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is 35 prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to the solution. The reaction mixture in the Erlenmeyer flask A is poured over 1 minute 30 seconds into the methanolic sodium acetate solution at a temperature in - 44 the region of 4'C, with magnetic stirring. After stirring for 5 minutes, the suspension is left to settle for 30 minutes. The clear part of the supernatant is separated out and discarded (400 ml). 5 400 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 1 hour 20 minutes. The supernatant is separated out and discarded (250 ml). 250 ml of methanol are added to the 10 sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate in suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is drained and then dried under 15 reduced pressure (6 kPa) at a temperature in the region of 40'C. After drying, 5.39 g of depolymerized heparin (benzyl ester, sodium salt) are obtained. The yield obtained is 69%. 20 Saponification: 5 g (0.0076 mol) of the depolymerized heparin (benzyl ester, sodium salt) obtained above and 35 ml of water are placed in a 50 ml Erlenmeyer flask. The solution is filtered through a No. 3 sintered glass and rinsed with 25 twice 10 ml of water. The filtrate obtained is placed in a 250 ml Erlenmeyer flask. 1 ml (0.01 mol) of 30% caustic soda is added with magnetic stirring, at a temperature in the region of 4 0 C. After addition, the mixture is stirred for 2 hours. The solution is 30 neutralized by adding 1N HCl and 6 g of sodium chloride are added. 42 ml of methanol are added to the reaction medium. After stirring for 15 minutes, 104 ml of methanol are added, followed by stirring for 15 minutes. Stirring is then stopped and the suspension is 35 left to sediment for 1 hour at a temperature in the region of 40C. The supernatant is then separated out and discarded (140 ml). 140 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment - 45 for about 45 minutes. The supernatant is separated out and discarded (160 ml). 160 ml of methanol are added to the sedimented precipitate and the mixture is stirred for- 5-minutes. The precipitate in suspension is then 5 filtered through a No. 3 sintered glass. The cake obtained is then washed with 100 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 40*C. After drying for 48 hours, 2.7 g of crude 10 depolymerized heparin (sodium salt) are obtained. The yield obtained is 59%. Purification: 2.6 g of crude depolymerized heparin obtained above and 15 25 ml of distilled water are placed in a 50 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.15 ml of 20 aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 200C, the mixture is neutralized by adding 1N HCl, followed by addition of 3 g of sodium chloride. The solution is then filtered through a 0.45 pm 25 membrane and 21 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 200C and stirred for 15 minutes. 54 ml of methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is 30 left to sediment for 20 minutes. The supernatant is then separated out and discarded (50 ml). 50 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 20 minutes. The 35 supernatant is separated out and discarded (50 ml) . The precipitate in suspension is then filtered through a No. 3 sintered glass. The white cake obtained is then washed with 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a - 46 temperature in the region of 400C. After drying for 18 hours, 2.35 g of pure depolymerized heparin (sodium salt) are obtained. The yield obtained is 90%. The- characteristics of the depolymerized heparin thus 5 obtained are as follows: mean molecular weight: 2400 daltons anti-Xa activity: 167.5 IU/mg anti-IIa activity: 1.1 IU/mg anti-Xa activity/anti-IIa activity ratio: 152 10 EXAMPLE 12 Depolymerization and conversion to the sodium salt (0.05% water): 140 ml of dichloromethane are placed in a Erlenmeyer 15 flask. 20 g (0.019 mol) of benzyl ester of heparin (degree of esterification: 75%, benzethonium salt) obtained as described in example A, are added slowly, with stirring. 20 g of 4 A molecular sieve are added to the reaction medium and the water content is brought to 20 0.05%, while stirring slowly for 48 h. The supernatant is transferred under an inert atmosphere to a Erlenmeyer flask A. After total dissolution, 3.5 ml (0.012 mol) of 2-tert-butylimino-2-diethylamino-1,3 dimethylperhydro-1,2,3-diazaphosphorine are added. The 25 mixture is stirred at a temperature in the region of 200C for 24 hours. During this time, a solution of 60 g of anhydrous sodium acetate in 600 ml of methanol is prepared in a Erlenmeyer flask B. After total dissolution, 10 g of Hyflo supercel Celite are added to 30 the solution. The reaction mixture in the Erlenmeyer flask A is poured over about 2 minutes into the methanolic sodium acetate solution at a temperature in the region of 40C, with magnetic stirring. After stirring for 15 minutes, the suspension is left to 35 settle for 1 hour. The clear part of the supernatant is separated out and discarded (420 ml). 420 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 15 minutes. The precipitate is left to resediment for about 1 hour. The supernatant - 47 is separated out and discarded (450 ml). 450 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 15 minutes. The suspension is -t-h-en filtered through a No. 3 sintered glass. The 5 cake obtained is then washed with 200 ml of methanol. The pale yellow wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 500C. After drying for 16 hours, 5.36 g of crude depolymerized heparin are obtained, as the sodium 10 salt in Celite (10 g). The yield obtained is 68.6%. Saponification: 5.36 g (0.00817 mol) of the sodium salt of crude depolymerized heparin in Celite (10 g) obtained above 15 and 50 ml of water are placed in a 50 ml Erlenmeyer flask. The suspension is filtered through a No. 3 sintered glass and rinsed with 4 times 15 ml of water. The filtrate obtained is placed in a 150 ml Erlenmeyer flask. 1.01 ml (0.0122 mol) of 35% caustic soda are 20 added with magnetic stirring, at a temperature in the region of 40C. After addition, the mixture is stirred for 3 hours. The solution is neutralized by adding 1N HCl and 11 g of sodium chloride are added. 77 ml of methanol are added to the reaction medium. After 25 stirring for 15 minutes, 200 ml of methanol are added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 1 hour at a temperature in the region of 4 0 C. The supernatant is then separated out and discarded 30 (240 ml). 240 ml of methanol are added to the sedimented precipitate and the mixture is stirred for 10 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and discarded (225 ml). 225 ml of methanol are added to the 35 sedimented precipitate and the mixture is stirred for 10 minutes. The suspension is then filtered through a No. 3 sintered glass. The cake obtained is then washed with twice 50 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a - 48 temperature in the region of 40 0 C. After drying for 18 hours, 2.65 g of crude depolymerized heparin (sodium salt) are obtained. The yield obtained is 53.7%. 5 Purification: 2.65 g of crude depolymerized heparin obtained above and 26.5 ml of distilled water are placed in a 100 ml Erlenmeyer flask. The mixture is brought to 400C with magnetic stirring. The pH is brought to 9.7 ± 0.1 by 10 addition of 1N sodium hydroxide. The reaction medium is filtered through a 0.45 pm membrane and 0.25 ml of aqueous 30% hydrogen peroxide solution is added. After stirring for about 2 hours at a temperature in the region of 20*C, the mixture is neutralized by adding 15 1N HCl, followed by addition of 3 g of sodium chloride. The solution is then filtered through a 0.45 pm membrane and 21 ml of methanol are then poured in, at a temperature in the region of 100C. The solution is then brought to 200C and stirred for 15 minutes. 54 ml of 20 methanol are then added, followed by stirring for 1 hour. Stirring is then stopped and the suspension is left to sediment for 45 minutes. The supernatant is then separated out and discarded (46 ml). 46 ml of methanol are added to the sedimented precipitate and 25 the mixture is stirred for 5 minutes. The precipitate is left to resediment for about 30 minutes. The supernatant is separated out and discarded (50 ml). 50 ml of methanol are added and the precipitate in suspension is then filtered through a No. 4 sintered 30 glass. The white cake obtained is then washed with 2 portions of 10 ml of methanol. The wet solid is drained and then dried under reduced pressure (6 kPa) at a temperature in the region of 500C. After drying for 18 hours, 2.363 g of pure depolymerized heparin (sodium 35 salt) are obtained. The yield obtained is 89.1%. The characteristics of the depolymerized heparin thus obtained are as follows: mean molecular weight: 2500 Daltons anti-Xa activity: 192 IU/mg - 49 anti-IIa activity: 1.3 IU/mg anti-Xa activity/anti-IIa activity ratio: 148
Claims (2)
- 2-tert-butylimino-2-diethylamino-1,3-dimethylperhydro 1, 3,2-diazaphosphorine or tert-butyliminotri (pyrrolidino)-phosphorane. 20 12) The method of preparation as claimed in claim 7 or 8, characterized in that the strong base/ester mol ratio is between 0.2 and 5, and preferably between 0.6 and 2. 25 13) A method for preparing oligosaccharides as claimed in any one of claims 1 to 6 from heparins, in which the following operations are carried out: a) transalification of sodium heparin by the action of 30 benzethonium chloride, b) esterification of benzethonium heparinate obtained by the action of benzyl chloride, c) transalification of the benzyl ester obtained and obtaining of the quaternary ammonium salt, - 53 d) depolymerization of the quaternary ammonium salt of the benzyl ester of heparin by the method as defined in claim 10 or 11, e) conversion of the quaternary ammonium salt to a 5 sodium salt, f) optionally saponification of the heparin by the action of a base such as sodium hydroxide, g) optionally purification in particular by the action of an oxidizing agent such as hydrogen peroxide. 10 14) The method as claimed in claim 13, characterized in that the reaction of step a) is carried out by the action of benzethonium chloride in excess, on sodium heparin, at a temperature in the region of 15 to 250C, 15 with a salt/sodium heparin molar ratio of between 3 and
- 4. 15) The method as claimed in claim 13, characterized in that the esterification of step b) is carried out in a 20 chlorinated organic solvent such as chloroform or methylene chloride, at a temperature of between 25 and 450C, preferably between 30 and 400C, and the ester in the form of a sodium salt is then recovered by . precipitation by means of sodium acetate at 10% by 25 weight in an alcohol such as methanol in a proportion of 1 to 1.2 volumes of alcohol per volume of reaction medium. 16) The method as claimed in claim 13 or 15, 30 characterized in that the degree of esterification of the quaternary ammonium salt of the benzyl ester of heparin is between 50 and 100%, and preferably between 70 and 90%. 35 17) The method as claimed in claim 13, 15 or 16, characterized in that 0.5 to 1.5 parts by weight of benzyl chloride per 1 part by weight of benzethonium salt of heparin are used with a reaction time which will be between 10 and 35 h. - 54 18) The method as claimed in claim 13, characterized in that the transalification of step c) is carried out by means-of a quaternary ammonium chloride, and preferably 5 by means of benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride, in an aqueous medium at a temperature between 10 and 25*C. 19) The method as claimed in claim 18, characterized in 10 that the quaternary ammonium chloride/sodium salt of the benzyl ester of heparin mol ratio is between 2 and 3. 20) The method as claimed in claim 13, characterized. in 15 that the conversion to a sodium salt of the quaternary ammonium salt of the benzyl ester of depolymerized heparin (step e) is carried out by treating the reaction medium with an alcoholic solution of sodium acetate, and preferably with a 10% solution of sodium 20 acetate in methanol (weight/volume), at a temperature between 15 and 250C. 21) The method as claimed in claim 13, characterized in that the saponification (step f) is carried out by 25 means of an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in an aqueous medium, at a temperature between 0 and 200C, and preferably 0 and 10C. 30 22) The method as claimed in claim 21, characterized in that 1 to 5 molar equivalents of alkali metal hydroxide and preferably 1 to 2 molar equivalents of sodium hydroxide are used. 35 23) The method as claimed in claim 13, characterized in that the purification (step g) is carried out by means of hydrogen peroxide, in an aqueous medium, at a temperature of 10 to 500C and preferably between 20 and 400C. - 55 24) As a medicament, the oligosaccharides as claimed in any one of claims 1 to 6. 5 25) As a medicament having an antithrombotic activity, the oligosaccharides as claimed in any one of claims 1 to 6. 26) The medicaments as claimed in claim 24 or 25, for 10 the prevention or treatment of venous and arterial thromboses, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, the proliferation of smooth 15 muscle cells, atheriosclerosis and arteriosclerosis, cancer by modulating angiogenesis and growth factors, and diabetic disorders such as diabetic retinopathies and nephropathies. 20 27) A pharmaceutical composition containing at least one medicament as defined in claim 24 and one or more pharmaceutically inert excipients or vehicles or additives. 25 28) The pharmaceutical composition as claimed in claim 27, characterized in that it consists of solutions for injection by the subcutaneous or intravenous route. 29) The pharmaceutical composition as claimed in claim 30 28, characterized in that it consists of a formulation for inhalation intended for the pulmonary route. 30) The pharmaceutical composition as claimed in claim 28, characterized in that it consists of a formulation 35 for administration intended for the oral route. 31) A mixture of polysaccharides as defined in any one of claims 1 to 6, which can be obtained by the method as defined in claim 13.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| FR0212584 | 2002-10-10 | ||
| FR0212584A FR2845686B1 (en) | 2002-10-10 | 2002-10-10 | MIXTURES OF HEPARIN-DERIVED POLYSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| PCT/FR2003/002960 WO2004033503A1 (en) | 2002-10-10 | 2003-10-08 | Heparin-derived polysaccharide mixtures, preparation thereof and pharmaceutical compositions containing same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| AU2003300161A1 AU2003300161A1 (en) | 2004-05-04 |
| AU2003300161B2 true AU2003300161B2 (en) | 2009-08-27 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| AU2003300161A Ceased AU2003300161B2 (en) | 2002-10-10 | 2003-10-08 | Heparin-derived polysaccharide mixtures, preparation thereof and pharmaceutical compositions containing same |
Country Status (35)
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| EP (1) | EP1556414B1 (en) |
| JP (2) | JP5021898B2 (en) |
| CN (6) | CN101817940A (en) |
| AT (1) | ATE548393T1 (en) |
| AU (1) | AU2003300161B2 (en) |
| BR (1) | BR0315149A (en) |
| CA (1) | CA2501546C (en) |
| CY (1) | CY1112808T1 (en) |
| DK (1) | DK1556414T3 (en) |
| EC (1) | ECSP055722A (en) |
| ES (1) | ES2383597T3 (en) |
| FR (1) | FR2845686B1 (en) |
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| HN (1) | HN2003000312A (en) |
| IL (1) | IL167868A (en) |
| MA (1) | MA27398A1 (en) |
| MX (1) | MXPA05003321A (en) |
| MY (1) | MY142376A (en) |
| NI (1) | NI200300087A (en) |
| NO (1) | NO20052170L (en) |
| NZ (1) | NZ539229A (en) |
| OA (1) | OA12940A (en) |
| PA (1) | PA8584201A1 (en) |
| PE (1) | PE20040507A1 (en) |
| PT (1) | PT1556414E (en) |
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| UA (1) | UA81925C2 (en) |
| UY (1) | UY28016A1 (en) |
| WO (1) | WO2004033503A1 (en) |
| ZA (1) | ZA200502787B (en) |
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|---|---|---|---|---|
| FR2845686B1 (en) * | 2002-10-10 | 2013-08-30 | Aventis Pharma Sa | MIXTURES OF HEPARIN-DERIVED POLYSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| FR2857971B1 (en) * | 2003-07-24 | 2005-08-26 | Aventis Pharma Sa | MIXTURES OF HEPARIN DERIVED OLIGOSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| CA2652205A1 (en) * | 2006-05-25 | 2007-12-06 | Mallik Sundaram | Low molecular weight heparin composition and uses thereof |
| WO2008068854A1 (en) | 2006-12-05 | 2008-06-12 | Glycoscience Laboratories, Inc. | Therapeutic agent for degenerative arthritis |
| MX2011008119A (en) | 2009-02-02 | 2011-08-24 | Otsuka Chemical Co Ltd | Low-molecular polysulfated hyaluronic acid derivative and medicine containing same. |
| EP2233145A1 (en) | 2009-03-19 | 2010-09-29 | Sanofi-Aventis | A dose of AVE5026 for the treatment of venous thromboembolism in patients with severe renal impairment |
| EP2399590A1 (en) | 2010-06-25 | 2011-12-28 | Aventis Pharma S.A. | Semuloparin for the prevention of a mortality and/or morbidity event in a patient undergoing major orthopaedic surgery |
| EP2399592A1 (en) | 2010-06-25 | 2011-12-28 | Aventis Pharma S.A. | Semuloparin for use as an antithrombotic treatment in hip replacement surgery with improved safety in terms of clinically relevant bleedings and major bleedings |
| EP2399591A1 (en) | 2010-06-25 | 2011-12-28 | Aventis Pharma S.A. | Semuloparin for the extended prevention of a mortality and/or morbidity event in a patient having undergone hip fracture surgery |
| EP2399593A1 (en) | 2010-06-28 | 2011-12-28 | Aventis Pharma S.A. | Semuloparin for use as an antithrombotic treatment in orthopaedic surgery with improved benefit-risk profile |
| JP2012046511A (en) * | 2010-07-30 | 2012-03-08 | Otsuka Chem Co Ltd | Pharmaceutical agent containing low molecular weight polysulfated hyaluronic acid derivative |
| CN103209997B (en) * | 2010-09-14 | 2016-03-16 | 国立大学法人宫崎大学 | high-purity heparin and preparation method thereof |
| EP2446891A1 (en) | 2010-10-28 | 2012-05-02 | Aventis Pharma S.A. | Semuloparin for use as an antithrombotic treatment in major abdominal surgery with improved safety in terms of clinically relevant bleedings and major bleedings |
| WO2012055843A1 (en) | 2010-10-28 | 2012-05-03 | Aventis Pharma S.A. | Semuloparin for the prevention of major venous thromboembolism in a patient undergoing major abdominal surgery |
| WO2012072799A1 (en) | 2010-12-02 | 2012-06-07 | Aventis Pharma S.A. | New methods for the in vitro measurement of the biological activity of an ultra low molecular weight heparin sample |
| AR085961A1 (en) * | 2011-04-11 | 2013-11-06 | Sanofi Sa | POLISACARIDS THAT HAVE TWO SITES OF UNION TO ANTHROMBIN III, METHOD TO PREPARE THEM AND PHARMACEUTICAL COMPOSITIONS THAT CONTAIN THEM |
| EP2548561A1 (en) | 2011-07-22 | 2013-01-23 | Aventis Pharma S.A. | Semuloparin for improving the survival of patients with locally advanced cancer |
| WO2013044793A1 (en) * | 2011-09-26 | 2013-04-04 | Xu Meiying | High-purity heparin benzyl ester salt, preparation method therefor and application thereof |
| CN102633908A (en) * | 2012-05-02 | 2012-08-15 | 雷晓刚 | Method for preparing high-quality LMW (low molecular weight) heparins |
| ES2445494B1 (en) * | 2012-08-02 | 2015-03-06 | Rovi Lab Farmaceut Sa | Procedure for obtaining low and very low molecular weight heparins |
| US9873712B2 (en) * | 2014-10-03 | 2018-01-23 | Amphastar Pharmaceuticals, Inc. | Method of purifying idraparinux sodium |
| EP3398971A4 (en) * | 2015-12-30 | 2019-09-25 | Shenzhen Hepalink Pharmaceutical Group Co., Ltd. | Sulfated heparin oligosaccharide and preparation method and application thereof |
| CZ308106B6 (en) * | 2016-06-27 | 2020-01-08 | Contipro A.S. | Unsaturated derivatives of polysaccharides, preparing and using them |
| CN110917208B (en) * | 2019-12-27 | 2021-02-12 | 浙江医院 | Application of sulfated mannoglucuronhexaose in preventing and treating atherosclerosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| IL61201A (en) * | 1979-10-05 | 1984-09-30 | Choay Sa | Oligosaccharides having no more than 8 saccharide moieties,their obtention from heparin and pharmaceutical compositions containing them |
| FR2482611B1 (en) * | 1980-05-14 | 1986-03-07 | Pharmindustrie | NOVEL SULFATED POLYSACCHARIDES, METHODS FOR THEIR PREPARATION AND THEIR USE AS MEDICAMENTS |
| FR2504928A1 (en) * | 1981-04-29 | 1982-11-05 | Choay Sa | SHORT CHAIN OLIGOSACCHARIDES HAVING BIOLOGICAL PROPERTIES, PREPARATION THEREOF AND APPLICATIONS THEREOF AS MEDICAMENTS |
| FR2584606A1 (en) * | 1985-07-12 | 1987-01-16 | Dropic | USE OF POLY- AND OLIGOSACCHARIDES FOR THE PRODUCTION OF ACTIVE MEDICAMENTS IN THE PATHOLOGIES OF CONNECTIVE TISSUE |
| FR2584728B1 (en) * | 1985-07-12 | 1987-11-20 | Choay Sa | PROCESS FOR THE SULFATION OF GLYCOSAMINOGLYCANS AND THEIR FRAGMENTS |
| JP4897991B2 (en) * | 1999-07-23 | 2012-03-14 | ラボラトリオス ファルマセウティコス ロビ ソシエダッド アノニマ | Ultra low molecular weight heparin composition |
| ES2161615B1 (en) * | 1999-07-23 | 2003-03-16 | Rovi Lab Farmaceut Sa | COMPOSITIONS OF HEPARINS OF VERY LOW MOLECULAR WEIGHT. |
| FR2807043B1 (en) * | 2000-03-28 | 2002-11-22 | Aventis Pharma Sa | PHARMACEUTICAL COMPOSITION CONTAINING OLIGOSACCHARIDES, NEW OLIGOSACCHARIDES AND THEIR PREPARATION |
| FR2811992B1 (en) * | 2000-07-21 | 2003-07-04 | Aventis Pharma Sa | MIXTURES OF HEPARIN-DERIVED POLYSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| FR2845686B1 (en) * | 2002-10-10 | 2013-08-30 | Aventis Pharma Sa | MIXTURES OF HEPARIN-DERIVED POLYSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
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