ZA200600657B - Heparin-derived oligosaccharide mixtures, preparations thereof and pharmaceutical compositions containing said mixtures - Google Patents
Heparin-derived oligosaccharide mixtures, preparations thereof and pharmaceutical compositions containing said mixtures Download PDFInfo
- Publication number
- ZA200600657B ZA200600657B ZA200600657A ZA200600657A ZA200600657B ZA 200600657 B ZA200600657 B ZA 200600657B ZA 200600657 A ZA200600657 A ZA 200600657A ZA 200600657 A ZA200600657 A ZA 200600657A ZA 200600657 B ZA200600657 B ZA 200600657B
- Authority
- ZA
- South Africa
- Prior art keywords
- activity
- molecular weight
- mixture
- oligosaccharide mixture
- vlmwh
- Prior art date
Links
- 239000000203 mixture Substances 0.000 title claims description 166
- 229920001542 oligosaccharide Polymers 0.000 title claims description 54
- 150000002482 oligosaccharides Chemical class 0.000 title claims description 53
- 229920000669 heparin Polymers 0.000 title claims description 41
- 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 description 40
- 238000002360 preparation method Methods 0.000 title claims description 26
- 229960002897 heparin Drugs 0.000 title claims description 19
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 10
- OKKJLVBELUTLKV-UHFFFAOYSA-N methanol Natural products OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 275
- 239000000243 solution Substances 0.000 claims description 91
- 238000000034 method Methods 0.000 claims description 79
- 230000000694 effects Effects 0.000 claims description 75
- -1 alkaline-earth metal salt Chemical class 0.000 claims description 64
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 60
- 159000000000 sodium salts Chemical class 0.000 claims description 57
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 50
- 229940127215 low-molecular weight heparin Drugs 0.000 claims description 39
- 239000003055 low molecular weight heparin Substances 0.000 claims description 34
- 230000001858 anti-Xa Effects 0.000 claims description 31
- 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 30
- 230000032050 esterification Effects 0.000 claims description 29
- 238000005886 esterification reaction Methods 0.000 claims description 29
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 24
- 239000001632 sodium acetate Substances 0.000 claims description 23
- 235000017281 sodium acetate Nutrition 0.000 claims description 23
- 238000006243 chemical reaction Methods 0.000 claims description 19
- 239000002585 base Substances 0.000 claims description 17
- 150000003242 quaternary ammonium salts Chemical class 0.000 claims description 17
- 239000011734 sodium Substances 0.000 claims description 16
- GKTNLYAAZKKMTQ-UHFFFAOYSA-N n-[bis(dimethylamino)phosphinimyl]-n-methylmethanamine Chemical class CN(C)P(=N)(N(C)C)N(C)C GKTNLYAAZKKMTQ-UHFFFAOYSA-N 0.000 claims description 12
- 238000000746 purification Methods 0.000 claims description 12
- 238000007127 saponification reaction Methods 0.000 claims description 11
- 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
- 238000004519 manufacturing process Methods 0.000 claims description 10
- 229960003872 benzethonium Drugs 0.000 claims description 8
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical compound ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 claims description 8
- 229940073608 benzyl chloride Drugs 0.000 claims description 8
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 8
- 206010047249 Venous thrombosis Diseases 0.000 claims description 7
- 239000007858 starting material Substances 0.000 claims description 7
- 108010074860 Factor Xa Proteins 0.000 claims description 6
- 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
- 150000008044 alkali metal hydroxides Chemical class 0.000 claims description 6
- 150000002016 disaccharides Chemical class 0.000 claims description 6
- 150000002148 esters Chemical class 0.000 claims description 6
- 229920002401 polyacrylamide Polymers 0.000 claims description 6
- 239000012429 reaction media Substances 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
- 229910052783 alkali metal Inorganic materials 0.000 claims description 5
- 150000001340 alkali metals Chemical class 0.000 claims description 5
- 229910052784 alkaline earth metal Inorganic materials 0.000 claims description 5
- 229960000610 enoxaparin Drugs 0.000 claims description 5
- 229940126601 medicinal product Drugs 0.000 claims description 5
- 229910052708 sodium Inorganic materials 0.000 claims description 5
- 150000004044 tetrasaccharides Chemical class 0.000 claims description 5
- 229920000936 Agarose Polymers 0.000 claims description 4
- 201000001320 Atherosclerosis Diseases 0.000 claims description 4
- 206010003658 Atrial Fibrillation Diseases 0.000 claims description 4
- 206010051055 Deep vein thrombosis Diseases 0.000 claims description 4
- 208000007342 Diabetic Nephropathies Diseases 0.000 claims description 4
- 206010012689 Diabetic retinopathy Diseases 0.000 claims description 4
- 206010028980 Neoplasm Diseases 0.000 claims description 4
- 208000010378 Pulmonary Embolism Diseases 0.000 claims description 4
- 230000033115 angiogenesis Effects 0.000 claims description 4
- 239000002870 angiogenesis inducing agent Substances 0.000 claims description 4
- 230000002785 anti-thrombosis Effects 0.000 claims description 4
- 239000012736 aqueous medium Substances 0.000 claims description 4
- 210000001367 artery Anatomy 0.000 claims description 4
- 201000011510 cancer Diseases 0.000 claims description 4
- 230000004663 cell proliferation Effects 0.000 claims description 4
- 238000004587 chromatography analysis Methods 0.000 claims description 4
- 239000000470 constituent Substances 0.000 claims description 4
- 206010012601 diabetes mellitus Diseases 0.000 claims description 4
- 208000033679 diabetic kidney disease Diseases 0.000 claims description 4
- 201000010099 disease Diseases 0.000 claims description 4
- 208000035475 disorder Diseases 0.000 claims description 4
- 239000003102 growth factor Substances 0.000 claims description 4
- 208000010125 myocardial infarction Diseases 0.000 claims description 4
- 230000002093 peripheral effect Effects 0.000 claims description 4
- 210000000329 smooth muscle myocyte Anatomy 0.000 claims description 4
- CTSLUCNDVMMDHG-UHFFFAOYSA-N 5-bromo-3-(butan-2-yl)-6-methylpyrimidine-2,4(1H,3H)-dione Chemical compound CCC(C)N1C(=O)NC(C)=C(Br)C1=O CTSLUCNDVMMDHG-UHFFFAOYSA-N 0.000 claims description 3
- 206010002388 Angina unstable Diseases 0.000 claims description 3
- 206010003178 Arterial thrombosis Diseases 0.000 claims description 3
- 206010003210 Arteriosclerosis Diseases 0.000 claims description 3
- 208000007814 Unstable Angina Diseases 0.000 claims description 3
- 230000001476 alcoholic effect Effects 0.000 claims description 3
- 230000003024 amidolytic effect Effects 0.000 claims description 3
- 208000011775 arteriosclerosis disease Diseases 0.000 claims description 3
- 239000000872 buffer Substances 0.000 claims description 3
- NEUSVAOJNUQRTM-UHFFFAOYSA-N cetylpyridinium Chemical compound CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 NEUSVAOJNUQRTM-UHFFFAOYSA-N 0.000 claims description 3
- 229960004830 cetylpyridinium Drugs 0.000 claims description 3
- RLGQACBPNDBWTB-UHFFFAOYSA-N cetyltrimethylammonium ion Chemical class CCCCCCCCCCCCCCCC[N+](C)(C)C RLGQACBPNDBWTB-UHFFFAOYSA-N 0.000 claims description 3
- 239000003593 chromogenic compound Substances 0.000 claims description 3
- 229940087051 fragmin Drugs 0.000 claims description 3
- 150000004676 glycans Chemical class 0.000 claims description 3
- 229940095443 innohep Drugs 0.000 claims description 3
- 201000004332 intermediate coronary syndrome Diseases 0.000 claims description 3
- 208000031225 myocardial ischemia Diseases 0.000 claims description 3
- 150000007530 organic bases Chemical class 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 239000005017 polysaccharide Substances 0.000 claims description 3
- SYRHIZPPCHMRIT-UHFFFAOYSA-N tin(4+) Chemical compound [Sn+4] SYRHIZPPCHMRIT-UHFFFAOYSA-N 0.000 claims description 3
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 2
- 239000008118 PEG 6000 Substances 0.000 claims description 2
- 229920002584 Polyethylene Glycol 6000 Polymers 0.000 claims description 2
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 2
- 239000002253 acid Substances 0.000 claims description 2
- 229910052791 calcium Inorganic materials 0.000 claims description 2
- 239000011575 calcium Substances 0.000 claims description 2
- 150000001720 carbohydrates Chemical group 0.000 claims description 2
- 125000004432 carbon atom Chemical group C* 0.000 claims description 2
- 238000012691 depolymerization reaction Methods 0.000 claims description 2
- 238000010253 intravenous injection Methods 0.000 claims description 2
- 159000000003 magnesium salts Chemical class 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 229910052700 potassium Inorganic materials 0.000 claims description 2
- 239000011591 potassium Substances 0.000 claims description 2
- 230000002685 pulmonary effect Effects 0.000 claims description 2
- 238000010254 subcutaneous injection Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims 6
- 230000002265 prevention Effects 0.000 claims 4
- 238000009472 formulation Methods 0.000 claims 2
- 206010002383 Angina Pectoris Diseases 0.000 claims 1
- 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 1
- 239000004698 Polyethylene Substances 0.000 claims 1
- 239000000654 additive Substances 0.000 claims 1
- 150000003863 ammonium salts Chemical class 0.000 claims 1
- 230000000747 cardiac effect Effects 0.000 claims 1
- 125000000623 heterocyclic group Chemical group 0.000 claims 1
- 208000028867 ischemia Diseases 0.000 claims 1
- 229920000573 polyethylene Polymers 0.000 claims 1
- 238000006116 polymerization reaction Methods 0.000 claims 1
- 239000003981 vehicle Substances 0.000 claims 1
- 239000006228 supernatant Substances 0.000 description 50
- 239000013049 sediment Substances 0.000 description 48
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 32
- 239000000725 suspension Substances 0.000 description 30
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 22
- 239000012528 membrane Substances 0.000 description 17
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 16
- 239000011541 reaction mixture Substances 0.000 description 16
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 15
- 239000007787 solid Substances 0.000 description 15
- 238000003756 stirring Methods 0.000 description 12
- 239000011780 sodium chloride Substances 0.000 description 11
- 239000002244 precipitate Substances 0.000 description 9
- 108090000935 Antithrombin III Proteins 0.000 description 8
- 150000003839 salts Chemical class 0.000 description 8
- 235000017557 sodium bicarbonate Nutrition 0.000 description 8
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 8
- 102100022977 Antithrombin-III Human genes 0.000 description 7
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 7
- 229960000583 acetic acid Drugs 0.000 description 7
- 239000000047 product Substances 0.000 description 7
- 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 description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 6
- 239000012362 glacial acetic acid Substances 0.000 description 6
- 239000002609 medium Substances 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000000926 separation method Methods 0.000 description 4
- 239000012300 argon atmosphere Substances 0.000 description 3
- 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 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 238000005194 fractionation Methods 0.000 description 2
- IIPYXGDZVMZOAP-UHFFFAOYSA-N lithium nitrate Chemical compound [Li+].[O-][N+]([O-])=O IIPYXGDZVMZOAP-UHFFFAOYSA-N 0.000 description 2
- YRNOSHBJMBLOSL-UHFFFAOYSA-N n-[tert-butylimino-bis(dimethylamino)-$l^{5}-phosphanyl]-n-methylmethanamine Chemical compound CN(C)P(N(C)C)(N(C)C)=NC(C)(C)C YRNOSHBJMBLOSL-UHFFFAOYSA-N 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000000870 ultraviolet spectroscopy Methods 0.000 description 2
- WFIYPADYPQQLNN-UHFFFAOYSA-N 2-[2-(4-bromopyrazol-1-yl)ethyl]isoindole-1,3-dione Chemical compound C1=C(Br)C=NN1CCN1C(=O)C2=CC=CC=C2C1=O WFIYPADYPQQLNN-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- 102000004411 Antithrombin III Human genes 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 150000001342 alkaline earth metals Chemical class 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
- 238000003556 assay Methods 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 229960001927 cetylpyridinium chloride Drugs 0.000 description 1
- YMKDRGPMQRFJGP-UHFFFAOYSA-M cetylpyridinium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+]1=CC=CC=C1 YMKDRGPMQRFJGP-UHFFFAOYSA-M 0.000 description 1
- WOWHHFRSBJGXCM-UHFFFAOYSA-M cetyltrimethylammonium chloride Chemical compound [Cl-].CCCCCCCCCCCCCCCC[N+](C)(C)C WOWHHFRSBJGXCM-UHFFFAOYSA-M 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-M hydroxide Chemical compound [OH-] XLYOFNOQVPJJNP-UHFFFAOYSA-M 0.000 description 1
- 125000001841 imino group Chemical group [H]N=* 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 230000014508 negative regulation of coagulation Effects 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 229940093429 polyethylene glycol 6000 Drugs 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- XOFYZVNMUHMLCC-ZPOLXVRWSA-N prednisone Chemical compound O=C1C=C[C@]2(C)[C@H]3C(=O)C[C@](C)([C@@](CC4)(O)C(=O)CO)[C@@H]4[C@@H]3CCC2=C1 XOFYZVNMUHMLCC-ZPOLXVRWSA-N 0.000 description 1
- 238000002953 preparative HPLC Methods 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
- ILXAOQAXSHVHTM-UHFFFAOYSA-M sodium;2-amino-2-(hydroxymethyl)propane-1,3-diol;chloride Chemical compound [Na+].[Cl-].OCC(N)(CO)CO ILXAOQAXSHVHTM-UHFFFAOYSA-M 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 229940061706 sulfated mucopolysaccharides Drugs 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
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
-
- 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
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/04—Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
- A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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Description
® ® WO 20057010051 -1- PCT/FR2004/001943
HEPARIN-DERIVED OLIGOSACCHARIDE MIXTURES, PREPARATION
THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING SAID
MIXTURES
The present invention relates to oligosaccharide mixtures derived from heparin, with an average molecular weight of from 1800 to 2400 daltons, characterized by high anti-Xa (aXa) activity and an absence of anti-IIa (alla) activity, to a process for preparing them and to pharmaceutical compositions containing them.
Heparin is a mixture of sulfated mucopolysaccharides of animal origin, used especially for its anticoagulant and antithrombotic properties.
However, heparin has drawbacks that limit its conditions of use. In particular, its substantial anticoagulant activity (aIla) can cause hemorrhaging (Seminars in Thrombosis and Hemostasis, vol 5 sup. 3 (1999)).
Low molecular weight heparins, obtained especially by basic depolymerization of heparin esters, which are currently marketed, such as Enoxaparin, also show substantial alla activity.
More recently, very low molecular weight heparins have been described in the prior art. For example, in patent
US 6 384 021, the products show anti-Xa activity of between 100 and 120 IU/mg and anti-IIa activity of between 2 and 8 IU/mg. In the international applications WO 02/08295 and WO 2004/033503, the products have anti-Xa activities which are especially between 100 and 190 IU/mg for anti-IIa activities of less than 5 IU/mg. However, none of these very low molecular weight heparins effectively has anti-Xa activity of greater than 190 IU/mg while at the same time having no, or virtually no, anti-IIa activity. (IU = International Unit)
The expression “virtually no anti-IIa activity” (in other words, showing virtually no anti-IIa activity) means an activity of less than 0.2 IU/mg.
One subject of the invention is oligosaccharide mixtures with very selective activity toward activated factor X (factor Xa) while at the same time showing no, or virtually no, anti-IIa activity.
One subject of the present invention is thus oligosaccharide mixtures having the general structure of the constituent polysaccharides of heparin and having the following characteristics: - they have an average molecular weight of from 1800 to 2400 daltons, anti-Xa activity of between 190 IU/mg and 450 IU/mg and have no, or virtually no, anti-IIla activity, - the constituent oligosaccharides of the mixtures - contain from 2 to 1l€ saccharide units, - have a 4,5-unsaturated uronic acid 2-0O-sulfate unit at one of their ends, - and contain the hexasaccharide of the following formula:
® » he
Na Na Na o) 0 o 0=570 0=5=0 0=570
Na O 0 Na oO o} Na wa 0 0} 0 0} 0] 0 0} “oH “NH “oH 0. NH 0 © 0 NH — 0 SPC SI 0 Na O u a 0 Na ©O
Alla Hs Is in the form of an alkali metal or alkaline-earth metal salt.
The hexasaccharide AIIa-IIs-Is contained in the oligosaccharide mixture described in the present invention is a sequence with high affinity for ATIII and 1s characterized by aXa activity of greater than 740 IU/mg.
Alkali metal or alkaline-earth metal salts that are preferred are the sodium, potassium, calcium and magnesium salts.
The average molecular weight 1s determined by high- pressure liquid chromatography using two columns in series, for example those sold under the name
TSK G3000 XL and TSK G2000 XL. The detection is performed by refractometry. The eluent used is lithium nitrate and the flow rate is 0.6 ml/minute. The system is calibrated with standards prepared by fractionation of Enoxaparin by chromatography on agarose- polyacrylamide gel (IBF). This preparation is performed 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 using the GPC6t software (Perkin Elmer).
® oC
The anti-Xa activity 1s measured via the amidolytic method on a chromogenic substrate according to the principle described by Teien et al, Thromb. Res., 10, 399-410 (1977). The assays are performed according to the method described in the monograph on low molecular weight heparins of the European pharmacopea in force, except for the reconstitution buffer : the alubumin in the tris-NaCl buffer, pH 7.4, is replaced aby polyethylene glycol 6000(PEG 6000). The anti Xa activity 1s measured relative to a standard Very Low
Molecular Weight Heparin (VLMWH) which measures from 140 to 180 U/mg (dry weight)
The activity of the standard VLMWH is measured relataive to the international low molecular weight heparin standard. This standard VLMWH was prepared according to the teaching of patent applications
WO WO 02/08 295 and in particular of WO 2004/033503.
The activity of the standard VLMWH is measured relative to the international low molecular weight heparin standard.
The anti-IIa activity is measured via the amidolytic method on a chromogenic substrate according to the method described in the moncgraph on low molecular weight heparins of the Zuropean pharmacopea in force.
The alla activities are measured relative to a standard
Very Low Molecular Weight Heparin (VLMWH) with a measured activity of 2.1 IU/mg. The activity of the standard VLMWH is measured relative to the international low molecular weight heparin standard.
According to one preferred embodiment, the oligosaccharide mixture according tc the invention contains from 20% to 100% of a hexasaccharide fraction.
In particular, this mixture contains from 30% to 60% of hexasaccharide fraction.
_ ®
Moreover, the mixtures according to the invention contain from 20% to 70% of the hexasaccharide AIIa-IIs-
Is in the hexasaccharide fraction of the oligosaccharide mixture. In particular, this fraction
Alla-IIs-Is is present in the hexasaccharide fraction to a proportion of 25% to 50%.
The percentage of the hexasaccharide fraction may be determined analytically by high-pressure liquid chromatography on TSK G3000 XL and TSK G2000 XL columns or by preparative separation of the hexasaccharide fraction.
In this case, the mixture is chromatographed on columns filled with gel of polyacrylamide agarose type. The mixture 1is eluted with a sodium hydrogen carbonate solution. Preferably, the sodium hydrogen carbonate solution is a solution of from 0.1 mol/l to 1 mol/l.
Even more preferably, the separation is performed at a concentration of 1 mol/l. The detection is performed by
UV spectrometry (254 nm). After fractionation, the hexasaccharide fraction in solution in the 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 of greater than 30% by weight. The hexasaccharide fraction 1s precipitated by addition of 3 to 5 volumes of methanol. The hexasaccharide fraction is recovered by filtration through a No. 3 sinter funnel. The hexasaccharide mixture obtained may be analyzed by high performance liquid chromatography (HPLC) to determine its content of hexasaccharide AIIa-IIs-Is. The hexasaccharide AIla-IIs-Is may be isolated by preparative HPLC or by affinity chromatography on an antithrombin III sepharose column according to the techniques used by those skilled in the art (M. Hook,
I. Bjork, J. Hopwood and U. Lindahl, F.E.B.S letters, vol 656(1) (1976)).
®
Preferably, the mixtures according to the invention have an average molecular weight of between 1900 and 2200 daltons and in particular from 1950 to 2150 daltons.
According to one preferred embodiment, the oligosaccharide mixture according to the invention is characterized in that it has anti-Xa activity of between 190 IU/mg and 410 IU/mg and no, or virtually no, anti-IIa activity. Most particularly, the anti-Xa activity is between 200 and 300 IU.
A subject of the invention is thus, most particularly, mixtures having the following characteristics: - an average molecular weight of between 1950 and 2150 daltons, - anti-Xa activity of between 190 IU/mg and 410 IU/mg and no, or virtually no, anti-IIa activity, - they contain from 30 to 60% of hexasaccharide fraction, which contains from 25% to 55% of AIla-IIs-Is fraction.
The activity of the oligosaccharide mixtures according to the invention is obtained by means of a very particular process that is described hereinbelow. It is well known to those skilled in the art that the physicochemical characteristics of polysaccharide mixtures and the activity deriving therefrom are linked to the production process (J. Med. Chem. 33(6) 1639- 2093 (19%0)).
The oligosaccharide mixtures according to the invention are prepared by depolymerization of a quaternary ammonium salt of the benzyl ester of a Very Low
Mclecular Weight Heparin (VLMWH) in organic medium, this (VLMWH) itself being prepared according to the
_ ® nT teaching of patent applications WO 02/08285 and
WO 2004/033503. It is generally a matter of re- depolymerizing a very low molecular weight heparin which has itself been specifically obtained by depolymerization of esterified heparin in the presence of a strong base, preferably in dichloromethane, and in the presence of a percentage of water of less than 3%.
The VLMWHs used as starting material in this invention were prepared in particular according to the processes described in patent applications WO 02/08295 and
WO 2004/033503.
The VLMWHs used as starting material especially have aXa activity of greater than 140 IU/mg, alla activity of less than 5 IU/mg and average molecular masses of between 2000 and 3000 daltons. The aXa activities are measured relative to a standard VLMWH with a measured activity of 158 IU/mg. The activity of the standard
VLMWH is measured relative to the international low molecular weight heparin standard.
The starting VLMWHs obtained according to the process as described above are re-depolymerized using a strong organic base with a pKa value preferably of (greater than ZC (preferred phcsprazene-family-related properties defined, for example, by Schwesinger et al,
Angew. Chem. Int. Ed. Engl. 26, 1167-1169 (1987) or R.
Schwesinger et al, Angew. Chem. 105, 1420 (19%3)).
Next, the quaternary ammonium salt of the benzyl ester of the depolymerized VLMWH is converted into the sodium salt, the residual esters are saponified and the product obtained is optionally purified. The reaction scheme below illustrates the present invention:
- 8 =~
OOM (SO, Na 0 0 ~~ H20 TA a—En >a om | + ~ONS _ 0 -
OISONa)Y NHSO MaiZ “ Step a
VLMWH " benzethonum chioride me-4 (Hy Cr) n= X+Y+Z (overall degree of sulfatation of the mean disaccharide)
X= degree of sulfatation of the site, the remainder is represented by the H radical
Y=degree of sutfatation of the site, the remainder is represented by the H radical
Z=degree of sulfatation of the site, the remainder is represented by the COCHa radical a On OB O(SO Max On OAD CSO Nex e 0 0 0 1 .CHCL ° Ooh PuriBcation .A40 LJ) SR YVVN nb O50, NY NNSONOIZ |, OISO,NalY NHSONaIZ_ |r, ep be degree of esterification b= degree of esterificaton
Crude benzyl ester Pure benzyl ester, sodium salt v Oa OBO® _ O(SO,HyN
Pure benzyl ester, Ye PL Tram ° ] > 0~h sodium sak ° a” Step ©
Benzethonium chloride ep OSOHYIY NH(SOHYIZ . (wen) "
Pure benzyl ester, benzethonum sat rd
CiLk v TE ono TORE om ° 0 ° Saponification 0 0 o] {o] H NaOH «°C swore ET) pikinblaiiind - 2 O(SO;NajY O(SO,Na)Y NASON
Step dande Crude cigosacchan . m=-~3
Oa ONS QC Nap ONa (30 Na)X Du ONe O50 Nake OMe usoMext ° ° ° H202 0 o 0 o-+—OH 0 He fo) 0 lo] H
O(SO,N8)Y NY(SOa) O(SONe)Y NH(SO,Ne)Z Swp 2 3 Ne s ™ O(SO,Na)Y NSO OfSO,Na)Y NH(SONa)2 m=~3 nea
Crude oligosaccharide modures
Pure oligosaccharide mixtures
A subject of the invention 1s thus also a process for preparing the oligosaccharide mixtures as defined above, wherein a very low molecular weight heparin with aXa activity of greater than 140 IU/mg, alla activity of less than 5 IU/mg and an average molecular mass of between 2000 and 3000 daltons is subjected to the following chemical reactions: a) transsalification by the action of benzethonium { chloride to obtain benzethonium heparinate,
D) esterification o©f the Dbenzethonium heparinate obtained by the action of benzyl chloride, and treatment with alcoholic sodium acetate solution
® ® to obtain the sodium salt of the benzyl ester of the very low molecular weight heparin, c) transsalification of the benzyl ester obtained and production of the quaternary ammonium salt, preferably as the benzethonium, cetylpyridinium or cetyltrimethylammonium salt, d) depolymerization by means of a strong organic base with a pKa value preferably of greater than 20, so as to obtain a depolymerized very low molecular weight heparin, e) conversion of the quaternary ammonium salt of the depolymerized very low molecular weight heparin into the sodium salt, f) saponification of the residual esters and optional purification.
In the present invention, the high selectivity of the phosphazene base during the depolymerization step (step d) is most particularly used to enrich, unexpectedly, the oligosaccharide mixture in sequences with affinity for ATIII. Preferably, the strong base/ester mole ratio is between 0.2 and 5 and more particularly between 0.6 and 2.
For optimum selectivity and maximum preservation of the sequences with affinity for ATIII, it is preferable tc work at water contents of less than 0.3% when working with 1 molar equivalent of phosphazene base relative to the benzyl ester of the VLMWH, benzethonium salt.
The bases of the phosphazene family are preferably those of formula: ‘
_ ® - 10 -
R3
Ra—N R4 \ /
R1—N=—=P-—N—RS5 \
N—R6 rR? in which the radicals R; to Ry; which are identical or different, represent linear, branched or cyclic alkyl radicals containing from 1 to 6 carbon atoms it being possible for R; and Ry, where appropriate, to form with the -N-P-N-group which carries them, a 6-membered } heterocycle. In particular, one subject of the invention is the process as defined above, wherein the base used in the depolymerization step d) is 2-tert- butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2- diazaphosphorine (official momenclature: 1,3,2- diazaphosphorin-2-amine, 2-((l,1-dimethylethyl) imino] -
N,N-diethyl-1,2,2,2,3,5,6-octahydro-1, 3-dimethyl).
The reaction of the transsalification step a) is preferably performed by the action of excess benzethonium chloride on the sodium VLMWH, at a temperature in the region of 15 to 25°C.
Advantageously, the salt/sodium heparin mole ratio is between 2.5 and 3.5.
The esterification step b) is preferably performed in an organic chlorinated solvent (such as chloroform or dichloromethane), at a temperature of between 25 and 45°C and preferably between 30 and 40°C. The ester in the form of the sodium salt 1s then recovered by precipitation using 10% by weight of sodium acetate in an alcohol such as methanol. 1 to 1.2 volumes of alcohol per volume of reaction medium are generally f used. The amount of benzyl chloride and the reaction time are adapted to obtain a degree of esterification of between 40% and 100% and preferably between 70% and
® - 11 - 90%. 0.5 to 1.5 parts by weight of benzyl chloride per 1 part by weight of the benzethonium salt of heparin are preferably used. Similarly, the reaction time will preferably be between 10 and 35 hours.
Consequently, the process according to the invention uses a degree of esterification of the quaternary ammonium salt of the benzyl ester of heparin of between 40% and 100% and preferably between 70 and 90%.
The conversion of the quaternary ammonium salt of the benzyl ester of the depolymerized heparin into the sodium salt is generally performed by treating the reaction medium with alcoholic sodium acetate solution and preferably with a 10% solution of sodium acetate in methanol (weight/volume), at a temperature of between 15 and 25°C.
The weight equivalent of acetate added is preferably 3 times as great as the mass of quaternary ammonium salt of the benzyl ester of heparin subsequently used in the depolymerization reaction. The quaternary ammonium salt of the benzyl ester of the VLMWH obtained is preferably the benzethonium, cetylpyridinium or cetyltrimethylammonium salt.
The transsalification step cc) 1s performed using a quaternary ammonium chloride, preferably using benzethonium chloride, cetylpyridinium chloride or cetyltrimethylammonium chloride, in aqueous medium, at a temperature of between 10 and 25°C. Advantageously, the quaternary ammonium chloride/sodium salt of the benzyl ester of heparin mole ratio is between 2.5 and 3.5. ‘
The saponification 1s generally performed using an : alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in aqueous medium, at a temperature of between 0 and 20°C and
®
PY - 12 - preferably between 0 and 10°C. From 1 to 5 molar equivalents of alkali metal hydroxide will generally be used. The saponification will preferably be performed in the presence of 1 to 2 molar equivalents of alkali metal hydroxide.
The final product may optionally be purified by any known method for purifying depolymerized heparins (for example EP 0 037 319 Bl). Preferably, the purification is performed using hydrogen peroxide, in aqueous medium, at a temperature of from 10 to 50°C. This operation will preferably be performed at between 20 and 40°C.
The mixtures according to the invention in sodium salt form may be converted into a salt of another alkali metal or alkaline-earth metal. The conversion from one salt to the other is optionally performed using the method described in patent FR 73/13580.
The present invention especially allows a high enrichment in hexasaccharide AIIa-IIs-Is. When a low molecular weight heparin is re-depolymerized by the process that generated it, it is well known to those skilled in the art that the anti-Xa activity cf the product obtained decreases greatly until it is zero. In the case where the processes for cbtaining Enoxaparin,
Fraxiparin, Fragmin, Innohep (or Logiparin), Normiflo,
Embollex (cr Sandoparin), Fluxum (or Minidalton),
Clivarin and Hibor are used, this phenomenon may be observed if these LMWHs are re-depolymerized with their original process. This 1s the consequence of the low selectivity of these processes with respect to the preservation of the ATIII sites. ‘
In the present invention, 1f a VLMWH derived from the rnosphazene depolymerization process is used as starting material, exactly the reverse phenomencn takes place. The aXa activity of the oligosaccharide mixture
® - 13 - increases and even exceeds that of the heparin that was used to prepare the VLMWH. This 1s the probable consequence of the noteworthy selectivity of the phosphazene bases on the preservation of the sequences with affinity for ATIII.
This characteristic of the process is also observed through the average molecular masses of the oligosaccharide mixture obtained. By way of example, when a VLMWH with an average molecular mass of 2400 daltons is depolymerized, an oligosaccharide mixture with an average molecular mass of 2000 Da is obtained. It is found that the sequences with affinity for ATIII (hexasaccharides and octasaccharides) are preserved from the action of the phosphazene base, which results in the destruction and elimination of the other sequences, and consequently an average molecular mass that tends toward the average molecular mass of the non-depolymerizable species, ie the hexasaccharide
AIIa-IIs-Is (1834 g/mol). It should be pointed out that, in the step of heparin depolymerization with a phosphazene base, the average molecular mass changes from about 15 000 Da to about 2400 Da.
As an alternative method, the process according to the inventicn for increasing the activity and selectivity toward factor Xa is also appiicable to low molecular weight heparins in general. By way of example, mention will be made, for example, c¢f Enoxaparin, Fraxiparin,
Fragmin, Innohep (or Logiparin), Normiflo, Embcllex (or
Sandoparin), Fluxum (or Minidalton), Clivarin and
Hibor. They may also be certain very low molecular weight heparins as described in US 6 384 021 (2000-4000
Da) or WO 02/082%5 (1500 to 3000 Da), with anti-Xa activity of less than 140 IU/mg. (in particular between - 100 and 140 IU/mg).
This characteristic of the process is reflected by the production cf anti-Xa activities that are unexpected
®
PS - 14 - with regard to the average molecular weight of the oligosaccharide mixtures (190 IU/mg < aXa < 450 IU/mg; 1800 Da < MW < 2400 Da).
According to one particular embodiment of the invention, the selectivity toward factor Xa of the oligosaccharide mixtures may be further increased by removing the disaccharide and tetrasaccharide fractions (fractions not specifically binding to ATIII). In this case, the mixture is chromatographed on columns filled with gel of polyacrylamide agarose type or a polyacrylamide gel. The mixture is eluted with a sodium hydrogen carbonate solution. Preferably, the sodium hydrogen carbonate solution is a solution of from 0.1 mol/l to 1 mol/l. Even more preferably, the separation is performed at a concentration of 1 mol/l.
The detection is performed by UV spectrometry (254 nm).
After removal of the disaccharide and tetrasaccharide fractions, the oligosaccharide mixture 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 of greater than 20% by weight. The oligosaccharide mixture is precipitated by addition of 3 to 5 volumes of methanol. The high-affinity oligosaccharide mixture is recovered by filtration. If necessary, it may be purified by desalting on a suitable column. Example 6 illustrates this alternative method and allows very low molecular weight heparins with anti-Xa activity of greater than 400 IU/mg to be obtained.
A subject of the invention is thus also a process as defined above for preparing oligosaccharide mixtures having increased selectivity toward factor Xa of the - oligosaccharide mixture, wherein the disaccharide and tetrasaccharide fractions are also removed by chromatography, especially on columns filled with gel of polyacrylamide agarose type.
® ® - 15 -
The mixtures according to the invention may be used as medicinal products.
The oligosaccharide mixtures of the present invention may be used as antithrombotic agents. In particular, they are useful for treating or preventing venous and arterial thrombosis, 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 preventing and treating smooth muscle cell proliferation, atherosclerosis and arteriosclerosis, for treating and preventing cancer by modulating angiogenesis and growth factors, and for treating and preventing diabetic disorders such as diabetic retinopathy and diabetic nephropathy.
The present invention also relates to pharmaceutical compositions containing, as active principle, a mixture of formula (I) optionally combined with one or more inert excipients.
The pharmaceutical compositions are, for example, solutions for subcutaneous or intravenous injection.
Cther pharmaceutical compositions according to the invention are also used for pulmonary administration (inhalation) or oral administration.
The dosage may vary as a function of the age, weight and state of health of the patient. For an adult, it is generally between 20 and 100 mg per day via the intramuscular or subcutaneous route.
The examples thet follow illustrate the invention - without, however, limiting it.
® ® Preparation 1: Production of a starting very low molecular weight heparin having aXa activity equal to 158.8 IU/mg
The very low molecular weight heparin (VLMWH) used as starting material for example 1 is prepared according to patent application WO 2004/033503 from sodium heparin, by performing steps a to f as defined above, the depolymerization step being performed in the presence of 2-tert-butylimino-2-diethylamino-1, 3- dimethylperhydro-1,3,2-diazaphosphorine in the presence of a percentage of water of less than 0.6%.
Characteristics of the very low molecular weight heparin obtained
The characteristics of the depolymerized heparin thus obtained are as follows:
Average molecular weight: 2400 daltons
Anti-Xa activity: 158.8 IU/mg
Anti-IIa activity: 3.1 IU/mg
Anti-Xa activity/anti-IIa activity ratio: 51
Preparation 2: Production of a starting very low molecular weight heparin having aXa activity equal to 158 IU/mg
The very low molecular weight heparin (VLMWH) used as starting material for examples 2, 3, 4 and 5 is prepared according to patent application
WO 2004/033503, from sodium heparin, by performing steps a to f as defined above, the depolymerization step being performed ‘in the presence of 2-tert- butylimino-2-diethylamino-1, 3-dimethylperhydro-1, 3, 2- diazaphosphorine in the presence of a percentage of water of less than 0.6%.
Characteristics of the very low molecular weight . heparin obtained
The characteristics of the depolymerized heparin thus obtained are as follows:
Average molecular weight: 2450 daltons
PY - 17 -
Anti-Xa activity: 158 IU/mg
Anti-IIa activity: 2.1 IU/mg
Anti-Xa activity/anti-IIa activity ratio: 75
Preparation 3: VILMWH, benzethonium salt
Transsalification of the VLMWH to the benzethonium salt (corresponding to step a) of the process): 12.53 g (20.7 mmol) of the VLMWH sodium salt obtained according to preparation 1 are placed in a 500 ml conical flask A and dissolved in 85 ml of water (yellow solution). 31.62 g (70.5 mmol) of benzethonium chloride are placed in a 100 ml conical flask B with 250 ml of water (colorless solution).
The content of B is poured into A and the mixture is stirred for about 1 hour at room temperature. The resulting mixture is left to sediment for about 1 hour.
The supernatant is discarded and then replaced with the same volume of water (250 ml). The mixture is stirred for about 15 minutes and left to sediment for approximately 30 minutes. The supernatant is discarded and then replaced with the same vclume of water (250 ml). The mixture is stirred for about 15 minutes and then filtered. The cake is washed with 3 times 200 ml of water. The wet beige-cclored solid is drained by suction and then dried at 80°C for about 18 hours in an oven under reduced pressure (6 kPa). 35.56 g of
VLMWH benzethonium salt are obtained.
The yield obtained is 89%. 35 .
Preparation 4
Transsalification of the VLMWH to the benzethonium salt (corresponding to step a) of the process):
® - 18 - 17.93 g (30.2 mmol) of VLMWH sodium salt obtained according to preparation 2 are placed in a 1 1 conical flask A and dissolved in 120 ml of water (yellow solution). 45 g (0.1 mol) of benzethonium chloride are placed in a 500 ml conical flask B with 360 ml of water (colorless solution).
The content of B is poured into A and the mixture is stirred for about 1 hour at room temperature. The resulting mixture is left to sediment for about 1 hour.
The supernatant is discarded and then replaced with the same volume of water (500 ml). The mixture is stirred for about 15 minutes and left to sediment for approximately 30 minutes. The supernatant is discarded and then replaced with the same volume of water (500 ml). The mixture is stirred for about 15 minutes and then filtered. The cake is washed with 3 times 200 ml of water. The wet beige-colored solid is drained by suction and then dried at 80°C for about 48 hours in an oven under reduced pressure (6 kPa). 49.5 g of VLMWH benzethonium salt are obtained.
The yield obtained is 87%.
Example 1:
Very low molecular weight heparin (VLMWH) obtained by the process according to the invention, comprising a step of 77% esterification and a step of depolymerization with a base derived from phosphazene, in anhydrous medium .Esterification of the VIMWH (step b of the process): - 35.39 g (18.3 mmol) of VLMWH benzethonium salt obtained according to preparation 3 (with a water content of 0.20%) are dissolved in 183.3 g of dry dichloromethane and placed in a 500 ml three-necked flask. 29.5 ml
PY - 19 - (25.7 mmol) of benzyl chloride are added at a temperature of 30°C. The degree of esterification is 77% after about 23 hours of reaction at 30°C. After cooling to room temperature (22+3°C), the reaction mixture is poured into 490 ml of a 10% solution of sodium acetate in methanol. The mixture is stirred for about 1 hour and then left to sediment for approximately 1 hour. The supernatant is discarded and then replaced with the same volume of methanol (250 ml). The mixture is stirred for approximately 30 minutes and then left to sediment for about 45 minutes. The supernatant is discarded and then replaced with the same volume of methanol (250 ml).
This mixture is left to sediment for about 16 hours.
The supernatant is discarded and then replaced with the same volume of methanol (350 ml). The mixture is stirred for about 5 minutes and the suspension is filtered. The cake is washed with twice 50 ml of methanol, drained by suction and then dried at 40°C under reduced pressure (6 kPa) for about 18 hours. 34.48 g of crude VLMWH benzyl ester sodium salt with a degree of esterification of 77% are obtained. .Purification of the VIMWH benzyl ester sodium salt (step b) of the process):
The 34.48 g of crude VLMWH benzyl ester scdium salt are dissolved in 350 ml of aqueous 10% NaCl solution. The solution is poured into 1.57 1 of methanol. The suspension 1s stirred for about 40 minutes and is then left to sediment for about 16 hours. The supernatant is discarded and replaced with the same volume of methanol (1.5 1). This mixture is stirred for about 1 hour and is left to sediment for about 1.5 hours. The supernatant is discarded and replaced with the same volume of methanol (1.2 1). This mixture is stirred for - approximately 15 minutes and then filtered. The cake is washed with 3 times 50 ml of methanol. The wet white solid is drained by suction and then dried at 40°C
® ® - 20 - under reduced pressure (6 kPa) for about 18 hours. 6.07 g of VLMWH benzyl ester sodium salt are obtained.
The esterification yield is 50%. .Transsalification of the VLMWH benzyl ester sodium salt to the benzethonium salt (step c) of the process): 6 g (9.14 mmol) of VLMWH benzyl ester sodium salt are dissolved in 40 ml of water in a 250 ml conical flask A.
In parallel, 13.93 g (31 mmol) of benzethonium chloride are placed in 110 ml of water in a 250 ml conical flask B.
The content of B is poured into A. The suspension is stirred for about 1 hour at room temperature (22+3°C) and then left to sediment for 1 hour. The supernatant is discarded and replaced with the same volume of water (140 ml). This mixture is stirred for about 15 minutes and left to sediment for 1 hour. The supernatant is discarded and replaced with the same volume of water (140 ml). This mixture is stirred for approximately 15 minutes and left to sediment for about 3C minutes.
The supernatant is discarded and replaced with the same volume of water (140 ml). This mixture is stirred for about 5 minutes and then filtered. The cake is washed with 3 times 50 ml of water, drained by suction and then dried at 80°C under reduced pressure (6 kPa) for about 18 hours. 17.43 g of VLMWH benzyl ester, benzethonium salt are obtained.
The yield is 100%. -. .Depolymerization of the VLMWH benzyl ester, benzethonium salt in anhydrous medium: undetectable water content < 0.01% (step d) of the process)
®
PY - 21 - 17.43 g (9.14 mmol) of VLMWH according to preparation 2 are placed in a 250 ml three-necked flask with 122 ml of dry dichloromethane. 17.4 g of 4 A molecular sieves are added. The mixture is stirred for about 18 hours at room temperature (22+3°C) under an argon atmosphere.
The sieves are separated from the mixture by transferring the solution into a 250 ml three-necked flask. 2.64 ml (9.14 mmol) of 2-tert-butylimino-2- diethylamino-1, 3-dimethylperhydro-1, 3,2- diazophosphorine are added and the mixture is stirred for 24 hours at 22+3°C under an argon atmosphere. .Conversion of the quaternary ammonium salt into the sodium salt (step e) of the process)
In parallel, 730 ml of methanolic 10% sodium acetate solution are prepared in a 2 1 conical flask. 8.71 g of
Hyflo supercel Celite are added to the solution. The reaction mixture is poured into the methanolic solution, while maintaining the temperature at about 4°C. The suspension is stirred for about 15 minutes at this temperature. The mixture is left to sediment for approximately 45 minutes at room temperature and the supernatant 1s then discarded and replaced with the same amount of methanol (450 ml). This mixture is stirred for 15 minutes and left to sediment for approximately 45 minutes. The supernatant is again discarded and replaced with the same amount of methanol (420 ml). This mixture is stirred for about 15 minutes and then filtered through a No. 3 sinter funnel. The cake is washed with twice 70 ml of methanol, drained by suction and then dried for about 18 hours at 50°C under reduced pressure (6 kPa). 4.35 g of crude depclymerized
VLMWH (sodium salt) in Celite (8.71 g) are obtained. -
The yield is 72.5%.
®
PS - 22 - .Saponification of the crude depolymerized VLMWH, sodium salt (step f1) of the process): 4.35 g (6.63 mmol) of crude depolymerized VLMWH (sodium salt) in Celite are dissolved in 46 ml of water and then filtered through a No. 3 sinter funnel. The Celite is rinsed with 2 portions of 30 ml of water. The filtrate is placed in a 500 ml conical flask. 823 nl (9.94 mmol) of 35% sodium hydroxide solution are introduced at a temperature in the region of 4°C. This mixture is stirred for about 3 hours at this temperature. The medium is neutralized by adding 1N HCl solution, followed by addition of 11.5 g of NaCl and 80 ml of methanol. After stirring for approximately 15 minutes, 210 ml of methanol are added. The suspension is stirred for about 1 hour and then left to sediment for 30 minutes. The supernatant is discarded and replaced with the same amount of methanol (230 ml).
This mixture is stirred for about 15 minutes and left to sediment for 30 minutes. The supernatant is discarded and replaced with the same amount of methanol (210 ml). This mixture is stirred for approximately 15 minutes and then filtered. The cake 1s washed with twice 9 ml of methanol, drained by suction and then dried for abcut 18 hours at 50°C under reduced pressure € kPa). 2.95 g of crude depclymerized VLMWH (scdium salt) are obtained.
The yield is 73.7%. f) Purification of the crude depolymerized VLMWH, sodium salt (step £2) of the process): 1.5 g of crude depolymerized VLMWH, sodium salt, are placed in a 50 ml three-necked flask with 16 ml of - water. The solution is maintained at 40°C for about 10 minutes. The pH is brought to about 9.7 by addition cf O0.1N sodium hydroxide solution. The solution is filtered through a 0.45 pm membrane, and 84 pl of
®
PY - 23 - aqueous 30% hydrogen peroxide solution are then added.
The mixture is stirred for 2 hours at room temperature, while keeping the pH constant at 9.7 * 0.1 by adding 0.1N sodium hydroxide solution. The reaction mixture is then neutralized with 0.1N HCl, and 2 g of NaCl are then added. After stirring for about 10 minutes, the solution is filtered through a 0.45 pm membrane. 14 ml of methanol are added at a temperature in the region of 4°C. The solution is stirred for approximately minutes at room temperature. 36 ml of methanol are then added and the suspension is stirred for about 1 hour. The stirring is then stopped and the mixture is left to sediment for about 30 minutes. The supernatant is then taken up and discarded (40 ml). 40 ml of 15 methanol are added to the sedimented precipitate and this mixture 1s stirred for about 10 minutes. The precipitate is left to resediment for approximately 30 minutes. The supernatant is taken up and discarded (45 ml). 45 ml of methanol are added and the precipitate in suspension is then filtered off. The white cake obtained is then washed with 2 portions of 3 ml of methanol. The wet solid is drained by suction and then dried under reduced pressure (6 kPa) at a temperature in the region of 50°C. After drying for about 18 hours, 1.303 g of pure depclymerized VLMWH (sodium salt) are obtained.
The yield obtained is 86.8%. g) Characteristics of the depolymerized VIMWH thus obtained
Average molecular weight: 1950 daltons
Polydispersity index: 1.1
Anti-Xa activity: 283 U/mg alla activity: undetectable (<0.2 U/mg) -
Example 2:
Very low molecular weight heparin (VLMWH) obtained by the process according to the invention, comprising a step of 49% esterification and a step of depolymerization with a base derived from phosphazene, in anhydrous medium 5 .Esterification of the VIMWH (step b) of the process): 13.29 g (7.6 mmol) of VLMWH benzethonium salt obtained according to preparation 4 are dissolved in 70.43 g of anhydrous dichloromethane and placed in a 100 ml three- necked flask (the water content of the reaction medium is 0.073%). 12.3 ml (107 mmol) of benzyl chloride are added at a temperature of 30°C. The degree of esterification is 49% after reaction for about 7 hours at 30°C. After cooling, the reaction mixture is poured into 160 ml of a 12% solution of sodium acetate in methanol. The mixture is stirred for 1 hour at room temperature and then left to sediment for about 16 hours. The supernatant is discarded and then replaced with the same volume of methanol (100 ml).
This mixture is stirred for about 1 hour and left to sediment for about 1 hour. The supernatant is again discarded and replaced with the same volume of methanol (100 ml). This mixture is stirred for about 5 minutes and then filtered. The cake 1s washed with 2 x 40 ml of methanol, drained by suction and then dried in an oven at 40°C under reduced pressure (€&€ kPa) for about 18 hours. 3.90 g of crude VLMWH benzyl ester sodium salt with a degree of esterification of 49% are obtained. .Purification of the VLMWH benzyl ester (49% esterified) sodium salt (step b) of the process) 3.90 g of crude VLMWH benzyl ester sodium salt are dissolved in 39 ml of aqueous 10% NaCl solution. The . solution 1s poured into 176 ml of methanol. The suspension 1s stirred for about 15 minutes and then left to sediment for 2 hours. The mixture is filtered.
The cake 1s resuspended in 175 ml of methanol and
®
PS - 25 - stirred for 10 minutes. The mixture is filtered and the cake is washed with 2 portions of 10 ml of methanol.
The wet white solid is drained by suction and dried in an oven at 40°C under reduced pressure (6 kPa) for 5S about 18 hours. 2.62 g of VLMWH benzyl ester sodium salt are obtained.
The overall yield for the esterification phase is 57.3%. 10 .Transsalification of the VILMWH benzyl ester to the benzethonium salt (step c) of the process): 2.62 g (4.37 mmol) of VLMWH benzyl ester sodium salt 15 are dissolved in 20 ml of water (conical flask “A”). In parallel, 5.92 g (13.2 mmol) of benzethonium chloride are placed in 60 ml of water in a conical flask “B”.
The content of “B” is poured into “A”. The suspension 20 is stirred for about 1 hour at room temperature and then left to sediment for 1 hour. The supernatant is discarded and replaced with the same volume of water (70 ml). This mixture is stirred for about 15 minutes and left to sediment for 1 hour. The supernatant is 25 discarded and replaced with the same volume of water (70 ml). This mixture 1s stirred for a further minutes approximately and filtered. The cake 1s washed with 3 portions of 50 ml of water, drained by suction and then dried in an oven at 80°C under reduced pressure (6 kPa) for about 18 hours. 6.85 g of VLMWH benzyl ester benzethonium salt are obtained.
The yield is 99%. The water content of the benzethonium salt is 0.6%. - .Depolymerization of the VIMWH benzyl ester, benzethonium salt:
®
PY - 26 - 6.80 g (4.3 mmol) of VLMWH are placed in a 100 ml three-necked flask with 54 ml of dry dichloromethane.
The mixture is brought to 30°C and then stirred until dissolution is complete. The estimated water content of the reaction mixture is about 0.05%. 1.25 ml (4.3 mmol) of 2-tert-butylimino-2-diethylamino-1, 3- dimethylperhydro-1,3,2-diazaphosphorine are added and the mixture is stirred for 24 hours at 30°C under an inert atmosphere. .Conversion of the quaternary ammonium salt into the sodium salt (step e) of the process)
In parallel, 270 ml of methanolic 10% sodium acetate solution are prepared in a 1 1 conical flask. The reaction mixture is poured into the methanolic solution, while maintaining the temperature at about 4°C. The suspension is stirred for about 1 hour at room temperature. This mixture is left to sediment for 1 hour. The supernatant is discarded and then replaced with the same amount of methanol (165 ml). This mixture is stirred for about 1 hour and left to sediment for 1 hour. The supernatant is again discarded and replaced with the same amount of methanol (170 ml). This mixture is stirred for about 15 minutes and filtered. The cake is washed with 2 portions of 4C ml of methanol, draired by suction and then dried for about 18 hours in an oven at 50°C under reduced pressure (6 kPa). 2.29 g of crude depolymerized VLMWH, sodium salt, are obtained.
The yield obtained is 89%. .Saponification of the crude VIMWH, sodium salt (step fl) of the process):
Ee 2.2% g (3.8 mmol) of crude depolymerized VLMWH, sodium a salt, are dissolved in 23 ml of water. The solution is filtered through a 0.8 pm membrane and then placed in a 100 ml three-necked flask. 57% pl (5.73 mmol) of 30%
_
PY - 27 - sodium hydroxide solution are introduced at a temperature in the region of 3°C. The mixture is stirred for about 2 hours at this temperature.
Half of the reaction mixture is neutralized by adding glacial acetic acid, followed by addition of 367 mg of solid sodium acetate and 13 ml of methanol. The solution is stirred for about 15 minutes and 65 ml of methanol are then added. The suspension obtained is stirred for about 30 minutes and is then left to sediment for about 16 hours. The supernatant is discarded and replaced with the same amount of methanol (36 ml). This mixture 1s stirred for a further 30 minutes approximately and is left to sediment for about 30 minutes. The supernatant is discarded and replaced with the same amount of methanol (16 ml). This mixture is stirred for about 15 minutes and filtered through a 0.22 pum membrane. The cake is washed with twice 5 ml of methanol, drained by suction and then dried under reduced pressure (6 kPa) for about 18 hours in an oven at 50°C. 563 mg of crude depolymerized VLMWH (sodium salt) are obtained.
The yield is 52.6%. .Purification of the crude depolymerized VIMWH (sodium salt) precipitated with NaOAc (step £2) of the process) : 560 mg of crude depolymerized VLMWH (sodium salt) are placed in a 100 ml three-necked flask with 5.6 ml of water. The brown solution is maintained at 40°C for 10 minutes. The pH is brought to 9.7 by adding O0.1N sodium hydroxide solution. The solution is filtered through a 0.45 pm membrane and 28 ul of aqueous 30% oo hydrogen peroxide solution are added. The mixture is . stirred for 2 hours at room temperature, while keeping the pH constant at 9.5 * 0.1 by adding 0.1N sodium hydroxide solution. The reaction mixture is neutralized
_
PY - 28 - with O0.1N HCl and 620 mg of NaCl are added. After stirring for 10 minutes, the solution is filtered through a 0.45 um membrane. 4.35 ml of methanol are added at a temperature in the region of 4°C. The solution is stirred for 15 minutes at room temperature. 11.2 ml of methanol are added. The suspension is stirred for 1 hour. The stirring is then stopped and the mixture is left to sediment for 1 hour. The supernatant is then taken up and discarded (13.5 ml). 13.5 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 30 minutes. The supernatant is taken up and discarded (13 ml). 13 ml of methanol are added and the precipitate in suspension is then filtered off. The white cake obtained is then washed with 2 portions of 5 ml of methanol. The wet solid is drained by suction and then dried under reduced pressure (6 kPa) at a temperature in the region of 50°C. After drying for 18 hours, 376 mg of pure depolymerized VLMWH (sodium salt) are obtained. The yield obtained is 67%.
Characteristics of the depolymerized VLMWH thus obtained
Anti-Xa activity: 191 IU/mg
Rverage molecular weight: 2100 Da
Example 3:
Very low molecular weight heparin (VLMWH) obtained by the process according to the invention, comprising a step of 73% esterification and a step of depolymerization with a base derived from phosphazene, in anhydrous medium 35 .Esterification of the VIMWH (step b) of the process): oe 13.7 g (7.3 mmol) of VLMWH benzethonium salt obtained according to preparation 4 are dissolved in 73.67 g of arhydrous dichloromethane and placed in a 100 ml three-
®
PS - 29 - necked flask (the water content of the reaction medium is assayed as 0.23%). 13 ml (113 mmol) of benzyl chloride are added at a temperature of 30°C. The degree of esterification 1s 73% after reaction for about 20 hours at 30°C. After cooling to room temperature, the reaction mixture is poured into 210 ml of a 12% solution of sodium acetate in methanol. The mixture is stirred for 30 minutes at room temperature and then left to sediment for about 1.5 hours. The supernatant is discarded and then replaced with the same volume of methanol (140 ml). This mixture is stirred for minutes and the suspension is filtered. The cake is washed with 2 portions of 100 ml of methanol, drained by suction and then dried for approximately 18 hours in 15 an oven at 40°C under reduced pressure (6 kPa). 13.3 g of crude VLMWH benzyl ester sodium salt, with a degree of esterification of 73%, are obtained. .Purification of the VILMWH benzyl ester (73% esterified), sodium salt (step b) of the process):
The 13.3 g of crude VLMWH benzyl ester sodium salt are dissolved in 133 ml of aqueous 10% NaCl solution. The solution is poured into 600 ml of methanol. The suspension 1s stirred for about 15 minutes and then left to sediment for approximately 1 hour. The supernatant is discarded and then replaced with the same volume of methanol (400 ml). This mixture is stirred for about 5 minutes and then filtered. The cake is washed with 3 times 100 ml of methanol. The wet white solid is drained by suction and then dried for approximately 18 hours in an oven at 40°C under reduced pressure (6 kPa). 2.33 g of VLMWH benzyl ester sodium salt are obtained. -
The esterification yield is 49.6%. .Transsalification of the VIMWH benzyl ester, benzethonium salt (step c) of the process):
PS - 30 - 2.27 g (3.53 mmol) of VLMWH benzyl ester sodium salt are dissolved in 15 ml of water in a 100 ml conical flask “A”. In parallel, 5.22 g (11.6 mmol) of benzethonium chloride are placed in 55 ml of water in a 100 ml conical flask “B”.
The content of “B” 1s poured into “A”. The suspension is stirred for about 1 hour at room temperature and then left to sediment for approximately 1 hour. The supernatant is discarded and replaced with the same volume of water (50 ml). The mixture is stirred for about 15 minutes and left to sediment for approximately 1 hour. The supernatant is discarded and replaced with the same volume of water (50 ml). The mixture is stirred for a further 5 minutes and then filtered. The cake is washed with 3 portions of 50 ml of water, drained by suction and then dried for about 18 hours in an oven at 80°C under reduced pressure (6 kPa). 5.67 g of VLMWH benzyl ester benzethonium salt are obtained.
The yield obtained is 98%. The water content of the product obtained is 1%. 25 .Depolymerization of the VLMWH benzyl ester, benzethonium salt (step d) of the process): 5.45 g (3.3 mmol) of VLMWH are placed in a 100 ml three-necked flask with 40 ml of dry dichloromethane.
The estimated water content of the mixture is about 0.1%. The mixture is brought to 30°C. 958 pl (3.3 mmol) of 2-tert-butylimino-2-diethylamino-1, 3- dimethylperhydro-1, 3,2-diazaphosphorine are added and the mixture is stirred for 24 hours at 30°C under an 23 argon atmosphere. - .Conversion of the quaternary ammcnium salt into the sodium salt (step e) of the process)
® ® - 31 -
In parallel, 200 ml of methanolic 10% sodium acetate solution are prepared in a 500 ml conical flask. The reaction mixture is poured into the methanolic solution, while maintaining the temperature at about 4°C. The suspension is stirred for about 1 hour at room temperature. This mixture is left to sediment for approximately 1 hour. The supernatant is discarded and then replaced with the same amount of methanol (150 ml). This mixture is stirred for about 30 minutes and left to sediment for about 30 minutes. The supernatant is again discarded and replaced with the same amount of methanol (150 ml). This mixture is stirred for approximately 15 minutes and then filtered.
The cake is washed with 3 times 50 ml of methanol, drained by suction and then dried for about 18 hours at 50°C under reduced pressure (6 kPa). 1.40 g of crude depolymerized VLMWH, sodium salt, are obtained. The yield obtained is 65.8%. 20 .Saponification of the crude depolymerized VLMWH, sodium salt (step fl) of the process): 1.40 g (2.18 mmol) of crude depolymerized VLMWH (sodium salt) are dissolved in 14 ml of water. The solution is placed in a 100 ml three-necked round-bottomed flask. 351 pl (3.85 mmol) of 30% scdium hydroxide solution are introduced at a temperature in the region of 4°C. This mixture is stirred for about 2 hours at this temperature. The solution is neutralized by adding glacial acetic acid (100%). 7 g of solid sodium acetate and 130 ml of methanol are then added. The suspension is stirred for 30 minutes and is then left to sediment for about 1 hour. The supernatant is discarded and replaced with the same amount of methanol (80 ml). This mixture is stirred for a further 30 minutes - approximately and is left to sediment for approximately a 16 hours. The supernatant is discarded and replaced with the same amount of methanol (80 ml). This mixture is stirred for about 15 minutes and is then filtered
®
PY - 32 - through a 0.45 pm membrane. The cake is washed with twice 10 ml of methanol, drained by suction and then dried for about 18 hours at 50°C under reduced pressure (6 kPa). 1.15 g (yield: 895.4%) of crude depolymerized
VLMWH (sodium salt) are obtained.
The yield obtained is 89.4%. .Purification of the crude depolymerized VIMWH, sodium salt (step £2) of the process): 373 mg of crude depolymerized VLMWH (sodium salt) are placed in a 10 ml three-necked flask with 3.7 ml of water. The solution is maintained at 40°C for 10 minutes. The pH is brought to about 9.5 by adding 1N sodium hydroxide solution. The solution is filtered through a 0.45 pm membrane and 18 pl of aqueous 30% hydrogen peroxide solution are then added. The mixture is stirred for about 2 hours at room temperature, while keeping the pH constant at 9.5 + 0.1 by adding O0.1N sodium hydroxide solution. The reaction mixture is neutralized with 0.1N HCl and 430 mg of NaCl are then added. After stirring for about 10 minutes, the solution is filtered through a 0.45 pm membrane. 3 ml of methanol are added at a temperature in the region of 4°C. The soluticn is stirred for 15 minutes at room temperature. 7.7 ml of methanol are then added. The suspension is stirred for about 1 hour. The stirring is then stopped and the mixture is left to sediment for approximately 40 minutes. The supernatant is then taken up and discarded (10 ml). 10 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 30 minutes. The supernatant is taken up and discarded (10 ml). 10 ml of methanol are - added and the precipitate in suspension is then : filtered ofr on a 0.45 um membrane. The white cake obtained is washed with 4 portions of 5 ml of methanol.
The wet solid is drained by suction and then dried
_
PY - 33 - under reduced pressure (6 kPa) at a temperature in the region of 50°C. After drying for about 18 hours, 199 mg of pure depolymerized VLMWH (sodium salt) are obtained.
The yield obtained is 54%.
Characteristics of the depolymerized VLMWH thus obtained
Average molecular weight: 2000 daltons.
Polydispersity index: 1.1
Anti-Xa activity: 252 IU/mg
Example 4:
VLMWH obtained by the process according to the invention, comprising a step of 96% esterification and a step of depolymerization with BEMP .Esterification of the VIMWH (step b) of the process) 14.45 g (7.7 mmol) of VLMWH benzethonium salt obtained according to preparation 4 are dissolved in 75.79 g of anhydrous dichloromethane and placed in a 250 ml three- necked flask (the water content of the reaction medium is 0.20%). 12.4 ml (108 mmol) of benzyl chloride are added at a temperature of 30°C. The degree of esterification is 96% after reaction for about 26 hours at 30°C. After «cooling to room temperature, the reaction mixture is poured into 180 ml of a 12% solution of sodium acetate in methanol. The mixture is stirred for about 30 minutes at room temperature and is then left to sediment for approximately 30 minutes. The supernatant is discarded and then replaced with the same volume of methanol (150 ml). This mixture is stirred for about 15 minutes and then filtered. The - cake 1s washed with 2 portions of 100 ml of methanol, ’ drained by suction and then dried for about 18 hours at 40°C under reduced pressure (6 kPa). 3.67 g of crude
®
PN - 34 -
VLMWH benzyl ester, sodium salt, with a degree of esterification of 96%, are obtained.
Purification of the VLMWH benzyl ester (96% esterified) sodium salt (step b) of the process):
The 3.67 g of crude VLMWH benzyl ester, sodium salt are dissolved in 37 ml of aqueous 10% NaCl solution (3.7 g of NaCl in 37 ml of water). The solution is poured into 167 ml of methanol. The suspension is stirred for about minutes and 1s then left to sediment for approximately 1 hour. The supernatant is discarded and then replaced with the same volume of methanol (38 ml).
This mixture is stirred for about 5 minutes and 15 filtered. The cake is washed with twice 30 ml of methanol. The wet white solid is drained by suction and dried for about 18 hours at 40°C under reduced pressure (6 kPa). 2.76 g of VLMWH benzyl ester, sodium salt are obtained.
The esterification yield is 54.4%. . Transsalification of the VILMWH benzyl ester to the benzethonium salt (step ¢) of the process): 2.83 g (4.2% mmol) of VLMWH benzvl ester, sodium salt, are dissolved in 20 ml of water in a 100 ml conical flask “a”. In parallel, 6.35 g (14.2 mmol) of benzethonium chloride are placed in 50 ml of water in a 100 ml conical flask “B”.
The content of “B” is poured into “A”. The suspension is stirred for about 1 hour at room temperature and is then left to sediment for approximately 1 hour. The supernatant 1s discarded and replaced with the same ~ volume of water (60 ml). This mixture is stirred for about 15 minutes and left to sediment for approximately 1 hour. The supernatant is discarded and replaced with the same volume of water (60 ml). This mixture is
_ ® - 35 - stirred for a further 5 minutes and then filtered. The cake is washed with 4 times 50 ml of water, drained by suction and then dried for about 18 hours at 80°C under reduced pressure (6 kPa). 7.0 g of VLMWH benzyl ester,
Dbenzethonium salt, are obtained.
The observed yield is about 100%. The water content is 0.23%. 10 .Depolymerization of VLMWH benzyl ester, benzethonium salt (step d) of the process): 3.67 g (2.3 mmol) of VLMWH are placed in a 50 ml three- necked round-bottomed flask with 27 ml of dry dichoromethane. The mixture is brought to 30°C. 676 nl (2.3 mmol) of 2-tert-butylimino-2-diethylamino-1,3- dimethylperhydro-1, 3,2~-diazaphosphorine are added and the mixture is stirred for 24 hours at 30°C. 20 .Conversion of the quaternary ammonium salt into the sodium salt (step e) of the process):
In parallel, 150 ml of methanolic 10% sodium acetate solution are prepared in a 250 ml conical flask. The reaction mixture is poured inte the methanolic solution, while maintaining the temperature at about 4°C. The suspension is stirred for about 1 hour at room temperature. This mixture 1s left to sediment for approximately 1 hour. The supernatant is discarded and then replaced with the same amount of methanol (100 ml). This mixture is stirred for 30 minutes and left to sediment for 30 minutes. The supernatant is again discarded and replaced with the same amount of methanol (100 ml). This mixture 1s stirred for 15 minutes and filtered. The cake 1s washed with 3 times 40 ml of methanol, drained by suction and dried for about 18 hours at 50°C under reduced pressure (6 kPa). 966 mg of depolymerized VIMWH, sodium salt, are obtained.
® ® / 2308/0065%
The yield obtained is 64%. .Saponification of the VIMWH benzyl ester, sodium salt (step £1) of the process): 942 mg (1.43 mmol) of depolymerized VLMWH sodium salt are dissolved in 9.5 ml of water. The solution is placed in a 100 ml three-necked round-bottomed flask. 236 pl (2.35 mmol) of 30% sodium hydroxide solution are introduced at a temperature in the region of 4°C. This mixture is stirred for about 2 hours at room temperature. The solution is neutralized by addition of glacial acetic acid (100%). 4.5 g of solid sodium acetate and 85 ml of methanol are then added. The suspension is stirred for about 30 minutes and then left to sediment for about 1 hour. The supernatant is discarded and replaced with the same amount of methanol (40 ml). This mixture is stirred for a further 30 minutes approximately and is left to sediment for about 16 hours. The supernatant 1s discarded and replaced with the same amount of methanol (40 ml). This mixture is stirred for about 30 minutes and filtered through a 0.45 pm membrane. The cake is washed with twice 10 ml of methanol, drained by suction and then dried for about 18 hours at 50°C under reduced pressure (6 kPa). 776 mg of crude depolymerized VLMWH (sodium salt) are obtained.
The yield obtained is 91.4%. .Purification of the crude depolymerized VLMWH, sodium salt (step £2) of the process): 758 mg of crude depolymerized VLMWH (sodium salt) are placed in a 25 ml three-necked flask with 7.6 ml of water. The solution is maintained at 40°C for 10 minutes. The pH is brought to about 9.5 by addition of 0.1N sodium hydroxide solution. The sclution is
®
PY - 37 - filtered through a 0.45 pm membrane, and 38 pl of aqueous 30% hydrogen peroxide solution are then added.
The mixture is stirred for about 2 hours at room temperature, while keeping the pH constant at 9.5 % 0.1
S by addition of O0.1N sodium hydroxide solution. The reaction mixture is neutralized with IN HCl and 880 mg of NaCl are added. After stirring for about 10 minutes, the solution is filtered through a 0.45 pm membrane. 6.2 ml of methanol are added at a temperature in the region of 4°C. The solution is stirred for approximately 15 minutes at room temperature. 16 ml of methanol are added and the suspension is stirred for about 1 hour. The stirring is then stopped and the suspension is filtered. The «cake is washed with 2 portions of 15 ml of methanol. The wet solid is drained by suction and then dried under reduced pressure (6 kPa) at a temperature in the region of 50°C. After drying for approximately 18 hours, 490 mg of pure depolymerized VLMWH (sodium salt) are obtained. The yield obtained is 65%.
Characteristics of the depolymerized VLMWH thus obtained
Average molecular weight: 2000 daltons
Polydispersity index: 1.1
Anti-Xa activity: 205 IU/mg
Example 5:
VLMWH obtained by the process according to the invention, comprising a step of 96% esterification and a step of depolymerization with tert- butyliminotris(dimethylamino)phosphorane .Depolymerization of VIMWE benzyl ester, benzethonium salt (step d) of the process): 3.67 g (2.3 mmol) of VLMWH benzyl ester, benzethonium salt, obtained according to example 4 (96% esterified), with a water content of 0.23%, are placed in a 50 ml
®
PS - 38 - three-necked flask with 30 ml of dry dichloromethane.
The mixture is brought to 30°C. 595 pl (2.3 mmol) of tert-butyliminotris(dimethylamino)phosphorane are added and the mixture is stirred for 24 hours at 30°C. .Conversion of the quaternary ammonium salt into the sodium salt (step e) of the process):
In parallel, 160 ml of methanolic 10% sodium acetate solution are prepared in a 250 ml conical flask. The reaction mixture is poured into the methanolic solution, while maintaining the temperature at about 4°C. The suspension is stirred for approximately 1 hour at room temperature. This mixture is left to sediment for about 1 hour. The supernatant is discarded and then replaced with the same amount of methanol (120 ml).
This mixture is stirred for about 30 minutes and left to sediment for 30 minutes. The supernatant is again discarded and replaced with the same amount of methanol (125 ml). This mixture is stirred for about 15 minutes and filtered. The cake is washed with 3 times 40 ml of methanol, drained by suction and dried for about 18 hours at a temperature in the region of 50°C under reduced pressure (6 kPa). 982 mg of crude depolymerized
VLMWH, sodium salt, are obtained. The yield obtained is 65%. .Saponification of the crude depolymerized VLMWH, sodium salt (step £1) of the process): 980 mg (1.49 mmol) of crude depolymerized VLMWH, sodium salt, are dissolved in 10 ml of water. The solution is placed in a 100 ml three-necked round-bottomed flask. 246 pl (2.45 mmol) of 30% sodium hydroxide solution are introduced at a temperature in the region of 4°C. This mixture is stirred for about 2 hours at this temperature. The solution 1s neutralized by adding glacial acetic acid (100%). 4.9 g of solid sodium acetate and 95 ml of methanol are then added. The
_
PY - 39 - suspension is stirred for 30 minutes and then left to sediment for about 1 hour. The supernatant is discarded and then replaced with the same amount of methanol (60 ml). This mixture 1s stirred for a further 30 minutes approximately and is then left to sediment for about 16 hours. The supernatant is discarded and then replaced with the same amount of methanol (60 ml).
This mixture is stirred for about 30 minutes and filtered through a 0.45 pm membrane. The cake is washed with twice 10 ml of methanol, drained by suction and then dried for approximately 18 hours at 50°C under reduced pressure (6 kPa). 809 mg of crude depolymerized
VLMWH, sodium salt, are obtained.
The reaction yield is 91.6%. .Purification of the crude depolymerized VLMWHE, sodium salt (step £2) of the process): 792 mg of crude depolymerized VLMWH (sodium salt) are placed in a 25 ml three-necked flask with 8 ml of water. The solution is maintained at 40°C for 10 minutes. The pH is brought to about 9.5 by adding 0.1N sodium hydroxide solution. The solution is filtered through a 0.45 pm membrane, and 39.6 pl of agueous 30% hydrogen peroxide solution are then added.
The mixture Is stirred for 2 hours at rcom temperature, while keeping the pH constant at 9.5 + 0.1 by adding 0.1N sodium hydroxide solution. The reaction mixture is neutralized with 1IN HCl ‘and 1.04 g of NaCl are added.
After stirring for about 10 minutes, the solution is filtered through a 0.45 pm membrane. 7.3 ml of methanol are added at a temperature in the region of 4°C. The solution is stirred for 15 minutes at room temperature. 18.8 ml of methanol are added and the suspension is } stirred for about 1 hour. The stirring is then stopped and the mixture is filtered. The cake is washed with 3 portions of 15 ml of methanol. The wet solid is drained by suction and then dried under reduced
®
PS - 40 - pressure (6 kPa) at a temperature in the regicn of 50°C. After drying for 18 hours, 538 mg of pure depolymerized VLMWH (sodium salt) are obtained. The yield obtained is 67.9%.
Characteristics of the depolymerized VLMWH thus obtained
Average molecular weight: 2100 daltons
Polydispersity index: 1.1
Anti-Xa activity: 209 IU/mg
Example 6:
VLMWH obtained by the process according to the invention, comprising an additional step of separation by chromatography to remove the disaccharide and tetrasaccharide fractions
The oligosaccharide mixture described in example 1 (286 mg) is dissolved in 20 ml of mobile phase (aqueous sodium bicarbonate solution at a concentration of 0.2 mol/l).
The chromatographic conditions are as follows: - Mobile phase: sodium bicarbonate solution at a concentration of 0.2 mol/l - Stationary phase: biogel P6 gel - Column: length 1 m, diameter 5 cm - Detection wavelength: 240 nm.
The fractions greater than or equal to a hexasaccharide are collected and pooled. They are neutralized with acetic acid and then concentrated until a solution containing 200 g/l of sodium acetate is obtained. 5 volumes of methanol are added to the solution obtained with stirring. The suspension is stirred for about 18 hours and then filtered through a 0.45 um membrane. The cake 1s dried for about 6 hours at a temperature in the region of 40°C under reduced pressure (6 kPa). The product obtained is
® St reprecipitated and then dissolved in a minimum amount of water and desalified on a Sephadex G10 column. After concentrating the desalified fractions and then freeze- drying, 109 mg of product are obtained. The yield is 38%.
The characteristics of the oligosaccharide mixture thus obtained are as follows:
Anti-Xa activity: 403 IU/mg
The oligosaccharide percentage is as follows:
Mw Poly- Tetra Hexa Octa Deca >Deca (Da) dispersity % 3 % % % iso [10 Jo fo [snes [2.8 [10s [as
Pharmacological activity of the compounds according to the invention:
Examples Average Anti-Xa Anti-IIa molecular activity activity weight IU/mg 1 253 <0.2 2100 191 c 2000 252 0 a 2000 20s Jo 2100 200 Jo 6 [e150 aos Jo
Percentage of hexasaccharide A IIa-IIs-Is in the compounds according to the invention:
_
PY - 42 -
Examples Percentage of Percentage of hexasaccharide hexasaccharide A IIa- fraction IIs-Is in the hexasaccharide fraction
I TT ET
6 | s3es | 0 aes
Claims (44)
- PS WO 20057010051 - 43 - PCT/FR2004/001943 CLAIMS 1) An oligosaccharide mixture having the general structure of the constituent polysaccharides of heparin and having the following characteristics: - an average molecular weight of from 1800 to 2400 daltons, - anti-Xa activity of between 190 IU/mg and 450 IU/mg - no, or virtually no, anti-IIa activity, - it being understood that the constituent oligosaccharides of the mixtures - contain from 2 to 16 saccharide units, : - have a 4,5-unsaturated uronic acid 2-0O-sulfate unit at one of their ends, ~ and contain the hexasaccharide of the following formula: Na Na Na i é o 0) j ! 0=s=0 0=5=0 o=5=0 Na, © 0 Na © O Na © 0 0 o o—4 oO oO 0} Oo —0 Oo BH Ymapm{OH Yu gmdQH Y=0m{ 9505 0H =on OH »wOH “oH “NH oH oN 00 0, NH — 0-8, oS QS, 0 Na © i O Na O Na Alla Ills Is in the form of an alkali metal or alkaline-earth metal salt.
- ® oH 2) The oligosaccharide mixture as claimed in claim 1, wherein the alkali metal or alkaline-earth metal salts are the sodium, potassium, calcium and magnesium salts.
- 3) The oligosaccharide mixture as claimed in claim 1 or 2, which contains from 20% to 100% and in particular from 30% to 60% of hexasaccharide fraction.
- 4) The oligosaccharide mixture as claimed in claim 3, wherein the hexasaccharide fraction contains from 20% to 70% and in particular from 25% to 50% of the hexasaccharide AIIa-IIs-Is as defined in claim 1.
- 5) The oligosaccharide mixture as claimed in any one of claims 1 to 4, which has an average molecular weight of between 1900 and 2200 and in particular from 1950 to 2150 daltons.
- 6) The oligosaccharide mixture as claimed in any one of claims 1 to 5, which has anti-Xa activity of between 190 IU/mg and 410 IU/mg and in particular between 200 and 300 IU/mg, while at the same time having no, or virtually no, anti-IIa activity.
- 7) The oligosaccharide mixture as claimed in any one cof claims 1 tc €, which has the following characteristics: - an average molecular weight of between 1950 and 2150 daltons, - anti-Xa activity of between 190 IU/mg and 410 IU/mg and no, or virtually no, anti-IIa activity, - it contains from 30% to 60% of hexasaccharide fraction, which contains from 25% to 55% of AIIa-IIs-Is fraction.
- 8) A process for preparing the oligosaccharide mixture as claimed in any one of claims 1 to 7, wherein a very® ® - 45 -low molecular weight heparin with aXa activity of greater than 140 IU/mg, alla activity of greater thanIU/mg and an average molecular mass of between 2000 and 3000 daltons is subjected to the following chemical reactions:a) transsalification by the action of benzethonium chloride to obtain benzethonium heparinate,b) esterification of the benzethonium heparinate obtained by the action of benzyl chloride, and treatment to obtain the sodium salt of the benzyl ester of the very low molecular weight heparin,c) transsalification of the benzyl ester obtained and production of the quaternary ammonium salt,d) depolymerization by means of a strong organic base with a pKa value preferably of greater than 20, so as to obtain a depolymerized very low molecular weight heparin,e) conversion of the quaternary ammonium salt of the depolymerized very low molecular weight heparin into the sodium salt,f) saponification of the residual esters and optional purification.
- 9) The preparation process as claimed 1in claim 8,wherein the strong base/ester mole ratio used during the polymerization step d) is between 0.2 and 5 and preferably between 0.6 and 2.
- 10) The preparation process as claimed in claim 8 or 9,wherein the depolymerization step d) is performed using a phosphazene derivative as base.
- 11) The preparation process as claimed in any one of claims 8 to 10, wherein the depolymerization step d) is performed with water contents of less than 0.3% when the process is performed with 1 molar equivalent of phosphazene base relative to the benzyl ester of theVLMWH, benzethonium salt.®®
- 12) The preparation process as claimed in any one of claims 8 to 11, wherein the bases of the phosphazene family used during the depolymerization step d) are preferably those of formula: R3 R2—N R4 RN Ags \ N—RS RY in which the radicals R; to R;, which are identical or different, represent linear, branched or cyclic alkyl radicals containing from 1 to 6 carbon atoms, it being possible for R; and Rs, where appropriate, to form, with the -N-P-N group which carries them, a 6-membered heterocycle.
- 13) The preparation process as claimed in any one of claims 8 to 12, wherein the base of the family of phosphazenes used in the depolymerization step is 2-tert-butylimino-2-diethylaminoc-1, 3-dimethylperhydro- 1,3,2-diazaphosphorine.
- 14) The preparation process as claimed in claim 8, wherein the degree of esterification cf the guaternary ammonium salt of the benzyl ester of heparin during step b) is between 40% and 100% and preferably between 70% and 90%.
- 15) The preparation process as claimed in claim 8, wherein the conversion of the quaternary ammonium salt of the benzyl ester of the very low molecular weight heparin as prepared according to step b) of the process as claimed in claim 8, into the sodium salt, is perfcrmed by treating the reaction medium with alcoholic sodium acetate solution and preferably with a 10% solution of sodium acetate in® © - 47 -methanol (weight/volume), at a temperature of between and 25°C.
- 16) The preparation process as claimed in claim 15, wherein the weight equivalent of sodium acetate added during the esterification step b) is 3 times as great as the mass of quaternary ammonium salt of the benzyl ester of heparin used in the depolymerization reaction.
- 17) The process as claimed in claim 8 for preparing oligosaccharide mixtures, wherein the quaternary ammonium salt of the benzyl ester of the very low molecular weight heparin obtained during step c¢) is preferably the benzethonium, cetylpyridinium or cetyltrimethylammonium salt.
- 18) The preparation process as claimed in claim 8, wherein the saponification (according to step f) is performed using an alkali metal hydroxide such as sodium hydroxide, potassium hydroxide or lithium hydroxide, in aqueous medium, at a temperature of between 0 and 20°C and preferably between 0 and 10°C.
- 19) The preparation process as claimed in claim 18, wherein frecm 1 tc 5 mclar equivalents of alkali metal hydroxide and mcre particularly from 1 to 2 molar equivalents of alkali metal hydroxide are used.
- 20) The process, as claimed in any one of claims 8 to 19, for preparing the oligosaccharide mixture as described in any one of claims 1 to 5 having increased selectivity toward factor Xa of the oligosaccharide mixture, wherein the disaccharide and tetrasaccharide fractions are also removed by chromatography, especially on columns filled with gel of polyacrylamide agarose type.
- 21) The process as claimed in any one of claims 8 to 20 for preparing the oligosaccharide mixture as claimed in®® any one of claims 1 to 7, wherein a low molecular weight heparin is used as starting material, or a very low molecular weight heparin (1500 to 3000 Da) with an anti-Xa activity of between 100 and 140 IU/mg instead of the very low molecular weight heparin as defined in claims 8.
- 22) The process as claimed in any one of claims 8 to 20 for preparing the oligosaccharide mixture as claimed in any one of claims 1 to 7, wherein a very low molecular weight heparin (1500 to 4000 Da) with an anti-Xa activity of between 100 and 140 IU/mg is used as starting material instead of the very low molecular weight heparin as defined in claim 8.
- 23) The preparation process as claimed in claim 21, wherein the low molecular weight heparin is chosen from Enoxaparin, Fraxiparin, Fragmin, Innohep (or Logiparin), Normiflo, Embollex (or Sandoparin), Fluxum (or Minidalton), Clivarin and Hibor.
- 24) The oligosaccharide mixture as defined in any one of claims 1 to 7, which may be obtained by the process as defined in any one of claims 7 to 23.
- 25) As a medicinal product, the oligosaccharide mixture as claimed in any one of claims 1 to 7.
- 26) As a medicinal product with antithrombotic activity, the oligosaccharide mixture as claimed in any one of claims 1 to 7.
- 27) The medicinal product as claimed in claim 25 or 26, for treating or preventing venous and arterial thromposis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac "ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis and arteriosclerosis,y ] - 49 - PCT/FR2004/001943 cancer by modulating angiogenesis and growth factors, and also diabetic disorders such as diabetic retinopathy and diabetic nephropathy.
- 28) A pharmaceutical composition containing at least one medicinal product as defined in claim 25 and one or more pharmaceutically inert excipients or vehicles or additives.
- 29) The pharmaceutical composition as claimed in claim 28, which conrsists of a solution for subcutaneous or intravenous injection.
- 30) The pharmaceutical composition as claimed in claim 28, which «consists of a pulmonary formulation for inhalation.
- 31) The pharmaceutical composition as claimed in claim 28, which consists of an oral formulation.
- 32) A method for determining the anti-Xa activity of the oligosaccharide mixture as claimed in any one of claims 1 to 7, wherein an amidolytic method is used on a chromogenic substrate in which the reconstitution buffer 1s Polyethylene Giycol 6000 (PEG 6000).
- 33) Use of the oligosaccharide mixture as claimea in any one of claims 1 to 7 1n the manuiacture oi a preparation with antithrombotic activity.
- 34) Use as claimed in claim 33, for treating or preventing venous and arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis and artericscleros:s, cancer by modulating angiogenesis and growth factors, and also AMENDED SEEETJ - 50 - PCT/FR2004/001943 J diabetic disorders such as diabetic retinopathy and diabetic nephropathy.
- 35) A substance or composition with antithrombotic activity, for use in a method of treatment or prevention, said substance or composition comprising the oligosaccharide mixture as claimed in any one of claims 1 to 7, and said method comprising administering said substance or composition.
- 36) A substance or composition for use in a method of treatment or prevention as claimed in claim 35, for treating or preventing venous and arterial thrombosis, deep vein thrombosis, pulmonary embolism, unstable: angina, myocardial infarction, cardiac ischemia, occlusive diseases of the peripheral arteries and atrial fibrillation, smooth muscle cell proliferation, atherosclerosis and arteriosclerosis, cancer by modulating angiogenesis and growth factors, and also diabetic disorders such as diabetic retinopathy and diabetic nephropathy.
- 37) An oligosaccharide mixture as claimed in any one of claims 1 to 7 or 24, substantially as herein described and ii:lustrated.
- 38) A process as claimed in any one of claims 8 to 23, substantially as herein described and illustrated.
- 39) A product as claimed in any one of claims 25 to 27, substantially as herein described and illustrated.
- 40) A composition as claimed in any one of claims 28 to 31, substantially as herein described and izlustrated.
- 41) A method as claimed in claim 32, substantially as herein described and illustrated.AMENDED SHEETD - 51 - PCT/FR2004/001943
- 42) Use as claimed in claim 33 or claim 34, substantially as herein described and illustrated.
- 43) A substance or composition for use in a method of treatment or prevention as claimed in claim 35 or claim 36, substantially as herein described and illustrated.
- 44) A new oligosaccharide mixture, a new process for preparing an oligosaccharide mixture, a new product, a new composition, a new use of an oligosaccharide mixture as claimed in any one of claims 1 to 7, or a substance or composition for a new use in a method of treatment or prevention, substantially as herein described.AMENDED SHEET
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FR0309041A FR2857971B1 (en) | 2003-07-24 | 2003-07-24 | MIXTURES OF HEPARIN DERIVED OLIGOSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
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US7589145B2 (en) | 2004-04-15 | 2009-09-15 | Exxonmobil Chemical Patents Inc. | Syndiotactic rich polyolefins |
CA2652205A1 (en) * | 2006-05-25 | 2007-12-06 | Mallik Sundaram | Low molecular weight heparin composition and uses thereof |
CN101711771B (en) * | 2008-10-07 | 2015-12-09 | 上海喜恩医药科技发展有限公司 | A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition |
EP3144325B1 (en) * | 2010-09-14 | 2020-11-04 | University Of Miyazaki | High purity heparin and production method therefor |
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 |
CN102558392A (en) * | 2010-12-14 | 2012-07-11 | 王芃 | Preparation method of high-FXa-resistant low-FIIa-resistant low-molecular heparin sodium |
CN102816255B (en) * | 2011-06-10 | 2015-11-25 | 王芃 | A kind of preparation method of ultra-low molecular heparin sodium |
WO2014153995A1 (en) * | 2013-03-26 | 2014-10-02 | 中国科学院昆明植物研究所 | Low molecular weight glycosaminoglycan derivative, pharmaceutical composition thereof, preparation method therefor and use thereof |
CN103864959A (en) * | 2014-04-03 | 2014-06-18 | 中国药科大学 | Application of heparin oligosaccharide dp12 in atherosclerosis resistance |
RU2639574C2 (en) * | 2016-05-23 | 2017-12-21 | Алексей Георгиевич Александров | Process for low molecular weight heparin preparation |
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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 |
FR2504928A1 (en) * | 1981-04-29 | 1982-11-05 | Choay Sa | SHORT CHAIN OLIGOSACCHARIDES HAVING BIOLOGICAL PROPERTIES, PREPARATION THEREOF AND APPLICATIONS THEREOF AS MEDICAMENTS |
AU7753898A (en) * | 1997-06-06 | 1998-12-21 | Hamilton Civic Hospitals Research Development, Inc. | Modified low molecular weight heparin that inhibits clot associated coagulation factors |
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. |
FR2800074B1 (en) * | 1999-10-22 | 2001-12-21 | Aventis Pharma Sa | NOVEL OLIGOSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
JP4585072B2 (en) * | 2000-02-29 | 2010-11-24 | 扶桑薬品工業株式会社 | Heparin depolymerization method, depolymerized heparin, derivatives thereof and pharmaceutical composition |
FR2807043B1 (en) * | 2000-03-28 | 2002-11-22 | Aventis Pharma Sa | PHARMACEUTICAL COMPOSITION CONTAINING OLIGOSACCHARIDES, NEW OLIGOSACCHARIDES AND THEIR PREPARATION |
US6969705B2 (en) * | 2000-07-21 | 2005-11-29 | Aventis Pharma S.A. | Compositions of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them |
FR2811992B1 (en) * | 2000-07-21 | 2003-07-04 | Aventis Pharma Sa | MIXTURES OF HEPARIN-DERIVED POLYSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
WO2003018032A1 (en) * | 2001-08-28 | 2003-03-06 | Leo Pharma A/S | Antithrombotic compositions comprising low molecular weight heparin and low molecular weight dermatan sulphate |
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