CN117064909A - Oligosaccharide mixture derived from heparan caprae seu ovis and preparation method and application thereof - Google Patents
Oligosaccharide mixture derived from heparan caprae seu ovis and preparation method and application thereof Download PDFInfo
- Publication number
- CN117064909A CN117064909A CN202310232108.2A CN202310232108A CN117064909A CN 117064909 A CN117064909 A CN 117064909A CN 202310232108 A CN202310232108 A CN 202310232108A CN 117064909 A CN117064909 A CN 117064909A
- Authority
- CN
- China
- Prior art keywords
- heparin
- ns6s
- ext
- oligosaccharide mixture
- oligosaccharide
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 302
- 150000002482 oligosaccharides Chemical class 0.000 title claims abstract description 298
- 229920001542 oligosaccharide Polymers 0.000 title claims abstract description 284
- 238000002360 preparation method Methods 0.000 title claims description 35
- 229920002971 Heparan sulfate Polymers 0.000 title claims description 29
- 241000283898 Ovis Species 0.000 title claims description 9
- 229920000669 heparin Polymers 0.000 claims abstract description 120
- 229960002897 heparin Drugs 0.000 claims abstract description 104
- 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 claims abstract description 83
- 230000000694 effects Effects 0.000 claims abstract description 37
- 230000002785 anti-thrombosis Effects 0.000 claims abstract description 6
- 239000000243 solution Substances 0.000 claims description 63
- 239000003055 low molecular weight heparin Substances 0.000 claims description 61
- 238000000034 method Methods 0.000 claims description 57
- IAJILQKETJEXLJ-UHFFFAOYSA-N Galacturonsaeure Natural products O=CC(O)C(O)C(O)C(O)C(O)=O IAJILQKETJEXLJ-UHFFFAOYSA-N 0.000 claims description 54
- 150000002016 disaccharides Chemical class 0.000 claims description 47
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 47
- AEMOLEFTQBMNLQ-HNFCZKTMSA-N L-idopyranuronic acid Chemical group OC1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-HNFCZKTMSA-N 0.000 claims description 36
- -1 heparin disaccharides Chemical class 0.000 claims description 35
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 30
- 239000002244 precipitate Substances 0.000 claims description 29
- 238000003756 stirring Methods 0.000 claims description 27
- 238000001035 drying Methods 0.000 claims description 26
- 230000004048 modification Effects 0.000 claims description 26
- 238000012986 modification Methods 0.000 claims description 26
- 239000002253 acid Substances 0.000 claims description 22
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 21
- 230000001858 anti-Xa Effects 0.000 claims description 20
- 230000019635 sulfation Effects 0.000 claims description 20
- 238000005670 sulfation reaction Methods 0.000 claims description 20
- MSWZFWKMSRAUBD-UHFFFAOYSA-N beta-D-galactosamine Natural products NC1C(O)OC(CO)C(O)C1O MSWZFWKMSRAUBD-UHFFFAOYSA-N 0.000 claims description 18
- 239000004593 Epoxy Substances 0.000 claims description 16
- 239000003146 anticoagulant agent Substances 0.000 claims description 15
- 238000006243 chemical reaction Methods 0.000 claims description 15
- 239000008213 purified water Substances 0.000 claims description 14
- 229960002442 glucosamine Drugs 0.000 claims description 13
- 239000011780 sodium chloride Substances 0.000 claims description 13
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 12
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 12
- 230000008569 process Effects 0.000 claims description 12
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 229940127215 low-molecular weight heparin Drugs 0.000 claims description 11
- 238000002156 mixing Methods 0.000 claims description 11
- 239000001632 sodium acetate Substances 0.000 claims description 11
- 235000017281 sodium acetate Nutrition 0.000 claims description 11
- 239000007787 solid Substances 0.000 claims description 11
- AEMOLEFTQBMNLQ-AQKNRBDQSA-N D-glucopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-AQKNRBDQSA-N 0.000 claims description 10
- 239000000470 constituent Substances 0.000 claims description 10
- 239000011734 sodium Substances 0.000 claims description 10
- 239000003513 alkali Substances 0.000 claims description 9
- 125000001033 ether group Chemical group 0.000 claims description 9
- 229940097043 glucuronic acid Drugs 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 9
- 229940127219 anticoagulant drug Drugs 0.000 claims description 8
- 229960001950 benzethonium chloride Drugs 0.000 claims description 8
- 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 8
- YRTWNFXASSUQEW-UHFFFAOYSA-M sodium;methanol;acetate Chemical compound [Na+].OC.CC([O-])=O YRTWNFXASSUQEW-UHFFFAOYSA-M 0.000 claims description 8
- 238000012691 depolymerization reaction Methods 0.000 claims description 7
- 150000008273 hexosamines Chemical class 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 claims description 6
- KCXMKQUNVWSEMD-UHFFFAOYSA-N benzyl chloride Chemical compound ClCC1=CC=CC=C1 KCXMKQUNVWSEMD-UHFFFAOYSA-N 0.000 claims description 6
- 229940073608 benzyl chloride Drugs 0.000 claims description 6
- NDKBVBUGCNGSJJ-UHFFFAOYSA-M benzyltrimethylammonium hydroxide Chemical compound [OH-].C[N+](C)(C)CC1=CC=CC=C1 NDKBVBUGCNGSJJ-UHFFFAOYSA-M 0.000 claims description 6
- 238000010438 heat treatment Methods 0.000 claims description 6
- 125000000896 monocarboxylic acid group Chemical group 0.000 claims description 6
- 239000003960 organic solvent Substances 0.000 claims description 6
- 230000001376 precipitating effect Effects 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 229960002233 benzalkonium bromide Drugs 0.000 claims description 5
- KHSLHYAUZSPBIU-UHFFFAOYSA-M benzododecinium bromide Chemical compound [Br-].CCCCCCCCCCCC[N+](C)(C)CC1=CC=CC=C1 KHSLHYAUZSPBIU-UHFFFAOYSA-M 0.000 claims description 5
- 238000007068 beta-elimination reaction Methods 0.000 claims description 5
- 239000008194 pharmaceutical composition Substances 0.000 claims description 4
- 238000007670 refining Methods 0.000 claims description 4
- 230000008684 selective degradation Effects 0.000 claims description 4
- 230000021736 acetylation Effects 0.000 claims description 3
- 238000006640 acetylation reaction Methods 0.000 claims description 3
- 230000002429 anti-coagulating effect Effects 0.000 claims description 3
- 229960004676 antithrombotic agent Drugs 0.000 claims description 3
- 102000004411 Antithrombin III Human genes 0.000 claims description 2
- 108090000935 Antithrombin III Proteins 0.000 claims description 2
- 229960005348 antithrombin iii Drugs 0.000 claims description 2
- 238000011161 development Methods 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000007515 enzymatic degradation Effects 0.000 claims description 2
- AEMOLEFTQBMNLQ-YMDCURPLSA-N D-galactopyranuronic acid Chemical group OC1O[C@H](C(O)=O)[C@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-YMDCURPLSA-N 0.000 claims 2
- 229940127090 anticoagulant agent Drugs 0.000 claims 2
- 125000001483 monosaccharide substituent group Chemical group 0.000 claims 1
- 238000011321 prophylaxis Methods 0.000 claims 1
- 230000035484 reaction time Effects 0.000 claims 1
- 230000009424 thromboembolic effect Effects 0.000 claims 1
- 239000003814 drug Substances 0.000 abstract description 18
- 241000699670 Mus sp. Species 0.000 abstract description 14
- 241001494479 Pecora Species 0.000 abstract description 12
- 238000010254 subcutaneous injection Methods 0.000 abstract description 9
- 239000007929 subcutaneous injection Substances 0.000 abstract description 8
- 231100000331 toxic Toxicity 0.000 abstract description 3
- 230000002588 toxic effect Effects 0.000 abstract description 3
- 238000005481 NMR spectroscopy Methods 0.000 description 49
- 238000004458 analytical method Methods 0.000 description 48
- 108010022901 Heparin Lyase Proteins 0.000 description 43
- 150000004044 tetrasaccharides Chemical group 0.000 description 41
- 238000004949 mass spectrometry Methods 0.000 description 39
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 36
- 238000004128 high performance liquid chromatography Methods 0.000 description 35
- 150000002772 monosaccharides Chemical class 0.000 description 25
- 239000000047 product Substances 0.000 description 25
- 239000000523 sample Substances 0.000 description 25
- 238000001514 detection method Methods 0.000 description 23
- QAOWNCQODCNURD-UHFFFAOYSA-L sulfate group Chemical group S(=O)(=O)([O-])[O-] QAOWNCQODCNURD-UHFFFAOYSA-L 0.000 description 17
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 16
- 150000004043 trisaccharides Chemical class 0.000 description 16
- 102100024025 Heparanase Human genes 0.000 description 15
- 230000010100 anticoagulation Effects 0.000 description 15
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 14
- IAJILQKETJEXLJ-RSJOWCBRSA-N aldehydo-D-galacturonic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-RSJOWCBRSA-N 0.000 description 13
- 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 13
- 229960001008 heparin sodium Drugs 0.000 description 13
- 239000008215 water for injection Substances 0.000 description 13
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 12
- 229940079593 drug Drugs 0.000 description 12
- 230000015556 catabolic process Effects 0.000 description 11
- 238000006731 degradation reaction Methods 0.000 description 11
- 108020004414 DNA Proteins 0.000 description 10
- 238000002474 experimental method Methods 0.000 description 10
- 108010037536 heparanase Proteins 0.000 description 10
- 238000000926 separation method Methods 0.000 description 10
- 230000007071 enzymatic hydrolysis Effects 0.000 description 9
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 description 9
- 238000002347 injection Methods 0.000 description 9
- 239000007924 injection Substances 0.000 description 9
- 241000283973 Oryctolagus cuniculus Species 0.000 description 8
- AEMOLEFTQBMNLQ-UHFFFAOYSA-N beta-D-galactopyranuronic acid Natural products OC1OC(C(O)=O)C(O)C(O)C1O AEMOLEFTQBMNLQ-UHFFFAOYSA-N 0.000 description 8
- 210000004369 blood Anatomy 0.000 description 8
- 239000008280 blood Substances 0.000 description 8
- 238000004587 chromatography analysis Methods 0.000 description 8
- 239000002158 endotoxin Substances 0.000 description 8
- 238000000338 in vitro Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- IAJILQKETJEXLJ-QTBDOELSSA-N aldehydo-D-glucuronic acid Chemical compound O=C[C@H](O)[C@@H](O)[C@H](O)[C@H](O)C(O)=O IAJILQKETJEXLJ-QTBDOELSSA-N 0.000 description 7
- 229960004969 dalteparin Drugs 0.000 description 7
- 229940018872 dalteparin sodium Drugs 0.000 description 7
- 102000004169 proteins and genes Human genes 0.000 description 7
- 108090000623 proteins and genes Proteins 0.000 description 7
- 238000011002 quantification Methods 0.000 description 7
- 241000894007 species Species 0.000 description 7
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 241000283690 Bos taurus Species 0.000 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 description 6
- 238000010521 absorption reaction Methods 0.000 description 6
- 238000001914 filtration Methods 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- 238000007689 inspection Methods 0.000 description 6
- 238000001819 mass spectrum Methods 0.000 description 6
- 229910052708 sodium Inorganic materials 0.000 description 6
- SRBFZHDQGSBBOR-IOVATXLUSA-N D-xylopyranose Chemical group O[C@@H]1COC(O)[C@H](O)[C@H]1O SRBFZHDQGSBBOR-IOVATXLUSA-N 0.000 description 5
- 101001047819 Homo sapiens Heparanase Proteins 0.000 description 5
- 238000003556 assay Methods 0.000 description 5
- 230000000593 degrading effect Effects 0.000 description 5
- 238000001425 electrospray ionisation time-of-flight mass spectrometry Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 238000009472 formulation Methods 0.000 description 5
- 238000004811 liquid chromatography Methods 0.000 description 5
- 210000000056 organ Anatomy 0.000 description 5
- 238000003753 real-time PCR Methods 0.000 description 5
- 239000013558 reference substance Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 108010049003 Fibrinogen Proteins 0.000 description 4
- 102000008946 Fibrinogen Human genes 0.000 description 4
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 150000007942 carboxylates Chemical class 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000012295 chemical reaction liquid Substances 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 238000001212 derivatisation Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- JQVDAXLFBXTEQA-UHFFFAOYSA-N dibutylamine Chemical compound CCCCNCCCC JQVDAXLFBXTEQA-UHFFFAOYSA-N 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 150000002118 epoxides Chemical class 0.000 description 4
- 229940012952 fibrinogen Drugs 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 239000008176 lyophilized powder Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000014508 negative regulation of coagulation Effects 0.000 description 4
- 210000002381 plasma Anatomy 0.000 description 4
- 239000000843 powder Substances 0.000 description 4
- 238000001556 precipitation Methods 0.000 description 4
- 239000011347 resin Substances 0.000 description 4
- 229920005989 resin Polymers 0.000 description 4
- 238000001228 spectrum Methods 0.000 description 4
- 238000012916 structural analysis Methods 0.000 description 4
- BDHFUVZGWQCTTF-UHFFFAOYSA-M sulfonate Chemical compound [O-]S(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-M 0.000 description 4
- 238000010998 test method Methods 0.000 description 4
- 238000005160 1H NMR spectroscopy Methods 0.000 description 3
- WVDDGKGOMKODPV-UHFFFAOYSA-N Benzyl alcohol Chemical compound OCC1=CC=CC=C1 WVDDGKGOMKODPV-UHFFFAOYSA-N 0.000 description 3
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 3
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 3
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 3
- 208000001435 Thromboembolism Diseases 0.000 description 3
- 230000002159 abnormal effect Effects 0.000 description 3
- PYMYPHUHKUWMLA-UHFFFAOYSA-N arabinose Natural products OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 3
- SRBFZHDQGSBBOR-UHFFFAOYSA-N beta-D-Pyranose-Lyxose Natural products OC1COC(O)C(O)C1O SRBFZHDQGSBBOR-UHFFFAOYSA-N 0.000 description 3
- 239000000872 buffer Substances 0.000 description 3
- VSGNNIFQASZAOI-UHFFFAOYSA-L calcium acetate Chemical compound [Ca+2].CC([O-])=O.CC([O-])=O VSGNNIFQASZAOI-UHFFFAOYSA-L 0.000 description 3
- 239000001639 calcium acetate Substances 0.000 description 3
- 229960005147 calcium acetate Drugs 0.000 description 3
- 235000011092 calcium acetate Nutrition 0.000 description 3
- 150000001875 compounds Chemical class 0.000 description 3
- 229940088679 drug related substance Drugs 0.000 description 3
- 238000001437 electrospray ionisation time-of-flight quadrupole detection Methods 0.000 description 3
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000012544 monitoring process Methods 0.000 description 3
- 238000000569 multi-angle light scattering Methods 0.000 description 3
- 229940045627 porcine heparin Drugs 0.000 description 3
- 239000003755 preservative agent Substances 0.000 description 3
- 239000012488 sample solution Substances 0.000 description 3
- 239000001509 sodium citrate Substances 0.000 description 3
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 3
- 239000002904 solvent Substances 0.000 description 3
- 239000007921 spray Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- AEMOLEFTQBMNLQ-VANFPWTGSA-N D-mannopyranuronic acid Chemical compound OC1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@@H]1O AEMOLEFTQBMNLQ-VANFPWTGSA-N 0.000 description 2
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical group CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- 208000032843 Hemorrhage Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- OVRNDRQMDRJTHS-RTRLPJTCSA-N N-acetyl-D-glucosamine Chemical compound CC(=O)N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-RTRLPJTCSA-N 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 239000008186 active pharmaceutical agent Substances 0.000 description 2
- AEMOLEFTQBMNLQ-VCSGLWQLSA-N alpha-L-iduronic acid Chemical compound O[C@@H]1O[C@@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-VCSGLWQLSA-N 0.000 description 2
- 239000004019 antithrombin Substances 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 229960003616 bemiparin Drugs 0.000 description 2
- MSWZFWKMSRAUBD-QZABAPFNSA-N beta-D-glucosamine Chemical compound N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-QZABAPFNSA-N 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 208000034158 bleeding Diseases 0.000 description 2
- 230000000740 bleeding effect Effects 0.000 description 2
- 230000023555 blood coagulation Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 239000005388 borosilicate glass Substances 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 239000012043 crude product Substances 0.000 description 2
- 238000004042 decolorization Methods 0.000 description 2
- 238000007599 discharging Methods 0.000 description 2
- 229960000610 enoxaparin Drugs 0.000 description 2
- 125000004185 ester group Chemical group 0.000 description 2
- 230000007717 exclusion Effects 0.000 description 2
- 229940087051 fragmin Drugs 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000005227 gel permeation chromatography Methods 0.000 description 2
- 101150055760 glcA gene Proteins 0.000 description 2
- 150000004676 glycans Polymers 0.000 description 2
- 238000005570 heteronuclear single quantum coherence Methods 0.000 description 2
- 230000000968 intestinal effect Effects 0.000 description 2
- 210000004347 intestinal mucosa Anatomy 0.000 description 2
- 231100000636 lethal dose Toxicity 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000005192 partition Methods 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000002335 preservative effect Effects 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 101150109655 ptsG gene Proteins 0.000 description 2
- 230000002685 pulmonary effect Effects 0.000 description 2
- 238000004451 qualitative analysis Methods 0.000 description 2
- 238000012372 quality testing Methods 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920006395 saturated elastomer Polymers 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 230000001180 sulfating effect Effects 0.000 description 2
- 230000001502 supplementing effect Effects 0.000 description 2
- 239000013076 target substance Substances 0.000 description 2
- 229960005062 tinzaparin Drugs 0.000 description 2
- 231100000820 toxicity test Toxicity 0.000 description 2
- 238000001291 vacuum drying Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- PGOHTUIFYSHAQG-LJSDBVFPSA-N (2S)-6-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-5-amino-2-[[(2S)-2-[[(2S)-2-[[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-5-amino-2-[[(2S)-1-[(2S,3R)-2-[[(2S)-2-[[(2S)-2-[[(2R)-2-[[(2S)-2-[[(2S)-2-[[2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-1-[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-4-methylsulfanylbutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]propanoyl]pyrrolidine-2-carbonyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-methylpentanoyl]amino]acetyl]amino]-3-hydroxypropanoyl]amino]-4-methylpentanoyl]amino]-3-sulfanylpropanoyl]amino]-4-methylsulfanylbutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-hydroxybutanoyl]pyrrolidine-2-carbonyl]amino]-5-oxopentanoyl]amino]-3-hydroxypropanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-5-yl)propanoyl]amino]-4-methylpentanoyl]amino]-3-hydroxybutanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-5-carbamimidamidopentanoyl]amino]-5-oxopentanoyl]amino]-3-hydroxybutanoyl]amino]-3-hydroxypropanoyl]amino]-3-carboxypropanoyl]amino]-3-hydroxypropanoyl]amino]-5-oxopentanoyl]amino]-5-oxopentanoyl]amino]-3-phenylpropanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-methylbutanoyl]amino]-4-methylpentanoyl]amino]-4-oxobutanoyl]amino]-5-carbamimidamidopentanoyl]amino]-3-(1H-indol-3-yl)propanoyl]amino]-4-carboxybutanoyl]amino]-5-oxopentanoyl]amino]hexanoic acid Chemical compound CSCC[C@H](N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCCCN)C(O)=O PGOHTUIFYSHAQG-LJSDBVFPSA-N 0.000 description 1
- AEMOLEFTQBMNLQ-XVYLPRKHSA-N (2r,3s,4r,5s)-3,4,5,6-tetrahydroxyoxane-2-carboxylic acid Chemical compound OC1O[C@@H](C(O)=O)[C@@H](O)[C@@H](O)[C@@H]1O AEMOLEFTQBMNLQ-XVYLPRKHSA-N 0.000 description 1
- PURMPUDWXOWORS-QTBDOELSSA-N (2s,3s,4s,5r)-2,3,4-trihydroxy-6-oxo-5-sulfooxyhexanoic acid Chemical compound OC(=O)[C@@H](O)[C@@H](O)[C@H](O)[C@@H](OS(O)(=O)=O)C=O PURMPUDWXOWORS-QTBDOELSSA-N 0.000 description 1
- 108010039209 Blood Coagulation Factors Proteins 0.000 description 1
- 102000015081 Blood Coagulation Factors Human genes 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 206010053567 Coagulopathies Diseases 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- YZCKVEUIGOORGS-OUBTZVSYSA-N Deuterium Chemical compound [2H] YZCKVEUIGOORGS-OUBTZVSYSA-N 0.000 description 1
- 108010074860 Factor Xa Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 241000694408 Isomeris Species 0.000 description 1
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- 241000283977 Oryctolagus Species 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 240000001462 Pleurotus ostreatus Species 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- YZCKVEUIGOORGS-IGMARMGPSA-N Protium Chemical compound [1H] YZCKVEUIGOORGS-IGMARMGPSA-N 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108010000499 Thromboplastin Proteins 0.000 description 1
- 102000002262 Thromboplastin Human genes 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 230000005856 abnormality Effects 0.000 description 1
- 125000002777 acetyl group Chemical group [H]C([H])([H])C(*)=O 0.000 description 1
- AEMOLEFTQBMNLQ-WAXACMCWSA-N alpha-D-glucuronic acid Chemical compound O[C@H]1O[C@H](C(O)=O)[C@@H](O)[C@H](O)[C@H]1O AEMOLEFTQBMNLQ-WAXACMCWSA-N 0.000 description 1
- 235000019270 ammonium chloride Nutrition 0.000 description 1
- 150000001450 anions Chemical class 0.000 description 1
- 229940127217 antithrombotic drug Drugs 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 235000019445 benzyl alcohol Nutrition 0.000 description 1
- 239000003114 blood coagulation factor Substances 0.000 description 1
- 229940064551 bovine heparin Drugs 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 238000007385 chemical modification Methods 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 230000035602 clotting Effects 0.000 description 1
- 230000001332 colony forming effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000011033 desalting Methods 0.000 description 1
- 229910052805 deuterium Inorganic materials 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000001647 drug administration Methods 0.000 description 1
- 230000009982 effect on human Effects 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 229960005153 enoxaparin sodium Drugs 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 125000002951 idosyl group Chemical group C1([C@@H](O)[C@H](O)[C@@H](O)[C@H](O1)CO)* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000000977 initiatory effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000010253 intravenous injection Methods 0.000 description 1
- 235000015141 kefir Nutrition 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 238000002514 liquid chromatography mass spectrum Methods 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- HEBKCHPVOIAQTA-UHFFFAOYSA-N meso ribitol Natural products OCC(O)C(O)C(O)CO HEBKCHPVOIAQTA-UHFFFAOYSA-N 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 239000002808 molecular sieve Substances 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 229950006780 n-acetylglucosamine Drugs 0.000 description 1
- 229960000899 nadroparin Drugs 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 230000005311 nuclear magnetism Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 229960004762 parnaparin Drugs 0.000 description 1
- 231100000915 pathological change Toxicity 0.000 description 1
- 230000036285 pathological change Effects 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 238000005220 pharmaceutical analysis Methods 0.000 description 1
- WVDDGKGOMKODPV-ZQBYOMGUSA-N phenyl(114C)methanol Chemical compound O[14CH2]C1=CC=CC=C1 WVDDGKGOMKODPV-ZQBYOMGUSA-N 0.000 description 1
- 230000000704 physical effect Effects 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 238000002953 preparative HPLC Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000006722 reduction reaction Methods 0.000 description 1
- 239000013557 residual solvent Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000002356 single layer Substances 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
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 229910001220 stainless steel Inorganic materials 0.000 description 1
- 239000010935 stainless steel Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- CIJQTPFWFXOSEO-NDMITSJXSA-J tetrasodium;(2r,3r,4s)-2-[(2r,3s,4r,5r,6s)-5-acetamido-6-[(1r,2r,3r,4r)-4-[(2r,3s,4r,5r,6r)-5-acetamido-6-[(4r,5r,6r)-2-carboxylato-4,5-dihydroxy-6-[[(1r,3r,4r,5r)-3-hydroxy-4-(sulfonatoamino)-6,8-dioxabicyclo[3.2.1]octan-2-yl]oxy]oxan-3-yl]oxy-2-(hydroxy Chemical compound [Na+].[Na+].[Na+].[Na+].O([C@@H]1[C@@H](COS(O)(=O)=O)O[C@@H]([C@@H]([C@H]1O)NC(C)=O)O[C@@H]1C(C[C@H]([C@@H]([C@H]1O)O)O[C@@H]1[C@@H](CO)O[C@H](OC2C(O[C@@H](OC3[C@@H]([C@@H](NS([O-])(=O)=O)[C@@H]4OC[C@H]3O4)O)[C@H](O)[C@H]2O)C([O-])=O)[C@H](NC(C)=O)[C@H]1C)C([O-])=O)[C@@H]1OC(C([O-])=O)=C[C@H](O)[C@H]1O CIJQTPFWFXOSEO-NDMITSJXSA-J 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 238000001269 time-of-flight mass spectrometry Methods 0.000 description 1
- 230000008791 toxic response Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 210000001364 upper extremity Anatomy 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/702—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- 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
- A61K31/7024—Esters of saccharides
-
- 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
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
-
- 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
- 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
- A61K31/737—Sulfated polysaccharides, e.g. chondroitin sulfate, dermatan sulfate
-
- 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
- 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)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Pharmacology & Pharmacy (AREA)
- Molecular Biology (AREA)
- Epidemiology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Dermatology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Diabetes (AREA)
- Materials Engineering (AREA)
- Hematology (AREA)
- Biochemistry (AREA)
- Polymers & Plastics (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- Cardiology (AREA)
- Heart & Thoracic Surgery (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
Ext> theext> inventionext> providesext> anext> oligosaccharideext> mixtureext> derivedext> fromext> heparanext>,ext> aext> preparationext> methodext> andext> applicationext> thereofext>,ext> whereinext> theext> weightext> averageext> molecularext> weightext> ofext> theext> oligosaccharideext> mixtureext> isext> 4000ext> Daext> -ext> 5000ext> Daext>,ext> theext> nonext> -ext> reducingext> endext> isext> aext> 4ext>,ext> 5ext> -ext> unsaturatedext> hexuronicext> acidext> structureext>,ext> theext> structureext> ofext> theext> oligosaccharideext> mixtureext> isext> obviouslyext> differentext> fromext> thatext> ofext> theext> existingext> medicalext> lowext> -ext> molecularext> heparinext> andext> refinedext> heparinext>,ext> andext> theext> oligosaccharideext> mixtureext> containsext> theext> characteristicsext> ofext> completeext> connectingext> areaext> structureext>,ext> highext> sulfationext> modificationext> degreeext>,ext> highext> contentext> ofext> highext> sulfationext> componentsext>,ext> 2.3ext> -ext> 2.5ext>%ext> ofext> Gext> -ext> Aext>,ext> highext> iduronicext> acidext> abundanceext> andext> theext> likeext> besidesext> aext> specialext> monosaccharideext> structureext>.ext> The oligosaccharide mixture has antithrombotic effect, has small toxic and side effects on mice due to subcutaneous injection, and can be developed into medicine. The heparin provided by the invention is derived from sheep, is simple and easy to obtain, expands the source of medical heparin, and has obvious economic effect.
Description
Technical Field
The invention belongs to the technical field of biological medicines, and relates to an oligosaccharide mixture derived from heparan caprae seu ovis, a preparation method and application thereof in treating and preventing thromboembolic diseases.
Background
Heparin (Heparin) and low molecular Heparin (Low Molecular Weight Heparin, LWMH) are important clinical anticoagulants and antithrombotics. Structurally, they mainly consist of a disaccharide repeating unit of one of glucosamine (D-glucosamine, glcN) and two kinds of uronic acid (iduronic acid (IdoA) and glucuronic acid (D-glucuronic, glcA)), and have sulfate groups randomly modified at different positions of the sugar unit. Their structure can be represented by a tetrasaccharide unit, a and B as shown below, while C shows the pentasaccharide structure of heparin binding to antithrombin, which is the core structure necessary to maintain anticoagulant activity.
From the above figures, heparin structure is various depending on the number and type of sugar units, the number and position of sulfate group modification, and the like. The main part of the heparin oligosaccharide chain is a regular region with ordered structure, namely a trisulfated disaccharide repeating unit with ordered structure, namely I2S-GlcNS6S (2-oxo-sulfate-iduronic acid-nitrogen-sulfate-6-oxo sulfate-glucosamine), and is shown as A; and B shows the low sulfation region, which is a "random region" of disordered structure, mainly some units containing IdoA (iduronic acid) and GlcA (glucuronic acid) (followed by N-acetyl-GlcN or N-sulfate-GlcN (GlcNAc, glcNS)) [ R.J.Linhardt, J.Med.Chem.46, (2003) 2551-2564 ], part B being smaller in heparin oligosaccharide chains. In addition, C shows the pentasaccharide structure of heparin and antithrombin binding site, bold indicates that it is necessary to maintain anticoagulant activity, if removed, the anticoagulant activity is reduced by 95%, and italics indicates that it is important that the anticoagulant activity is reduced by 25-50% after removal.
Compared with the large-molecular refined heparin with the weight average molecular weight of thousands to tens of thousands, the low-molecular heparin has small weight average molecular weight, generally has different weight average molecular weight of 3000Da to 8000Da and is more concentrated in distribution. The low molecular heparin has the advantages of good bioavailability and small side effects such as major bleeding risk. Currently, the main low molecular heparin for medical use in the mainstream market (U.S., china and europe) is mainly: natrexed heparin (Nadroparin), dalteparin (Dalteparin), enoxaparin (Enoxaparin), tinzaparin (Tinzaparin), pamaparin (Parnaparin), bemiparin (Bemiparin), and the like, All derived from pig intestine mucosa liver Element (A). Although these low molecular heparin are all derived from natural macromolecular heparin, the preparation process is different, and thus the molecular structure is also significantly different.
In the process of low molecular heparin production, heparin sugar chains tend to be selectively broken or modified, resulting in some specific structure. For example, when the depolymerization process is used to produce, the 6-position of glucosamine in the oligosaccharide chain is basic or heated, the desulphation ester group is further dehydrated to form an internal ether structure [ Mascellani, et al carbohydrate. Res. (2007) 342, 835-842 ] ], the action of the base also causes desulphation ester group at the 2-O position of idose [ IdoA (2S) ] and dehydration to form 2, 3-epoxy compound (2, 3 epoxy) or galacturonic acid (Galacturonic acid, galA) [ Mourier, P.A.J.; viskov, anal. Biochem. (2004), 332, 299-313.], heparin esters cause unsaturated uronic acid at the non-reducing end when depolymerization occurs in beta-elimination [ T.Toida, J.CarbohydrateChemistry, (1996) 15 (3), 351-360.], which are special structures that may be present in some low molecular weight heparin. In addition, heparin is a mixture of sugar chains of different sizes, different sugar chain molecules in the same stack of heparin, and monosaccharide composition sequences tend to be different, so that in depolymerization of low molecular heparin preparation, the breaking points of sugar chains are also various, and breaking tends to occur more easily in the polysulfated region [ casu b.et al elsevier ltd; (2006): 1-28], which affects the proportion of sulfation in each sugar chain molecule in low molecular heparin.
It is well known that heparin of other species or organ sources such as heparins and Niu Gansu (including bovine intestinal mucosal heparin and bovine pulmonary heparin) and the like, although similar in physicochemical and biological properties, differ to a different extent in the characteristics of the molecular structure and unit composition of the sugar chain and the like as compared to porcine intestinal mucosal heparin [ Zhang, f., et al Bioanal Chem (2011) 401:2793.], which is a source difference that can be categorized separately. If the low molecular heparin meeting the medical requirements can be prepared by using other source heparin, the low molecular heparin can be an important and beneficial supplement of the low molecular heparin of clinical pig source, and the source of the medical heparin can be enriched. However, this approach also necessarily brings about a unique structure due to the species or process, potentially bringing about a certain difference in biological activity. Non-swine source materials or products are generally clear and have great economic value.
The preparation and analysis characterization of low molecular heparin are complex, and the technological requirements for the process and analysis are extremely high. In the field, researchers have started research on low molecular heparin such as low molecular heparin prepared from non-porcine heparin (including bovine-derived heparin and sheep-derived heparin), and currently, mainly focused on some technological aspects, and few systematic researches are performed. Wherein [ Xinyue Liu, et al clinical and Applied Thrombosis/Hemostasis, (2017) 1-12] is one of the earliest studies on non-porcine low molecular heparin, wherein the prepared bovine low molecular heparin has remarkable difference in structure and physicochemical properties with clinical commercial medical porcine heparin.
Sheep-derived heparin is an important resource, and macromolecular sheep (fine) heparin has been used for clinical anticoagulation in some non-canonical markets in southeast asia. However, the international market is still lack of sheep-derived low molecular heparin or low molecular heparin drugs.
Disclosure of Invention
In view of the above-mentioned drawbacks of the prior art, the present invention aims to provide an oligosaccharide mixture derived from heparan caprae seu ovis for the preparation of new anti-thrombotic and halamic drugs.
The aim of the invention is achieved by the following technical scheme:
an oligosaccharide mixture is prepared from heparan Caprae Seu Ovis, and has the main structure shown in the following formula:
wherein: n=1-24; r is R 1 =h or SO 3 Na or COCH 3 ；R 2 =h or SO 3 Na；
The oligosaccharide mixture has a non-reducing end of a 4, 5-unsaturated uronic acid structure shown in the structural formula, and comprises 4, 5-unsaturated uronic acid-2-O-sulfate (delta U) 2S ) And 4, 5-unsaturated uronic acid (. DELTA.U).
The weight average molecular weight of the oligosaccharide mixture is 4000Da-5000 Da.
The structural features of the oligosaccharide mixture according to the invention are described in the detailed description below, and in the examples.
The oligosaccharide mixture has an anticoagulant and antithrombotic effect, and the anti-Xa activity depending on antithrombin III is 80-120U/mg.
The preparation method of the oligosaccharide mixture is prepared by derivatization of sheep intestinal mucosa heparin, and adopts a beta-elimination depolymerization chemical method.
The preparation method of the oligosaccharide mixture comprises the steps of derivatizing the heparan, initiating beta-elimination depolymerization reaction by using alkali, and forming a 4, 5-unsaturated uronic acid structure at the non-reducing end of a sugar chain of a product.
Preferably, the depolymerization reaction of beta-elimination is initiated by alkali action, one method is to dissolve the heparan derivative in an organic solvent system and initiate the depolymerization reaction by organic alkali action, and the other method is to dissolve the heparan derivative in an aqueous solution system and initiate the depolymerization reaction by inorganic alkali action.
The preparation method of the oligosaccharide mixture comprises the following steps: dissolving heparan in purified water, mixing with an aqueous solution prepared from benzethonium chloride or benzalkonium bromide with the mass of more than or equal to 2.5 times that of the heparan, collecting and separating out white solid insoluble matters, and drying to obtain heparin ammonium salt; dissolving heparin ammonium salt in an organic solvent with the mass of 5 times or more of the heparan, adding benzyl trimethyl ammonium hydroxide (Triton-B) with the mass of 0.5 times or more of the heparan, stirring, reacting and depolymerizing for more than 12 hours; adding sodium acetate methanol solution prepared from sodium acetate with the mass of 0.5 times or more of that of the heparan Caprae Seu Ovis after the reaction, collecting the precipitate, re-dissolving with saline, precipitating with ethanol, grading, collecting the component with the weight average molecular weight of 4000Da-5000Da, refining, and drying to obtain oligosaccharide mixture.
In one set of examples, but not limited to this example (example 1), the preparation method is as follows: dissolving heparan in 10 times of purified water, mixing with 2.5 times of aqueous solution prepared from benzethonium chloride, centrifuging, collecting white solid insoluble substances, and drying to obtain heparin ammonium salt; dissolving heparin ammonium salt in 20 times of dichloromethane, adding 0.5 times of benzyl trimethyl ammonium hydroxide, stirring and depolymerizing for 24 hours; preparing 1.8 times of sodium acetate and 18 times of methanol for dissolution, mixing the sodium acetate methanol solution with heparin reaction solution, separating out precipitate, centrifugally collecting, redissolving with 5 times of 5% saline, taking the fractional precipitate between 3 times and 6 times of ethanol, performing SEC-HPLC (SEC-HPLC) to control and check that the weight average molecular weight meets the regulation, drying, redissolving with 4 times of water for injection, and freeze-drying to obtain the oligosaccharide mixture.
The preparation method of the oligosaccharide mixture comprises the following steps: dissolving heparan in purified water, mixing with 2.5 times or more of aqueous solution prepared from benzethonium chloride or benzalkonium bromide, collecting the precipitated white solid insoluble substance, and drying to obtain heparin ammonium salt; dissolving heparin ammonium salt in an organic solvent with the weight of 5 times or more, adding benzyl chloride with the weight of 0.5 times or more, stirring and reacting for 12 hours or more, adding sodium acetate with the weight of 0.5 times or more to prepare sodium acetate methanol solution after the reaction is finished, collecting precipitated precipitate, and drying to obtain heparin benzyl ester obtained by derivatization; dissolving heparin benzyl ester in water, heating, adding alkali solution, stirring for depolymerization for more than 1 hr, precipitating with ethanol, re-dissolving precipitate with saline, precipitating with ethanol, grading, collecting fraction with weight average molecular weight of 4000-5000 Da, refining, and drying to obtain oligosaccharide mixture.
In one set of examples, but not limited to this example (example 3), the preparation method is as follows: dissolving heparan in 6 times of water, mixing with 2.5 times of benzethonium chloride and 10 times of purified water, centrifuging, collecting white precipitate, and lyophilizing to obtain heparin ammonium salt; dissolving heparin ammonium salt in 20 times of dichloromethane, stirring to dissolve, adding 4 times of benzyl chloride, and stirring to react for 20 hours; preparing 2 times of sodium acetate and 20 times of methanol for dissolution, mixing the sodium acetate methanol solution with heparin reaction liquid, separating out precipitate, centrifugally collecting, and drying to obtain heparin benzyl ester; dissolving heparin benzyl ester with 15 times of water, heating and maintaining at 55-58 ℃, adding 0.1 times of sodium hydroxide solid dissolved in advance (0.3 times of water), and stirring for reacting for 2 hours; cooling, adding 0.5 times of 30% hydrogen peroxide, standing for decolorizing for 2 hours, adding 0.5 times of sodium chloride, stirring for dissolving, filtering the reaction solution, adding methanol for fractional precipitation, taking 15-40 times of precipitate, performing SEC-HPLC (SEC-HPLC) central control to check that the weight average molecular weight meets the regulation, and drying to obtain oligosaccharide mixture.
The oligosaccharide mixture is applied to the prevention and treatment of the related thromboembolic diseases and developed into anticoagulant and antithrombotic medicines or halamic medicines.
A pharmaceutical composition comprising the oligosaccharide mixture described above and a pharmaceutically acceptable carrier.
The pharmaceutical composition containing the oligosaccharide mixture is applied to the prevention and treatment of related thromboembolic diseases and is developed into an anticoagulant and antithrombotic drug or an Islamic drug.
In one set of examples, but not limited to this example (example 17), the anticoagulation of the oligosaccharide mixture was examined in an in vitro test of human blood clotting. After blood plasma is separated, the influence of the oligosaccharide mixture on the blood coagulation routine, including APTT, TT, PT and the like, is examined according to an automatic coagulometer and a kit method, and the result shows that the oligosaccharide mixture obviously prolongs the APTT and the TT, but has no obvious effect on PT, and the anticoagulation effect is consistent with that of clinical commercial medical low-molecular heparin.
In one set of examples, but not limited to this example (example 18), the anticoagulation of the oligosaccharide mixture was performed in vivo in rabbits. After subcutaneous injection, rabbit blood was collected before and at various time points after administration, anticoagulated with 3.8% sodium citrate anticoagulant at a ratio of 1:9, and the effects on clotting routines, including anti-Xa, APTT, TT and PT, and other clotting factors were examined on-board. The results show that the oligosaccharide mixture remarkably prolongs APTT and TT, has no obvious effect on PT, has an anticoagulant effect in vivo, and is consistent with clinical commercial medical low-molecular heparin.
Pharmaceutical compositions containing the oligosaccharide mixture are administered by a route including, but not limited to, subcutaneous or intravenous injection, oral administration, external application, and pulmonary route.
The safety of the oligosaccharide mixture was experimentally examined in the present invention, and in one set of examples, but not limited to this example (example 19), the toxic response and death of ICR mice was examined with an aqueous solution of the oligosaccharide mixture by subcutaneous injection, resulting in LD 50 No pathological changes of organs and the like of the surviving mice in the experiment are abnormal at 3100 mg/Kg.
If the oligosaccharide mixture is used for substituting clinical commercial medical low-molecular heparin for anticoagulation treatment, the equivalent dose of the anti-Xa potency is required to be followed, so that the concentration of the human subcutaneous injection is about 1mg/Kg and is far lower than the LD of 3100mg/Kg 50 The values reflect that the oligosaccharide mixture is safe for subcutaneous injection.
The structure of the oligosaccharide mixture is characterized in detail, and the details are described below.
Oligosaccharide mixture belongs to low molecular heparin, is used as a complex molecular medicine with a non-single sugar chain structure, and has the structural characteristics which are difficult to analyze. The oligosaccharide mixture is a composition of sugar chains of different sizes, and cannot solve the problem by using only a Single Method (Single Method) or a Single layer (Single Level) for analysis such as HPLC and NMR, unlike a small molecule compound of a Single structure.
In the present invention, the process of characterizing the oligosaccharide mixture will be analyzed from a series of Multi-dimensions (Multi-dimentional Approach). This can be illustrated briefly as follows:
specifically, "Top-Down", i.e., from the whole oligosaccharide chain sequence to the constituent units, disaccharides, to monosaccharides, is carried out by subjecting the oligosaccharide mixture to partial or complete degradation with specific heparinases (Heparinase I, II, III, heparinases 1, 2, 3), either singly or in combination, which may be selectively to varying degrees, and separating the degraded components by chromatography, each component being then structurally resolved by mass spectrometry or the like. In addition, "Bottom-Up" refers to the sequence structure of a larger unit or complete oligosaccharide linkage, which is mapped and deduced in combination with detailed structural information obtained from analysis of monosaccharides, disaccharides, and the like. In this series of studies, it is necessary to use high pressure liquid chromatography (High Pressure Liquid Chromatography, HPLC), nuclear magnetic resonance (Nuclear Magnetic Resonance, NMR), mass Spectrometry (MS), rapid protein liquid chromatography (Fast Protein Liquid Chromatography, FPLC), enzymatic correlations, and the like, and to obtain Oligosaccharide (Oligosaccharide), disaccharide (Disaccharide), tetrasaccharide (Tetrasaccharide), hexasaccharide (Hexasaccharide), and the like in composition and sequence, and other effective information. The analyzed information is summarized to obtain the characteristics of the oligosaccharide mixture in different dimensions of each layer, so that the structure of the oligosaccharide mixture is effectively analyzed, and the difference between the oligosaccharide mixture and other low-molecular heparin, including the new structural characteristics, is clear.
Heparanase 1, 2, 3 is a site-specific selectively specific heparin degrading enzyme, heparanase selectivity [ Linhardt, et al, biochem. (1990), 29 (10), 2611-2617.][Product information，Heparinase]The structure is as follows:
selectivity of heparanase I, II, III
The partial characterization method for this oligosaccharide mixture is listed below:
TABLE 1 analysis method of sugar chain composition of oligosaccharide mixture
Mass Spectrometry (MS) and Nuclear Magnetic Resonance (NMR) are important tools for resolving polysaccharide structures. The mass spectrometry method is sensitive and efficient, and has been widely applied to structural analysis of sulfated polysaccharides [ Brustkern AM et al Anal chem. (2010), 82 (23), 9865-70 ] [ Doneanu CE, et al Analchem. (2009), 81 (9), 3485-99] [ HenriksenJ, et al Carbohydroles. (2006), 341 (3), 382-7]. The m/z values obtained in mass spectrometry can be used to calculate the weight average molecular weight of an analysis object for identifying its structure and chemical formula. It is also generally necessary to separate the individual components by chromatography/HPLC prior to mass spectrometry of the mixture of the components [ Teresa Cecchi, analytical Chemistry (2009) -215]. Chromatography mass spectrometry (e.g., IPRP-HPLC and ESI/TOF MS) has been used for structural analysis of some low molecular heparin sodium imitation pharmaceuticals. [ Jerkovich AD, et al LC-GC North Am, (2003), 21:600-610] [ Swartz me.j Liq Chromatogr Relat Technol, (2005), 28:1253-1263 ] [ L.Fu, et al J.of Pharmaceutical science, (2013), doi 10.1002/jps23501 ] [ Langslay D.J., et al journal of Chromatography A, (2013), 1292, 201-211 ]
The nuclear magnetic resonance, in particular, the two-dimensional nuclear magnetic resonance (HSQC-NMR) can analyze the structural information of low molecular heparin, and can analyze not only the abnormal peak signal but also the signal of the ring structure region [ Torri g., et al pharmaceutical analysis (2008), chapter 4, 407-428 ]. 2-O-sulfoglucuronic acid (GlcA 2S) is very low in natural heparin, which is generally absent in HSQC-NMR analysis, but present in some low molecular heparin [ Yamada S., et al j. Biol. Chem. (1995), 270, 8696-8705] [ guerrin M, et al, semin Thromb heat (2007) 33:478-487]. In addition, the HSQC-NMR can detect the epoxy compounds produced by depolymerization, the signals of H2/C2 and H3/C3 of which are 3.74/54.2ppm and 3.82/53.3ppm, respectively, and the epoxy compounds can further form galacturonic acid (L-galacturonic acid, galA) during depolymerization. Under alkaline treatment under different conditions, the epoxide opens to form α -L-iduronic acid or isomerises to galacturonic acid [ JasejaM, et al can J Chem (1989) 67:1449-1456]. Some of the GalA signals overlap with IdoA, in particular the H5/C5 signals, all at 4.69/74.5ppm, but were detectable by HSQC-NMR.
The structure of the oligosaccharide mixture is characterized by the following detailed analysis and examples section.
1. Two-dimensional nuclear magnetic (NMR-HSQC) analysis of monosaccharide compositions
There are several documents reporting HSQC-NMR spectra and analysis results of clinical commercial medical low molecular heparin products, using nuclear magnetism of 500MHz or more is sufficient to obtain high resolution results [ Capila I., et al methods for structural analysis of heparin and heparan sulfate, in: chemistry and Biology of Heparin and Heparan Sulphate, H.G.Garg, R.J.Linhardt, C.A.Hales, ed, (2005) Chapter 3, 55-71,Elsevier Inc.Oxford ] [ Guerrini m., et al heparin-A Century of Progress, handbook of Experimental Pharmacology 207].
In one example, but not limited to this example (example 7), we performed HSQC-NMR analysis of a multi-batch oligosaccharide mixture using the method reported in the literature, which showed the following characteristics: ext> theext> monosaccharideext> isext> mainlyext> aext> generalext> typeext> monosaccharideext> ofext> heparinext>,ext> containsext> aext> specialext> typeext> ofext> monosaccharideext> structureext>,ext> theext> contentext> ofext> eachext> monosaccharideext> componentext> isext> obviouslyext> differentext> fromext> otherext> heparinext>,ext> theext> sulfationext> modificationext> degreeext> isext> highext>,ext> theext> Gext> -ext> Aext> contentext> isext> 2.3ext>%ext> -ext> 2.5ext>%ext>,ext> andext> theext> iduronicext> acidext> abundanceext> isext> highext>.ext>
Monosaccharides (II): the monosaccharide type of the oligosaccharide mixture determined by HSQC-NMR and clinical commercial medical heparinSimilar to low molecular heparin, the species and amounts are listed in tables 2 and 3 below, and the quantitative signal of each monosaccharide component in the oligosaccharide mixture is significantly different from other heparin; also included in the oligosaccharide mixture are specific monosaccharide structures including internal ether structures including AnhydroΔIIS/AnhydroΔIIS, unsaturated uronic acid structures, epoxy (epoxy) and galacturonic acid (GlaA) structures epi AnhydroΔIS and AnhydroΔIS-IS epi ；
Sulfating modification: most of the N-position, 6-O-position and 2-O-position of iduronic acid (IdoA) of glucosamine (GlcN) are sulfation modification, and the acetylation modification of the N-position is only 4.9%, which is significantly less than that of fine heparin and other clinical commercial medical low molecular heparin (more than 10%;
ext> Gext> -ext> aext>:ext> Ext> Gext> -ext> Aext> (ext> betaext> -ext> Dext> -ext> glucuronicext> acidext> -ext> Next> -ext> sulfonicext> acidext> groupext> 3ext> -ext> Oext> -ext> sulfuricext> acidext> groupext> -ext> alphaext> -ext> Dext> -ext> glucosamineext>)ext> relatedext> toext> anticoagulationext> activeext> siteext> isext> containedext> inext> theext> oligosaccharideext> mixtureext> inext> theext> contentext> ofext> 2.3ext>%ext> -ext> 2.5ext>%ext>;ext>
Iduronic acid: the iduronic acid/glucuronic acid ratio (IdoA/GlcA, I/G) of the oligosaccharide mixture was 4.3, i.e. the uronic acid contained more iduronic acid.
Table 2 comparison of the average percentage of hexosamine monosaccharide components of the oligosaccharide mixture analyzed by HSQC-NMR with different clinical commercially available medical heparins
Remarks: * Data from literature [ Guerrini m., et al heparin-A Century of Progress, handbook of Experimental Pharmacology 207]; * HSQC-NMR measurements of multiple batches of oligosaccharide mixture samples.
Table 3 comparison of the average percentage of uronic acid monosaccharide fractions of the oligosaccharide mixture analyzed by HSQC-NMR with different clinical commercially available medical heparins
Remarks: * Data from literature [ Guerrini m., et al heparin-A Century of Progress, handbook of Experimental Pharmacology 207]; * HSQC-NMR measurements of multiple batches of oligosaccharide mixture samples.
2. Oligosaccharide composition and sequence analysis:
conventional chromatographic or mass spectrometry analysis can analyze the major component, but the oligosaccharide mixture has a large amount of other modified components such as acetylated fragments, linker Regions (LR) and the like, which tend to be masked by the major component and cannot be effectively analyzed in a single chromatographic or mass spectrometry method. By disaggregating the macromolecules to produce small fragments, some minor structural modifications can be detected.
The invention degrades the oligosaccharide mixture by using the single heparanase 1, the single heparanase 3 and the mixed enzyme of heparanase 1+2+3, effectively distinguishes the high sulfated oligosaccharide from the low sulfated oligosaccharide by using the specificity of the heparanase 1 and the heparanase 3, and then uses HPLC and ESI-TOF MS analysis to identify.
The results show that the oligosaccharide mixture shows very interesting structural characteristics, namely, a complete connecting region structure exists, the sulfation modification degree is high, the content of the high sulfation component is high, and the non-reducing end has a novel hexosamine structure. The method comprises the following steps:
connection region: the oligosaccharide mixture contains fragments of intact and abundant heparin-binding region with average content of 1.1%, including DeltaU-Gal-Gal-Xyl-Serine, deltaU 2, 1-U-Gal-Gal-Xyl-Serine, deltaU-Gal-Gal-Xyl-CH 2 COOH (detected in degradation solution of heparinase 3) and DeltaU 4,3,1-U-Gal-Gal-Xyl-CH 2 COOH (detected in degradation solution of heparinase 1);
sulfating modification: among the components of the oligosaccharide mixture after enzymatic degradation, including the conventional disaccharides Δu2,1 (detected in heparinase 1+2+3 degradation solution) and the tetrasaccharides Δu4,2,1, Δu4,3,1 and hexasaccharides Δu6,6,1 (detected in heparinase 1 degradation solution), the content is significantly less than that of other clinically commercially available medical heparins, which in turn reflects a higher degree of sulfation modification of the oligosaccharide mixture, as evidenced by the previous HSQC-NMR;
highly sulfated component: including non-reducing ends a (trisulfated hexosamine, ANS,3,6S), tetrasaccharides Δu4,5,0 and Δu4,6,0 and larger sulfate oligosaccharide chains (tetrasaccharides to dodecasaccharides, not degraded by heparanase 3, heparanase 3 selectively degrading low sulfate sequences or sites), which are present in higher abundance in the oligosaccharide mixture than clinically commercially available medical low molecular heparin;
Novel characteristic structure: at the non-reducing end, A3,4,0 (trisaccharide, containing four sulfate groups), A3,6,0 (trisaccharide, containing six sulfate groups) and A5, 0 (pentasaccharide, containing five sulfate groups), the hexosamines of these highly sulfated components are produced by the enzymatic hydrolysis of heparinase 3, which are absent or less present in general heparin or clinically commercially available medical low molecular heparin, but are present in the oligosaccharide mixture.
The foregoing summary is set forth in a set of embodiments, but not limited to, those embodiments (examples 8-10).
3. Disaccharide and basic building block structure and mass spectrometry:
in addition, specific analyses of the chromatography-mass spectrometry were also carried out on the disaccharides and the basic constituent units of the oligosaccharide mixtures according to the invention, after the enzymatic hydrolysis by heparinase, as follows:
the oligosaccharide mixture is degraded into disaccharide units by the heparinase 1+2+3 enzyme mixture. The degradation solution was subjected to HPLC to separate a plurality of disaccharide components. The structure of these disaccharides was then resolved by mass spectrometry. In one example, but not limited to this example (example 11), after complete enzymatic hydrolysis of the oligosaccharide mixture, the following characteristics were obtained by attributing and resolving mass spectra of the individual components in the LC-MS analysis using a combination of chromatography (shimeji LC-20A high performance liquid chromatography) and mass spectrometry (sameimer femto LTQ-Orbitrap mass spectrometry): disaccharides and basic building blocks are heparin disaccharides, containing other specific structural building blocks.
Disaccharide: disaccharides of oligosaccharide mixtures are predominantly common heparin disaccharides, accounting for 80% or more of the total components, including ΔIS (ΔUA2S-GlcNS 6S), ΔIIS (ΔUA-GlcNS 6S), ΔIIIS (ΔUA2S-GlcNS), ΔIVS (ΔUA-GlcNS), ΔIA (ΔUA2S-GlcNAc6S), ΔIIA (ΔUA-GlcNAc6S), ΔIIIA (ΔUA2S-GlcNAc) and ΔIVA (ΔUA-GlcNAc);
the special structure comprises the following components: the oligosaccharide mixture also contains other basic constituent units, the total amount of which is less than 20%, including 1N-position unsubstituted disaccharide (ΔIH, ΔUA2S-GlcN 6S), 2 kinds of 3-O-tetraose sulfate (ΔIIA-IIS) glu (DeltaUA-GlcNAc 6S-GlcA-GlcNS3S 6S) and DeltaIIS-IIS glu (DeltaUA-GlcNS 6S-GlcA-GlcNS3S 6S), 2 disaccharides containing a non-reducing terminal saturated structure (Disachride (2S) NRE (IdoA 2S-GlcNS) and Disachride (3S) NRE (IdoA 2S-GlcNS 6S)), 3 oligosaccharides containing a reducing terminal endo-ether structure (AnhydroDeltaIIS/AnhydroDeltaIIS) epi (DeltaUA-GlcNS-Anhydro/DeltaUA-ManNS-Anhydro), anhydroDeltaIS (DeltaUA 2-GlcNS-Anhydro) and AnhydroDeltaIS-IS epi Trisaccharide (2S) (DeltaUA-GlcNS 6S-HexA), trisaccharide (2S, 1 Ac) (DeltaUA-GlcNAc 6S-HexA 2S), trisaccharide (3S) (DeltaUA 2S-GlcNS 6S-HexA) and Trisaccharide (4S) (DeltaUA 2S-GlcNS6S-HexA 2S), 1 Epoxide (epoxy (DeltaUA 2S-GlcNS6S-GlcA-2, 3-carbohydrate-GlcNS)), 2 heparin protein binding domain oligosaccharides (Linkage (DeltaUA-Gal-Gal-Gal-Xyl-LinkO-Ser), and kage formed by a reduction reaction of 4 kinds of reducing ends ox (ΔUA-Gal-Gal-Xyl-O-Ser ox ))。
The variety of these disaccharides and basic building blocks is mutually evident from the monosaccharide analysis of HSQC-NMR as described above, where the reducing end contains a endo-ether structure and the non-reducing end contains an unsaturated uronic acid structure. In the specific example, but not limited to this example (example 11), the content of each basic constituent unit was also relatively quantified by extracting the peak area in the ion flow chromatogram (EIC).
4. Tetraose composition analysis:
the oligosaccharide mixture of the invention is also an important evaluation mode for direct sugar unit analysis, fingerprint analysis and direct oligosaccharide analysis. Short chain oligosaccharides retain more information on the cleavage reaction and they can be used to investigate the conditions of the depolymerization reaction. It is therefore important to conduct direct short-chain oligosaccharide analysis of these sugar mixtures [ s.lee, et al Nature biotechnology (2013), 31,3, 220-226 ].
The oligosaccharide mixture was first separated from the tetraose using FPLC. The separated tetrasaccharide mixture is analyzed by HSQC-NMR. Further, each tetraose component is separated by preparative HPLC and subjected to mass spectrometry or nuclear magnetic identification structure and approximate content determination.
4.1 separation of FPLC to prepare tetraose, hexaose and the like of oligosaccharide mixture
In one example, but not limited to this example (example 12), a plurality of oligosaccharides including tetraose, hexaose, octaose, and decaose, etc., were separated from the oligosaccharide mixture by molecular sieve-FPLC (SEC-FPLC). Each oligosaccharide peak contains a plurality of components, such as tetraose peak and HPLC separation, and 15 tetraose peaks can be separated.
4.2 analysis of the resulting tetrasaccharide mixture by NMR
In one example, but not limited to this example (example 13), a tetrasaccharide mixture of oligosaccharide mixture prepared by molecular sieve-FPLC (SEC-FPLC) separation was performed 1 H-NMR analysis.
In one example, but not limited to this example (example 14), HSQC-NMR relative quantitative analysis was performed on a plurality of tetrasaccharide mixtures separated by molecular sieve-FPLC (SEC-FPLC) to prepare oligosaccharide mixtures.
As shown in Table 3, the types and contents of the oligosaccharide mixture in the above section 1 (tables 2 and 3) were different from those of the oligosaccharide mixture in the type and content of the monosaccharides, and the epoxy structure (epoxy) and galacturonic acid (GalA) were not detected.
TABLE 4 HSQC-NMR analysis of the tetrasaccharide Components of oligosaccharide mixtures the percentage of monosaccharide components
Remarks: 1) # below the detection Limit (LOD), undetected; 2) The data in the table are averaged from HSQC-NMR measurements of oligosaccharide mixtures from multiple batches.
4.3 HPLC separation of the components in the tetraose
In one example, but not limited to this example (example 15), a mixture of tetrasaccharides from the oligosaccharide mixture prepared for SEC-FPLC was separated by HPLC to a further 15 tetrasaccharide peaks.
4.4 identification of the Components by NMR or MS
In one example, but not limited to this example (example 16), qualitative analysis was performed by NMR or MS for 15 tetrasaccharide peaks prepared, respectively.
The results show that the tetrasaccharides of the oligosaccharide mixture have the following characteristics: the components are all tetraose types commonly found in heparin, and the non-reducing end is 4, 5-unsaturated uronic acid-2-O-sulfate (delta U) 2S ) Structure is as follows.
Tetraose: the oligosaccharide mixture is not subjected to enzymolysis, and the four sugar components are separated, and all the four sugar components have 4, 5-unsaturated uronic acid-2-O-sulfate (delta U) 2S ) The structure is shown in Table 5.
TABLE 5 tetrasaccharide species/Structure of oligosaccharide mixtures
The tetrasaccharides are part of the composition of the oligosaccharide mixture, are essentially different from the disaccharide/tetrasaccharide composition after the enzymolysis of the heparinase, are structural modules of the oligosaccharide mixture after the enzymolysis of the heparinase, belong to long chain units and are obtained by degrading the oligosaccharide mixture by the heparinase, but the disaccharides in the raw material of the oligosaccharide mixture are little, and the tetrasaccharides are degraded into disaccharides during the enzymolysis. In this analysis, the oligosaccharide mixture is not enzymatically hydrolyzed but is a tetrasaccharide component due to the preparation process, and the non-reducing end contains a 4, 5-unsaturated uronic acid structure.
5. Chromatography-mass spectrometry analysis of sugar chain sequences
The analysis of sugar chain information spectrum or fingerprint spectrum of the whole sample by chromatography-mass spectrometry (LC-MS) is also a very effective method, and can obtain the information of species source, weight average molecular weight, chemical modification, abnormal structure and the like of the sample.
In one example, but not limited to this example (example 16), the oligosaccharide mixture was not subjected to heparinase enzymatic hydrolysis, but identified directly by HPLC with ESI-TOF MS analysis.
The results showed that the oligosaccharide mixture was characterized as follows: the main component is an oligosaccharide component common to medical low molecular heparin in clinical market, and the high weight average molecular weight component has higher sulfation modification degree and contains a endo-ether structure.
Oligosaccharide component: the oligosaccharide mixture is similar to the non-depolymerized component chromatograms of the clinical commercial medical low-molecular heparin, but has a certain difference in component types and relative contents;
sulfation modification of high weight average molecular weight components: the high weight average molecular weight components, including Δu6,9,0 (hexasaccharide, 9 sulfate groups), Δu8, 12,0 (octasaccharide, 12 sulfate groups) and Δu10, 14,0 (decasaccharide, 14 sulfate groups), are significantly more abundant in the oligosaccharide mixture than in the clinically commercially available low molecular weight heparin. These reflect a higher sulfation modification of the oligosaccharide mixture;
Internal ether structure: some oligosaccharide chains have a reduced end with a internal ether structure.
6. Analysis of weight average molecular weight and molecular weight distribution
The weight average molecular weight and molecular weight distribution of the oligosaccharide mixture were determined by SEC-HPLC analysis and calculated in combination with GPC software, specifically: the chromatographic column is a TSKgel G2000SW (7.8 mm. Times.30 cm,5 μm) and a TSKgel G3000SW (7.8 mm. Times.30 cm,5 μm) in series, the detector is a differential refraction detector (RID), the weight average molecular weight calculation standard is weight average molecular weight calibrator A RS and B RS of USP low molecular weight heparin, and the analysis software comprises HPLC workstation and GPC software of Agilent company.
The weight average molecular weight and molecular weight distribution of the mixture of batches of oligosaccharides are characterized by the following characteristics:
the weight average molecular weight (Mw) is 4000Da-5000Da, the number average molecular weight (Mn) is 2900Da-3200Da, and the polydispersity PD is 1.2-1.5.
In addition, the absolute weight average molecular weight of the oligosaccharide mixture is measured by SEC-MALLS, the average weight average molecular weight of the largest component is between 14500Da and 16500Da, and the n maximum in the general structure of the oligosaccharide mixture in the invention can be confirmed to be 24 according to the repeated disaccharide unit and the corresponding modification of sulfuric acid degree (2.4).
7. Examination and detection of other physicochemical indicators and biological Activity
Other physical and chemical indexes and biological activities of the oligosaccharide mixture (bulk drug) are detected, and the results comprise the following steps:
appearance of: white or light white powder;
appearance of solution: the solution should be clear and colorless; if turbidity is displayed, compared with the turbidity standard suspension No. 1, the turbidity is not required to be higher; if color development is performed, the color is not deeper than the yellow standard color solution of EP 4;
UV authentication: maximum absorption wavelength 231+ -2 nm;
sodium identification: meets the sodium identification reaction;
valency of: the potency of anti-Xa factor is 80-120U/mg (calculated as dry product);
loss on drying: no more than 8.0%;
pH:1.0g of 10mL of water dissolved in the carbon dioxide removal, the value is between 6.0 and 8.0;
a weight average molecular weight of 4000Da to 5000Da;
sulfonate/carboxylate ratio: not less than 2.2;
nitrogen content 1.8% -2.5% (calculated as dry product)
Sodium content:11.5% -13.5% (calculated as dry product);
the total weight of metal is less than 20ppm;
residual solvent (methanol): less than 3000ppm;
bacterial endotoxin:not higher than 0.01EU per anti-Xa unit;
microbial limit:the total number of aerobic colonies is less than or equal to 1000CFU/g, the total number of moulds/yeast is less than or equal to 100CFU/g, E.coli (E.coli) is not detected per 1g, salmonella (Salmonella) is not detected per 10g. In one example, but not limited to this example (examples 4-5, and the specific preparation examples), the results of quality testing of multiple batches of oligosaccharide mixtures are detailed, reflecting the high consistency in terms of the physical properties, biological anticoagulation activity (anti-Xa), and impurity residues of the various batches of oligosaccharide mixtures produced, and also demonstrating the uniformity and controllability of the production preparation of the oligosaccharide mixtures.
The invention analyzes the unit structure of the oligosaccharide mixture from molecular level to sugar sequence in detail by using orthogonal and multidimensional method, and the analysis of the molecular structure reveals that the oligosaccharide mixture consists of heparin disaccharide, wherein the structure of some characteristics is obviously different from that of refined heparin and other clinical commercial medical low molecular heparin. The oligosaccharide mixture prepared in each batch according to the preparation method of the invention is identical, and the deep structural analysis can ensure that the oligosaccharide mixture is consistent and controllable from the multi-dimensional aspect when being produced.
Advantageous effects of the invention：
The invention provides an oligosaccharide mixture derived from heparan, the non-reducing end of which is 4, 5-unsaturated uronic acid structure, the weight average molecular weight of which is 4000Da-5000Da, and the detail structural characteristics of the oligosaccharide mixture are obviously different from those of refined heparin and other clinical commercial medical low-molecular heparin. The oligosaccharide mixture has strong anticoagulation effect, and has small toxic and side effects on mice due to subcutaneous injection, and can be developed into medicine. The heparin provided by the invention is derived from sheep, is simple and easy to obtain, expands the source of medical heparin, can be developed into halal medicine, and has obvious economic effect.
Drawings
FIG. 1 is a schematic diagram of LC-MS comparison of three sets of oligosaccharide mixture samples subjected to heparinase 1+2+3 mixed enzymatic hydrolysis.
FIG. 2 is a schematic representation of LC-MS comparison of three sets of oligosaccharide mixture samples digested with heparinase 1.
FIG. 3 is a schematic representation of LC-MS comparison of three sets of oligosaccharide mixture samples digested with heparinase 3.
FIG. 4 is a schematic representation of the disaccharide or sugar unit content resolved by LC-MS after enzymatic hydrolysis of a sample of the oligosaccharide mixture.
FIG. 5 is a schematic diagram of several sets of Biogel P6 size exclusion-FPLC separation oligosaccharide mixture samples.
FIG. 6 separation of the tetrasaccharide component from the oligosaccharide mixture 1 H-NMR comparison scheme.
FIG. 7 is a schematic of HPLC analysis of different tetrasaccharides in a tetrasaccharide component of an oligosaccharide mixture.
FIG. 8 is a schematic diagram showing LC-MS spectra of three batches of oligosaccharide mixture samples (G12298, G12299 and G12300) and a clinically commercially available medical low molecular heparin control (G12292).
FIG. 9 is a graphical representation of the percentage of each oligosaccharide component in three batches of oligosaccharide mixture samples (G12298, G12299 and G12300) and a clinically commercially available medical low molecular heparin control (G12292).
FIG. 10 is a schematic diagram showing a comparison of anticoagulation experiments in rabbits between three batches of oligosaccharide mixture samples and a clinically commercially available medical low molecular heparin control.
Detailed Description
The present invention will be further described in detail with reference to the following specific embodiments, but is not intended to limit the scope of the present invention.
[ term/definition/abbreviation ]
A, ANS,3,6S: n-sulfonic acid group 3-O-sulfuric acid group-alpha-D-glucosamine
-Ac: acetyl group
an,1,6-an.,1, 6-anhydride: 1, 6-internal ether structure
APTT: time to activate partial thromboplastin
CFU: colony forming units
CV%: coefficient of variation
Da: dalton (daltons)
Dis-, disacchoride: disaccharides
DNA: deoxyribonucleic acid
EIC: ion extraction flow chromatography
EP: european pharmacopoeia
Epoxy, epoxy: epoxy structure
ESI-TOF MS: electrospray time-of-flight mass spectrometry
EU: bacterial endotoxin units
FPLC, fast Protein Liquid Chromatography: flash protein liquid chromatography
G, glcA, D-glucuronic Acid: glucuronic acid
Ext> Gext> -ext> aext>:ext> beta-D-glucuronic acid-N-sulfonic acid group 3-O-sulfuric acid group-alpha-D-glucosamine
Gal, galA, galacturonic Acid: galacturonic acid
GlcN, D-glucosamine: glucosamine, glucosamine
GlcNAc: n-acetyl-glucosamine
GlcNS6S nitrogen-sulfate-6-oxysulfate-glucosamine
GPC: gel permeation chromatography
-H: hydrogen radical
Heparin: heparin
Hexa-, hexa-sacchoride: hexasaccharide
HPLC, highPressure Liquid Chromatography: high pressure liquid chromatography
HSQC-NMR: heteronuclear single quantum coherence-nuclear magnetic resonance
I, idoA, L-iduronic Acid: iduronic acid
I/G, idoA/GlcA: iduronic acid/glucuronic acid (ratio)
I2S, ido2S: 2-oxo-sulfate-iduronic acid
IU: international units
LC-MS: chromatography mass spectrometry
LD 1 :1% lethal dose
LD 50 : half lethal dose
LOD, limit of Detection: quantitative limit
LOQ, limit of Quantification: detection limit
LR, linkage Region: connection region
LWMH, low Molecular Weight Heparin: low molecular heparin
M, manN, mannuronic Acid: mannuronic acid
Mn: number average molecular weight
MS, mass Spectrum: mass spectrometry
Mw: weight average molecular weight
NMR, nuclear Magnetic Resonance: nuclear magnetic resonance
Oligo-, oligo sacchoride: oligosaccharide
Pd: dispersity of
ppm: million percent
PT: prothrombin time
Q-PCR: quantitative polymerase chain reaction
RID: differential refraction detector
SEC-FPLC: size exclusion chromatography-flash protein liquid chromatography
SEC-MALLS: size exclusion chromatography-multi-angle laser light scattering
Serine: serine (serine)
Tetra-, tetra sacchoride: tetraose
TT: thrombin time
U: active unit
Δu:4, 5-unsaturated uronic acid
ΔU 2S :4, 5-unsaturated uronic acid-2-O-sulfate
USP: united states pharmacopoeia
UV: ultraviolet spectrophotometry
Xa: coagulation factor Xa
Xyl, xylose: xylose
Example 1: preparation of oligosaccharide mixture 1
(1) Preparation of raw material heparan Caprae Seu Ovis
The commercial crude heparin sodium is prepared by extracting a crude product from a by-product intestinal mucosa in the processing process of a sheep casing in a crude product factory. Animal DNA was detected by quantitative PCR (Q-PCR) technique, wherein the content of sheep DNA was 118370ppm, the content of pig DNA was 6.3ppm, the content of bovine DNA was 1.3ppm, and the total amount of pig DNA and bovine DNA was 0.01% based on the total DNA. The crude heparin was shown to be derived from sheep without heparin doping (< 0.5%) from porcine heparin and bovine heparin.
Taking 5 hundred million units of the crude heparin sodium, adding 150L of water, supplementing sodium chloride to 2%, and stirring at 40-50 ℃ until the crude heparin sodium is completely dissolved. 150g of protease is added for enzymolysis for 3 hours. The heparin sodium reaction solution after enzymolysis is filtered by a filter screen into a barrel containing 50Kg of anion resin, and stirred and adsorbed for 10 hours. The solution was then discarded and the resin was rinsed 3 times with 4% sodium chloride solution. The rinsed resin was eluted 3 times with 150L of 15% sodium chloride solution and the eluates were combined. Adding alcohol to a final concentration of 45%, stirring, standing, dehydrating and drying the precipitate.
Dissolving the dried heparin sodium in 20L of 3% sodium chloride solution, heating to 50 ℃ and keeping stirring, adding hydrogen peroxide to a final concentration of 0.5%, maintaining the temperature and pH during decolorization, monitoring the consumption condition of the hydrogen peroxide by using hydrogen peroxide test paper, and maintaining the hydrogen peroxide concentration of more than 0.1% for decolorization for 10 hours until the color of the solution is pale yellow. The solution was filtered, added with alcohol to a final concentration of 45%, stirred and allowed to stand. Taking the precipitate, re-dissolving and alcohol-precipitating for 2 times, and finally dehydrating and drying to obtain 2.37Kg of pure product heparin, wherein the anticoagulation activity measured by a sheep plasma method is 181U/mg, and the activity yield is 81.8%.
(2) Derivatization preparation of oligosaccharide mixtures
2.0Kg of the pure sheep heparin is dissolved in 20L of purified water; 5Kg of benzethonium chloride was prepared and dissolved in 25L of purified water. The two solutions are mixed uniformly under sufficient stirring, and a large amount of white insoluble matters are precipitated at the moment. And centrifuging to collect precipitate, and vacuum drying to obtain heparin ammonium salt. The heparin ammonium salt solid was dissolved in 40L of methylene chloride, 1Kg of benzyltrimethylammonium hydroxide was added and stirred at 30℃for 24 hours. In addition, 3.6Kg of sodium acetate is prepared, 36L of sodium acetate is dissolved in methanol, the sodium acetate methanol solution is dripped into heparin reaction liquid, a large amount of precipitate is separated out at the moment, the precipitate is centrifugally collected, 10L of 5% saline is added, stirring is carried out until the sodium acetate is fully dissolved, ethanol is used for fractional precipitation, 6L of ethanol is firstly added, the mixture is fully stirred and then is left for 6 hours, 6L of ethanol is added, the mixture is fully stirred and then is left for overnight, the supernatant is discarded, the precipitate is taken out, and the precipitate is subjected to SEC-HPLC (SEC-HPLC) central control inspection, wherein the weight average molecular weight of the precipitate is about 440 Da, and is dried in an oven under vacuum at 60 ℃. Dissolving the powder with 8L of water for injection, filtering with 0.22 mu m, loading the filtrate into a stainless steel freeze-drying plate, freeze-drying (program: pre-freezing at-40 ℃ for 4 hours, starting a cold trap cooling and vacuum pump (50 Pa), raising the temperature of a baffle plate to 0 ℃, maintaining the temperature for 2 hours, maintaining the temperature for 12 hours, maintaining the vacuum for 20+/-5 Pa, raising the temperature of the baffle plate to 40 ℃ for 2 hours, maintaining the temperature for 2 hours, limiting vacuum, discharging the vacuum after the end, and taking out of the box) to prepare 1.48Kg of low molecular heparin.
(3) Product inspection
The results were as follows: the appearance of the product is white powder; the drying weight loss is 3.7%; the weight average molecular weight is 4510Da; the anti-Xa activity is 106U/mg after being dried; 1.0g of the product is dissolved in 10mL of water, and the solution is clear and has a color degree shallower than Y6;1.0g of the product was dissolved in 10mL of water at pH 6.8; the sodium content is 12.8% after being dried; the aqueous solution has maximum absorption at 232nm wavelength; sulfonate/carboxylate ratio 2.5; the heavy metal residue is not more than 30ppm; ethanol in the solvent residue was 210ppm; bacterial endotoxin content, activity per anti-Xa unit is less than 0.01 bacterial Endotoxin Units (EU).
Example 2: preparation of oligosaccharide mixture 2
(1) Derivatization preparation of oligosaccharide mixtures
2.0Kg of purified heparan is taken and dissolved in 10L of purified water; another 6Kg of benzalkonium bromide was prepared and dissolved in 25L of purified water. The two solutions are mixed uniformly under sufficient stirring, and the white precipitate generated is collected by centrifugation and dried. The solid was dissolved in 40LN' N-dimethylformamide, 7.2Kg of benzyl chloride was added and stirred at 32℃for 20 hours. 4Kg of sodium acetate was prepared, dissolved in 40L of methanol, and the sodium acetate methanol solution was added to the heparin reaction solution and mixed, at which time a large amount of precipitate was precipitated. And (5) centrifuging to collect precipitate and drying. Adding 20L of water for injection, dissolving, heating and maintaining at 55-60 ℃, adding 100g of sodium hydroxide solid (prepared into about 30% concentrated solution by water for injection), stirring and reacting for 90 minutes, cooling the reaction liquid, adding hydrogen peroxide to a final concentration of 0.5%, standing and decoloring, and obtaining pale yellow reaction liquid after 3 hours. 1Kg of sodium chloride is supplemented, the mixture is stirred and dissolved, the reaction solution is filtered, ethanol is added for fractional precipitation, 0.6 to 1.2 times of precipitate is taken, the weight average molecular weight of the target substance is about 4500Da through SEC-HPLC (SEC-high performance liquid chromatography) central control inspection, the target substance is dried, then 6L of water for injection is used for dissolution, and the oligosaccharide mixture is prepared into 1.14Kg through freeze drying.
(2) Product inspection
The results were as follows: the appearance of the product is white powder; the loss on drying was 2.4%. The method comprises the steps of carrying out a first treatment on the surface of the The weight average molecular weight is 4435Da; the anti-Xa activity was 107U/mg after drying; 1.0g of the product is dissolved in 10mL of water, and the solution is clear and has a color degree shallower than Y6; 1.0g of the product was dissolved in 10mL of water at pH 7.1; the sodium content is 11.5% after being dried; the nitrogen content is 2.2% after being dried; the aqueous solution has maximum absorption at 232nm wavelength; sulfonate/carboxylate ratio 2.5; the heavy metal residue is not more than 30ppm; ethanol in the solvent residue was 140ppm; bacterial endotoxin content, activity per anti-Xa unit is less than 0.01 bacterial Endotoxin Units (EU).
Example 3: preparation of oligosaccharide mixture 3
1Kg of refined and decolorized heparin sodium is taken and dissolved in 6L of purified water; 2.5Kg of benzethonium chloride is weighed and dissolved in 10L of purified water; the two solutions were mixed with sufficient stirring, at which time a large amount of white precipitate was produced, and the produced white precipitate was collected by centrifugation and freeze-dried. The dried solid was transferred to a reaction kettle, and 20L of methylene chloride was added thereto and stirred until all was dissolved. 4Kg of benzyl chloride is added, the reaction is stirred for 20 hours at 35 ℃, a solution prepared from 2Kg of sodium acetate and 20L of methanol is added to stop the reaction, the mixture is fully stirred and then is left to stand, heparin benzyl ester precipitate is collected by centrifugation, and the heparin benzyl ester precipitate is dried. About 960g of dried heparin benzyl ester is dissolved by 15L of water for injection, heated and kept at 55-58 ℃, 100g of sodium hydroxide solid (prepared into about 30% concentrated solution by 0.3L of water for injection) is added, the reaction solution is stirred and reacted for 2 hours, 0.5L of 30% hydrogen peroxide is added, and the reaction solution is kept stand and decolored after stirring for 2 hours and is light yellow. Adding 0.5Kg of sodium chloride, stirring for dissolution, filtering a reaction solution, adding methanol for fractional precipitation, adding 15L of methanol, fully stirring, standing for 4 hours, removing a supernatant, adding 25L of methanol, fully stirring, standing overnight, discarding the supernatant, taking a precipitate, performing SEC-HPLC (SEC-HPLC) central control inspection, and performing vacuum drying on the precipitate in a 60 ℃ oven for 14 hours to obtain 0.63Kg of oligosaccharide mixture.
The product detection results are as follows: the loss on drying was 4.2%. The weight average molecular weight is 4650Da; the anti-Xa activity was 108U/mg after drying; 1.0g of the product is dissolved in 10mL of water, and the solution is clear and has a chromaticity not deeper than the No. 6 standard color; 1.0g of the product was dissolved in 10mL of water at pH 6.8; the sodium content is 12.1% after being dried; the aqueous solution has maximum absorption at 232nm wavelength; sulfonate/carboxylate ratio 2.5; the heavy metal residue is not more than 30ppm; methanol in the solvent residue was 621ppm; bacterial endotoxin content, activity per anti-Xa unit is less than 0.01 bacterial Endotoxin Units (EU).
Example 4: preparation of oligosaccharide mixture 4
Commercial crude heparin sodium products are subjected to Q-PCR detection, and the results show that the heparin sodium products are heparin, and the pig source DNA ratio of the heparin sodium products is below 0.1%.
The purification method of crude heparin sodium was the same as in example 1, with only batch size differences.
The oligosaccharide mixture was prepared as in example 3, but the batch was run in a process check batch with a 5Kg pure heparin sodium per batch and the final product was prepared by freeze-drying. Three batches were prepared in series, each batch having a quantity of product on the order of 3 Kg. The results of product quality testing are shown in Table 6 below:
TABLE 6 test results of three consecutive batches of 3Kg grade oligosaccharide mixture
/>
The data show that the quality inspection results of the oligosaccharide mixtures of different batches are highly consistent, and the controllability of the preparation process and quality is indirectly reflected.
Example 5: preparation of oligosaccharide mixture 5
Table 7 shows the quality test results of 10Kg grade production verification batches of three consecutive batches of product, with a batch dosage of 25Kg pure heparin sodium per batch.
TABLE 7 test results of three consecutive batches of 10Kg grade oligosaccharide mixture
/>
The results show that the production verification three batches meet the requirements of the established indexes, and the batches are consistent with each other.
EXAMPLE 6 preparation of oligosaccharide mixture preparation
This example examined oligosaccharide mixture formulations of different formulations, including injectable and lyophilized powder formulations that do not contain (and contain) preservatives, as well as their stability at high temperatures.
Prescription 1 (injection 1, no preservative): oligosaccharide mixture 3000000 anti-Xa units, water for injection added to 300mL; prescription 2 (injection 2, containing preservative): oligosaccharide mixture 3000000 anti-Xa units, 4.50g benzyl alcohol, water for injection to 300mL; prescription 3 (lyophilized powder): oligosaccharide mixture 3000000 anti-Xa units, water for injection was added to 300mL (water was subsequently removed during the freeze-drying process of preparation).
The preparation process comprises the following steps:
at room temperature, respectively adding the oligosaccharide mixture (and/or benzyl alcohol) with the prescription amount into a 500mL liquid preparation beaker, adding 250mL of precooled water for injection, stirring until the materials are completely dissolved, and then supplementing the water for injection until the total volume of the liquid medicine is 300mL;
Injection dosage form group (1, 2): the solution was sterilized (0.22 μm) filtered, manually filled into borosilicate glass tube injection bottles (penicillin bottles) of 1.0mL to 2mL, stoppered, capped.
Lyophilized powder formulation group: the solution was sterilized (0.22 μm) filtered, manually filled into borosilicate glass tube injection bottles (penicillin bottles) of 1.0mL to 2mL, half stoppered, freeze dried, procedure as follows: pre-freezing at-40deg.C for 4 hr, starting cold trap cooling and vacuum pump (50 Pa), raising the temperature of the partition plate to 0deg.C, maintaining at 0deg.C for 2 hr, and vacuum of 20+ -5 Pa for 12 hr; raising the temperature of the partition plate to 40 ℃ for 2 hours, maintaining the temperature at 40 ℃ for 2 hours, and carrying out extreme vacuum; and after the end, discharging vacuum, sealing by adding a plug at the pressure of 0.88mbar, and taking out the container.
Stability study at 40℃for 30 days: and (3) packaging in a sealing manner, placing in a stability test box with the temperature of 40+/-2 ℃ and the relative humidity of 75+/-5%, sampling in the test for 30 days, and detecting.
Analysis of results:
the test results are shown in Table 8.
TABLE 8 examination results of oligosaccharide mixture formulations
The results show that the oligosaccharide mixture preparation meets the preset requirements on each index and has good stability at high temperature.
Example 7: two-dimensional nuclear magnetic (NMR-HSQC) analysis of monosaccharide compositions
3 different batches of oligosaccharide mixture drug substances (lot numbers: G12300, G12299 and G12298) were taken as samples, and a solution of 20mg/mL was prepared with deuterium water, and HSQC-NMR analysis was performed, with the results shown in tables 9 to 12.
In particular, tables 9 and 10 reflect the monosaccharide composition of the oligosaccharide mixture, and the quantitative signal of hexosamine and uronic acid are relatively consistent with heparin and other oligosaccharide mixtures in the literature. But the species and the content have obvious characteristics in composition. The oligosaccharide mixture showed more sulfation modifications at the N-position, 6-O-position of GlcN and 2-O-position of IdoA. In another aspect, the oligosaccharide mixture has a reduced end having a endo-ether structure and a non-reduced end having an unsaturated structure. Ext> andext> theext> contentext> ofext> Gext> -ext> Aext> relatedext> toext> theext> anticoagulantext> activeext> siteext> inext> theext> oligosaccharideext> mixtureext> sampleext> isext> 2.3ext>%ext> -ext> 2.5ext>%ext>,ext> whichext> isext> slightlyext> lowerext> thanext> thatext> ofext> theext> clinicallyext> -ext> marketedext> medicalext> lowext> -ext> molecularext> heparinext>.ext>
Table 9 comparison of percent hexosamine in oligosaccharide mixture samples table (HSQC-NMR method)
/>
Remarks: * LOQ of the test method, limit of Quantification, method detection limit, G12300 of 1.4%, G12299 of 1.8% and G12298 of 3.0%; * LOD, limit of Detection, lowest limit of quantitation, G12300 of 0.4%, G12299 of 0.5% and G12298 of 0.9%; * CV% refers to the coefficient of variation of the assay data; n.a. =not Applicable, the method is Not Applicable.
Table 10 comparison of percentage uronic acid in oligosaccharide mixture samples table (HSQC-NMR method)
/>
Remarks: * LOQ of the test method, limit of Quantification, method detection limit, G12300 of 1.4%, G12299 of 1.8% and G12298 of 3.0%; * LOD, limit of Detection, lowest limit of quantitation, G12300 of 0.4%, G12299 of 0.5% and G12298 of 0.9%; * CV% refers to the coefficient of variation of the assay data.
TABLE 11 percentage of the structures of the connecting regions in the oligosaccharide mixture samples (HSQC-NMR method)
| Sample numbering | Connection region (LR) | LOQ** | LOD*** |
| G12300 | 1.1 | 1.5 | 0.4 |
| G12299 | 1.1 | 1.4 | 0.4 |
| G12298 | 1.1 | 1.0 | 0.3 |
| CV％* | 2.57 | / | / |
| Average value of | 1.1 | / | / |
| Standard deviation of | 0 | / | / |
Remarks: * CV% refers to the coefficient of variation of the detection method data; * LOQ, limit of Quantification, method limit of detection; * LOD, limit of Detection, lowest limit of quantitation.
Table 11 shows that the oligosaccharide mixture sample has a certain structure of the connecting region, and in combination with the embodiment of MS analysis after selective enzymolysis by heparinase, the structure of the connecting region of the oligosaccharide mixture is kept complete.
TABLE 12 iduronic acid/glucuronic acid (IdoA/GlcA, I/G) ratio in oligosaccharide mixture samples (HSQC-NMR method)
| Sample numbering | GlcA | IdoA | I/G ratio |
| G12300 | 15.0 | 64.4 | 4.3 |
| G12299 | 15.1 | 64.7 | 4.3 |
| G12298 | 15.0 | 64.3 | 4.3 |
| Average value of | 15.0 | 64.5 | 4.3 |
Table 12 reflects that in the oligosaccharide mixture samples the I/G ratio was on average 4.3, i.e.IdoA was more and GlcA was less.
In connection with the above results and analytical discussion, it was shown that these oligosaccharide mixture samples have the following characteristics:
Monosaccharide composition similar to heparin and clinical commercial low molecular heparin, but with a large difference in content,
the degree of sulfation modification is high,
ext> theext> averageext> valueext> ofext> theext> Gext> -ext> aext> contentext> isext> 2.4ext>%ext>,ext>
The iduronic acid is present in a high abundance,
monosaccharides (II): the monosaccharide types determined by HSQC-NMR of the oligosaccharide mixture are similar to those of clinically-marketed medical heparin and low-molecular heparin, the most predominant monosaccharides are IdoA2S and GlcNS6S, the quantitative signals of the monosaccharide components in the oligosaccharide mixture are significantly different from those of other heparin, and the monosaccharide types also comprise a endo-ether structure, an unsaturated uronic acid structure, epoxy (epoxy) and galacturonic acid (GlaA) structure;
sulfation modification: the oligosaccharide mixture showed more sulfation modifications at the N-position, 6-O-position and 2-O-position of iduronic acid (IdoA) of glucosamine (GlcN), wherein the major acetylation modification at the N-position was only on average 4.9%, significantly less than other heparins (all above 10%), reflecting a high degree of sulfation modification in the oligosaccharide mixture.
G-A*: ext> theext> contentext> ofext> Gext> -ext> aext> associatedext> withext> theext> anticoagulantext> activeext> siteext> wasext> 2.4ext>%ext> onext> averageext> inext> theext> oligosaccharideext> mixtureext> sampleext>;ext>
Iduronic acid: the iduronic acid/glucuronic acid ratio (IdoA/GlcA, I/G) of the oligosaccharide mixture was 4.3, and the oligosaccharide mixture contained more iduronic acid.
Example 8: heparinase 1+2+3 mixed enzymolysis, disaccharide and oligosaccharide composition and sequence LC-MS analysis
Three batches of oligosaccharide mixture bulk drug samples are taken to prepare 20mg/mL solutions respectively, heparinase 1+2+3, heparinase 1 and heparinase 3 are adopted to carry out selective degradation, and then the disaccharide and oligosaccharide composition is identified by HPLC (high Performance liquid chromatography) -TOF (time of flight) MS analysis.
1. Enzymolysis and sample treatment:
and (3) respectively diluting heparinase 1, heparinase 2 and heparinase 3 to 0.4IU/mL according to activity, and mixing according to the ratio of 1:1:1. The 20mg/mL solution of each oligosaccharide mixture sample was subjected to enzymolysis according to the following system: 20. Mu.L of the sample solution, 70. Mu.L of a calcium acetate buffer (pH 7.0, 20 mM) and 100. Mu.L of a heparinase 1+2+3 enzyme mixture were gently mixed, and then subjected to enzymolysis in a water bath at 25℃for more than 48 hours. Filtering with 0.22 μm after enzymolysis.
2. LC-MS method:
(1) HPLC method: chromatographic column: c18; mobile phase a:10mM dibutylamine, 10mM acetic acid in water; mobile phase B:10mM dibutylamine, 10mM acetic acid in methanol; flow rate: 0.15mL/min; gradient: 0-5min 10% mobile phase B,5-60min 80% mobile phase B,60-70min 90% mobile phase B,70-100min 10% mobile phase B; sample injection amount: 5. Mu.L; sample concentration: 2mg/mL; column temperature: 35 ℃; wavelength: 232nm.
(2) The MS method comprises the following steps: the device comprises: ESI-Q-TOF MS; capillary voltage: +3500V; spray needle pressure: 1.8bar; dry gas flow rate: 7.0L/min; drying gas temperature: 200 ℃; mass spectrum signal range: 140-2500m/z.
3. Experimental results:
analysis pattern as shown in fig. 1, thirty disaccharides or oligosaccharides were detected from oligosaccharide mixture samples, with some saccharide units being less or not present in clinically commercially available medical low molecular weight heparin.
The disaccharide or oligosaccharide composition species and MS data are presented in Table 13 below.
TABLE 13 heparinase 1+2+3 Mixed enzymatic hydrolysis of oligosaccharide mixture oligosaccharide constituent units
/>
Remarks: * These signals do not resolve the corresponding component structure; * These components are typically less detectable or undetectable in clinically commercially available medical low molecular weight heparin.
Example 9: heparanase 1 single enzymolysis, disaccharide and oligosaccharide composition and sequence LC-MS analysis
Three batches of oligosaccharide mixture bulk drug samples are taken to prepare 20mg/mL solutions respectively, heparinase 1 is adopted for selective degradation, and then the disaccharide and oligosaccharide composition is identified by HPLC (high performance liquid chromatography) and ESI-TOF (electronic stability index) MS (mass spectrometry) analysis.
1. Enzymolysis and sample treatment:
heparin enzyme 1 was diluted to 10IU/mL according to activity. The 20mg/mL solution of each oligosaccharide mixture was subjected to enzymolysis according to the following system: after 15. Mu.L of the sample solution and 85. Mu.L of calcium acetate buffer (pH 7.0, 20 mM) and 90. Mu.L of heparinase 1 were gently mixed, the mixture was subjected to enzymolysis in a water bath at 37℃for 48 hours or more. Filtering with 0.22 μm after enzymolysis.
2. LC-MS method: the device comprises: ESI-Q-TOF MS; capillary voltage: +3500V; spray needle pressure: 1.8bar; dry gas flow rate: 7.0L/min; drying gas temperature: 200 ℃; mass spectrum signal range: 140-2500m/z.
3. Experimental results:
the results are shown in fig. 2 and table 14, with about thirty disaccharides or tetrasaccharides detected from the oligosaccharide mixture samples.
TABLE 14 oligosaccharide mixture oligosaccharide constituent units for heparinase 1 enzymolysis
/>
Remarks: * These signals are not analyzed for the corresponding component structure.
Example 10: heparanase 3 single enzymolysis, disaccharide and oligosaccharide composition and sequence LC-MS analysis
Three batches of oligosaccharide mixture bulk drug samples are taken to prepare 20mg/mL solutions respectively, heparinase 3 is adopted for selective degradation, and then the disaccharide and oligosaccharide composition is identified by HPLC (high performance liquid chromatography) and ESI-TOF MS (electronic stability index) analysis.
1. Enzymolysis and sample treatment:
heparanase 3 was diluted to 0.4IU/mL according to activity. The 20mg/mL solution of each oligosaccharide mixture was subjected to enzymolysis according to the following system: 20. Mu.L of sample solution+120. Mu.L of calcium acetate buffer (pH 7.0, 20 mM) +50. Mu.L of heparinase 3, and after gently mixing, the mixture was subjected to enzymolysis in a water bath at 25℃for more than 48 hours. Filtering with 0.22 μm after enzymolysis.
2. LC-MS method: the device comprises: ESI-Q-TOF MS; capillary voltage: +3500V; spray needle pressure: 1.8bar: dry gas flow rate: 7.0L/min; drying gas temperature: 200 ℃; mass spectrum signal range: 140-2500m/z.
3. Experimental results:
analysis patterns as shown in fig. 3 and table 15, about thirty disaccharides or tetrasaccharides were detected from oligosaccharide mixture samples, some of which were typically absent or less present in clinically commercially available medical low molecular weight heparin.
In addition, MS analysis of the components such as general disaccharides is shown in Table 15:
TABLE 15 oligosaccharide mixture oligosaccharide constituent units enzymatically degraded with heparinase 3
/>
/>
/>
Remarks: * These signals do not resolve the corresponding component structure; * These components are typically less detectable or undetectable in clinically commercially available medical low molecular weight heparin.
In addition, the experiments and results of examples 8 and 9 and this example 10 show that the oligosaccharide mixture exhibits very interesting structural features as follows:
the presence of a complete structure of the connection region,
the degree of sulfation modification is high,
the high content of the sulphating component is high,
the non-reducing end has a novel hexosamine structure,
connection region: fragments DeltaU-Gal-Gal-Xyl-Serine and DeltaU 2, 1-U-Gal-Gal-Xyl-Serine, which are present in the heparin-binding region in their entirety, are detectable in the oligosaccharide mixture, and are less or not detectable in clinically commercially available medical low-molecular heparin; in addition, deltaU-Gal-Gal-Xyl-CH 2 COOH (detected in degradation solution of heparinase 3) and DeltaU 4,3,1-U-Gal-Gal-Xyl-CH 2 COOH (detected in the degradation solution of heparinase 1), both from oxidized heparin-binding domains, is more abundant in the oligosaccharide mixture;
sulfation modification: conventional disaccharides Δu2,1 (detected in heparinase 1+2+3 degradation solutions) and tetrasaccharides Δu4,2,1, Δu4,3,1 and hexasaccharides Δu6,6,1 (detected in heparinase 1 degradation solutions) were detected relatively less in the oligosaccharide mixture samples, which in turn reflects a higher degree of sulfation modification of the oligosaccharide mixture samples, consistent with HSQC-NMR analysis results in other examples;
highly sulfated component: including non-reducing ends a (trisulfated hexosamine, ANS,3,6S), tetrasaccharides Δu4,5,0 and Δu4,6,0 and larger sulfate oligosaccharide chains (tetrasaccharides to dodecasaccharides, not degraded by heparanase 3, heparanase 3 selectively degrading low sulfate sequences or sites), which are present in higher abundance in the oligosaccharide mixture sample;
novel feature structure: at the non-reducing end, A3,4,0 (trisaccharide, containing four sulfate groups), A3,6,0 (trisaccharide, containing six sulfate groups) anda5,5,0 (pentasaccharide, containing five sulfate groups), the hexosamines of these highly sulfated components, which are rarely found in other clinically commercially available medical low molecular weight heparins, are produced by the enzymatic hydrolysis of heparinase 3, but are present in the oligosaccharide mixture.
EXAMPLE 11 Shimadzu LC-20A high performance liquid chromatography and Siemens flight LTQ-Orbitrap Mass Spectrometry analysis of disaccharides and basic constituent Unit Structure
A sample of the oligosaccharide mixture drug substance (batch: 2016042) was taken and formulated as a 20mg/mL solution. The complete degradation is carried out by adopting heparinase 1+2+3, and the disaccharide and oligosaccharide composition is identified by HPLC and ESI-TOF MS analysis. The oligosaccharide mixture was analyzed by using Shimadzu LC-20A high performance liquid chromatography and Siemens FelTQ-Orbitrap mass spectrometry.
1. Qualitative analysis results:
the mass spectra of the components in the LC-MS analysis were assigned and resolved, and 23 disaccharides and basic building block structures were identified from oligosaccharide mixture samples, including 8 common heparin disaccharides (ΔIS (ΔUA2S-GlcNS 6S), ΔIIS (ΔUA-GlcNS 6S), ΔIIIS (ΔUA2S-GlcNS), ΔIVS (ΔUA-GlcNS), ΔIA (ΔUA2S-GlcNAc 6S), ΔIIA (ΔUA-GlcNAc 6S), ΔIIIA (ΔUA2S-GlcNAc) and ΔIVA (ΔUA-GlcNAc)), 1N-unsubstituted disaccharide (ΔIH), 2 types of 3-O-tetrasulfate (ΔIIA-IIS) glu And ΔIIS-IIS glu ) 2 disaccharides (2S) NRE and 3S NRE containing non-reducing terminal saturated structures, 3 oligosaccharides (AnhydroΔIIS/AnhydroΔIIS) containing reducing terminal internal ether structures epi AnhydroΔIS and AnhydroΔIS-IS epi ) Trisaccharides (Trisaccharide (2S), trisaccharide (2S, 1 Ac), trisaccharide (3S) and Trisaccharide (4S)), 1 Epoxide (DeltaUA 2S-GlcNS6S-GlcA-2, 3-carbohydrate-GlcNS), 2 heparin protein-binding domain oligosaccharides (Linkage and Linkage) ox ). The disaccharide and basic building block structures identified are summarized in Table 16.
Table 16 disaccharide and oligosaccharide identified in oligosaccharide mixtures and their relative content
/>
/>
/>
2. Relative quantitative analysis results:
the relative quantification of the peak areas of the components in the extracted ion flow chromatogram (EIC) was carried out, the classification is summarized in table 11 above, and the contents of the components in the oligosaccharide mixture samples were compared (fig. 4, x 1, ×10, ×50 in the chromatogram represent the expansion multiples of the original values), and the common heparin disaccharide was 84.6% of the total content, which is the main part.
3. Summarizing:
the oligosaccharide mixture samples after enzymolysis were characterized by LC-MS analysis:
the basic constituent unit is heparin disaccharide;
containing other specific structural building blocks, including some internal ether structures, linker structures, etc.
Example 12: separation of tetraose from oligosaccharide mixture by size exclusion-FPLC
Three batches of oligosaccharide mixture are taken as samples, FPLC is carried out by a molecular sieve column, oligosaccharides such as tetraose and the like are separated, and the separated tetraose mixture is analyzed by HSQC-NMR. Further, each of the tetraose components is separated by HPLC, and the structure and approximate content thereof are identified by mass spectrometry or HSQC-NMR analysis. The specific experiment is as follows:
1. the experimental process comprises the following steps:
taking 250-300mg of each sample, dissolving in 5mL of purified water, loading the solution onto a Biogel P6 resin column (95X 5cm,2 are connected in series), eluting with 0.25mol/L ammonium chloride solution at a rate of 1.8mL/min, monitoring the elution condition with UV 210/232nm, collecting each oligosaccharide peak component step by step, and concentrating under reduced pressure to 5mL at 35 ℃. The tetraose fraction of each group of samples was taken, desalted with a desalting column (TSK 40-S column, 10% ethanol solution as eluent), the targets were collected by UV 210/232nm monitoring, combined, concentrated to-5 mL under reduced pressure at 35℃and lyophilized, the lyophilized powder was used for further LC-MS or NMR analysis.
2. Experimental results:
the oligosaccharide mixture samples are separated for multiple times to obtain oligosaccharide components such as tetraose, hexaose and the like. See FIG. 5 (panel: I-tetraose, H-pentaose, G-hexaose, F-heptaose, E-octaose, D-decaose, C-dodecaose, B and A-macromolecular components), where G8620, G8621 and G8622 are oligosaccharide mixtures of three batches.
Example 13: 1 analysis of tetrasaccharide mixtures of oligosaccharide mixtures by H-NMR
In example 12, the hydrogen spectrum of the collected and post-treated tetrasaccharide fraction (fraction I) was analyzed by 500MHz nuclear magnetic resonance and the spectrum is shown in FIG. 6, in which G9013QA-I, G8626QA-I, G8804QA-I, G8811QA-I and G8620QA-I6 were each tetrasaccharide fractions of different oligosaccharide mixture samples. The results of the comparison showed that the tetraose component profiles of the respective oligosaccharide mixture samples were more consistent, but there was a certain difference in the response of each peak.
Example 14: HSQC-NMR analysis of tetrasaccharide mixtures of oligosaccharide mixtures
The four oligosaccharide component (component I) separation was performed on three oligosaccharide mixture samples (from three groups of oligosaccharide mixture samples: G8804QA, G8626QA and G9013 QA) according to the method of example 12, and the monosaccharide compositions such as hexosamine and uronic acid obtained therein were analyzed by HSQC-NMR, respectively, and listed in Table 17 and Table 18, and the analyses were as follows:
table 17 comparison table of hexosamine (HSQC-NMR method) for tetrasaccharide component mixtures
Remarks: * LOQ, limit of Quantification, method detection limit of the present test method; * LOD, limit of Detection, lowest limit of quantitation; # is below the limit of detection (LOD), undetected.
Table 18 uronic acid comparison table (HSQC-NMR method) for the tetrasaccharide component mixtures
Remarks: * L0Q, limit of Quantification of the test method, method detection limit; * LOD, limit of Detection, lowest limit of quantitation; # is below the limit of detection (LOD), undetected.
According to the data in tables 17 and 18, oligosaccharide mixture samples were taken of monosaccharides of the tetrasaccharide component, as A NS And I 2S Mainly, in addition to A 6S Is 89.2%, and further contains unsaturated uronic acid U 2S (4, 5-unsaturated uronic acid-2-O-sulfate). In comparison with the HSQC-NMR data of the mixture of oligosaccharides which were not isolated in example 7, the monosaccharides contained in the mixture were different to a certain extent, and the tetraose fraction was isolated without detection of the epoxides and GalAs.
Example 15: HPLC separation of the components of the tetrasaccharide mixture
A mixture of the tetrasaccharide components (component I) of the oligosaccharide mixture sample was isolated by the method of example 12 and analyzed by HPLC, the results of which are shown in FIG. 7.
HPLC can separate 15 tetraose compositions from the oligosaccharide mixture. Each tetrasaccharide was then qualitatively analyzed by NMR and MS for structure. These structures are part of the composition of the oligosaccharide mixture, which is essentially different from the disaccharide composition of examples 8-10 following heparinase hydrolysis, which is a structural module of the oligosaccharide mixture, which is obtained by degrading the oligosaccharide mixture by heparinase in the long chain units, but in which the disaccharide is very small. Tetraose also degrades into disaccharides during enzymatic hydrolysis.
The results showed that in the oligosaccharide mixture samples, the 15 tetrasugars detected were: deltaU 2S H NS6S U gal H NS6S (β)/I 2S H NS6S I 2S H NS6S 、ΔU 2S H NS6S U gal H NS6S (α)/I 2S H NS6S I 2S H NS6S 、ΔU 2S H NS6S I 2S H NS (α)、ΔU 2S H NS6S I 2S H NS (β)、ΔU 2S H NS6S I 2S Man NS (β)、ΔU 2S H NS6S I 2S Man NS (α)、ΔU 2S H NS I 2S H NS6S (α)、ΔU 2S H NS I 2S H NS6S (β)、ΔU 2S H NS6S I 2S Man NS (1，6anhydro)、ΔU 2S H NS6S I 2S H NS (1，6anhydro)、ΔU 2S H NS6S G 2S H NS 、ΔU 2S H NS6S GH NS6S (α)、ΔU 2S H NS6S GH NS6S (β)、ΔU 2S H NS6S I 2S H NS6S (α)、ΔU 2S H NS6S I 2S Man NS6S (β)、ΔU 2S H NS6S I 2S H NS6S (β)、ΔU 2S H NS6S I 2S Man NS6S (α)、ΔU 2S H NS6S G 2S H NS6S (alpha) and DeltaU 2S H NS6S G 2S H NS6S (beta). The non-reducing ends of these tetrasaccharides are each 4, 5-unsaturated uronic acid-2-O-sulfate (. DELTA.U) 2S ) Structure is as follows.
In addition, the experiment and results of examples 12 to 15 show that the oligosaccharide mixture has the following characteristics:
tetrasaccharides consist of a number of common tetrasaccharides of heparin,
non-reducing ends are all 4, 5-unsaturated uronic acid-2-O-sulfate (. DELTA.U) 2S ) The structure of the utility model is that,
example 16: chromatography-mass spectrometry analysis of sugar chain sequences
Different batches of oligosaccharide mixture drug substance samples (G12298, G12299 and G12300) and clinical commercial medical low molecular heparin (G12292, freeze-dried from the kefir, enoxaparin sodium injection) are prepared into a 5mg/mL solution by purified water, and the solution is directly identified by HPLC (high performance liquid chromatography) -TOF MS analysis without enzymolysis of heparinase. Comparative patterns and component and content analyses of the chromatin separation are shown in fig. 8 and 9:
the results are shown below after analysis:
the main component is an oligosaccharide component common to clinical commercial medical low molecular heparin;
-significant content differences of the components with clinically marketed medical low molecular heparin;
the degree of sulfation modification in the high weight average molecular weight component is high.
From the above specific results, it can be seen that: oligosaccharide mixtures consist mainly of common low molecular heparin oligosaccharides, but with significant content differences; among the high weight average molecular weight components, the content of components with polysulfated modifications such as DeltaU 6,9,0 (hexasaccharide, 9 sulfate groups) and the like in the oligosaccharide mixture is significantly more than that of common clinical commercial medical low molecular heparin; in addition, two of the three different batches of oligosaccharide mixture had nearly identical profiles, but the other batch had a small difference in integration.
Example 17: human blood anticoagulation assay of oligosaccharide mixtures
The experimental method comprises the following steps: platelet Poor Plasma (PPP) was isolated by anticoagulation with 3.8% sodium citrate anticoagulant at 1:9 and centrifugation at 3000rpm for 5min each time with 3 human peripheral venous blood 3 mL. The detection was performed on-line (fully automated coagulometer, stago Compact) according to the kit method. The experimental groups were as follows: the final concentrations of the three batches of oligosaccharide mixture sample groups and the dalteparin sodium reference substance group (clinical commercial medicines, fragmin, batch number: 74048A 93) are respectively-0.3 anti-Xa IU/mL, and physiological saline is used as a blank control in the experiment.
Results and analysis:
1) APTT, PT and TT
The experimental results are shown in table 19 below:
TABLE 19 in vitro effects on APTT, PT and TT
| Group of | APTT | PT | TT |
| Oligosaccharide mixture sample 1 | 114.1±8.5s | 13.7±0.6s | 157.4±32.1s |
| Oligosaccharide mixture sample 2 | 115.3±12.7s | 14.1±0.4s | 146.2±47.2s |
| Oligosaccharide mixture sample 3 | 112.5±10.7s | 13.4±0.3s | 135.7±41.6s |
| Reference substance of dalteparin sodium | 114.8±9.2s | 13.7±0.5s | 150.4±52.8s |
| Blank control | 37.1±6.3s | 13.1±0.6s | 21.3±0.7s |
From table 19 above, it can be seen that in vitro oligosaccharide mixtures similar to dalteparin can significantly prolong APTT and TT, but have less effect on PT.
2) AT and fibrinogen:
the experimental results are shown in table 20 below:
TABLE 20 in vitro effects on AT and fibrinogen
| Group of | AT | Fibrinogen | Time to recalcification |
| Oligosaccharide mixture sample 1 | 2.27±0.43g/L | 100.1±17.2s | 31.0±0.0s* |
| Oligosaccharide mixture sample 2 | 2.36±0.37g/L | 94.0±14.7s | 31.0±0.0s* |
| Oligosaccharide mixture sample 3 | 2.24±0.51g/L | 96.9±15.4s | 31.0±0.0s* |
| Reference substance of dalteparin sodium | 2.29±0.43g/L | 99.0±16.2s | 31.0±0.0s* |
| Blank control | 2.52±0.31g/L | 94.0±8.7s | 9.9±0.6s |
Remarks: * : all of which are beyond the detection range
As can be seen from table 20 above, similar to dalteparin, the oligosaccharide mixture had little effect on both AT and fibrinogen, but the recalcification time could be significantly prolonged, and the data was beyond the detection range (> 31.00 s).
All the above data reveal that the oligosaccharide mixture has good anticoagulation effect on human blood in vitro and is comparable to dalteparin.
Example 18: animal in vivo anticoagulation assay of oligosaccharide mixtures
The experimental method comprises the following steps: japanese white rabbits (2-3 Kg) were selected and administered subcutaneously in the front, back and near the upper limbs, respectively, according to body weight. The test groups were set as follows: three batches of oligosaccharide mixture sample groups and a dalteparin sodium reference substance group (clinical commercial medicines, fragmin, batch number: 74048A 93) are tested at 100 anti-Xa IU/Kg; the experiment was performed with normal saline as a blank. 3mL of blood was collected before and 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours and 8 hours after administration, respectively, anticoagulated with 3.8% sodium citrate anticoagulant 1:9, and tested on-machine (same as 1.1 in vitro anticoagulation assay) and anti-Xa activity.
Experimental results and analysis:
1) anti-Xa activity:
the experimental results are shown as 10-1 in FIG. 10, from which it can be seen that: after subcutaneous injection of the oligosaccharide mixture sample, the absorption and metabolism (or decay) curves of heparin in rabbit plasma are slightly different from that of dalteparin, the absorption peak is reached in about 2 to 4 hours, and almost all decays in 8 hours, compared with the clinically marketed standard of dalteparin sodium for medical use, the decay of which is more rapid.
2)APTT：
The experimental results are shown as 10-2 in FIG. 10, from which it can be seen that: the oligosaccharide mixture samples can obviously prolong the APTT, have equivalent effect on the APTT, and have similar maximum time and similar decay time of the APTT in rabbits.
3)PT：
The experimental results are shown as 10-3 in FIG. 10, from which it can be seen that: each group of samples had less effect on PT in rabbits. Furthermore, in the aforementioned 13-1, the oligosaccharide mixture and dalteparin did not significantly prolong PT in vitro, where the in vitro and in vivo effects exhibited relative consistency.
4)TT：
The experimental results are shown as 10-4 in FIG. 10, from which it can be seen that: the oligosaccharide mixture samples can obviously prolong TT, the time for reaching the maximum TT in rabbits is similar to that of a reference substance of the dalteparin sodium, and the decay time is also equivalent.
All the above data reveal that the oligosaccharide mixture samples have good anticoagulation effect in rabbits, no worse than commercially available dalteparin sodium.
Example 19: toxicity test of oligosaccharide mixture subcutaneously injected in mice
The experimental method comprises the following steps: ICR mice are adopted, the male and female mice in each group are 5, after single back subcutaneous injection, drinking water is freely taken, the mice are closely observed for 6 hours, and then the states of the mice are continuously observed for 7 days. Experimental samples: the oligosaccharide mixture was dissolved to the appropriate concentration with water for injection and filtered at 0.22 μm. Drug administration settings: (geometric series 0.8) 1280mg/Kg, 1600mg/Kg, 2000mg/Kg, 2500mg/Kg, 3125mg/Kg and 3906mg/Kg.
Experimental results:
(1) Within 7 days, 3, 5 and 8 mice died in 2500mg/Kg group, 3125mg/Kg group and 3906mg/Kg group, respectively, and no mice died in other low dose groups;
(2) Results statistics show that oligosaccharide mixture was singly LD subcutaneously in mice 50 (95% confidence limit) =3087 mg/Kg (2740-3601 mg/Kg), LD 1 (95% confidence limit) =1729 mg/Kg (976 to 2116 mg/Kg);
(3) The dead mice are dissected, and the frequent occurrence of the blood stasis from the neck to the back (injection position) and the black blood stasis of organs such as the lung, liver or heart can be judged to be dead due to bleeding side effects;
(4) Normal mice were sacrificed 7 days later and had some increase in body weight, no blood stasis at the injection site and in the organs, and no abnormality in the organs. The oligosaccharide mixture has low toxic and side effects.
The toxicity experiment above illustrates that: the oligosaccharide mixture has high LD 50 The safety is good.
There are, of course, many specific embodiments of the invention, not set forth herein. All technical solutions formed by equivalent substitution or equivalent transformation fall within the scope of the invention claimed.
Claims (12)
1. An oligosaccharide mixture characterized in that: is prepared by the derivative of the heparan caprae seu ovis, is a mixture composed of oligosaccharide chains with different sizes, has the main structural formula as follows,
wherein: n=1-24; r is R 1 =h or SO 3 Na or COCH 3 ；R 2 =h or SO 3 Na；
R 3 =h or
The weight average molecular weight of the oligosaccharide mixture is 4000Da-5000Da;
the non-reducing end of each oligosaccharide chain of the oligosaccharide mixture is a 4, 5-unsaturated uronic acid structure, including 4, 5-unsaturated uronic acid-2-O-sulfate (. DELTA.U) 2S ) And 4, 5-unsaturated uronic acid (. DELTA.U), as follows,
the reducing end of part of oligosaccharide chain of the oligosaccharide mixture is of an inner ether structure, comprising AnhydroΔIIS/AnhydroΔIIS epi AnhydroΔIS and AnhydroΔIS-IS epi The method comprises, as follows,
2. an oligosaccharide mixture as claimed in claim 1 wherein: has the following eight characteristics of the utility model,
(1) Containing specific monosaccharide structures, including: unsaturated uronic acid structure, internal ether structure, epoxy and galacturonic acid structure; wherein the unsaturated uronic acid structure is located at the non-reducing end of the oligosaccharide chain, the internal ether structure is located at the reducing end of a portion of the oligosaccharide chain, and the epoxy and galacturonic acid structures are located in the middle of the oligosaccharide chain;
(2) Disaccharides and basic constituent units, more than 80% of which are common heparin disaccharides, include: ΔIS (ΔUA2S-GlcNS 6S), ΔIIS (ΔUA-GlcNS 6S), ΔIIIS (ΔUA2S-GlcNS), ΔIVS (ΔUA-GlcNS), ΔIA (ΔUA2S-GlcNAc6S), ΔIIA (ΔUA-GlcNAc6S), ΔIIIA (ΔUA2S-GlcNAc) and ΔIVA (ΔUA-GlcNAc);
(3) The non-reducing ends of the tetraose all contain 4, 5-unsaturated uronic acid-2-O-sulfate (. DELTA.U) 2S ) The structure of the tetraose is that the tetraose is naturally existing in the oligosaccharide mixture and is not obtained through enzymolysis, and the types include: deltaU 2S H NS6S U gal H NS6S (β)/I 2S H NS6S I 2S H NS6S 、ΔU 2S H NS6S U ga1 H NS6S (α)/I 2S H NS6S I 2S H NS6S 、ΔU 2S H NS6S I 2S H NS (α)、ΔU 2S H NS6S I 2S H NS (β)、ΔU 2S H NS6S I 2S Man NS (β)、ΔU 2S H NS6S I 2S Man NS (α)、ΔU 2S H NS I 2S H NS6S (α)、ΔU 2S H NS I 2S H NS6S (β)、ΔU 2S H NS6S I 2S Man NS (1，6anhydro)、ΔU 2S H NS6S I 2S H NS (1，6anhydro)、ΔU 2S H NS6S G 2S H NS 、ΔU 2S H NS6S GH NS6S (α)、ΔU 2S H NS6S GH NS6S (β)、ΔU 2S H NS6S I 2S H NS6S (α)、ΔU 2S H NS6S I 2S Man NS6S (β)、ΔU 2S H NS6S I 2S H NS6S (β)、ΔU 2S H NS6S I 2S Man NS6S (α)、ΔU 2S H NS6S G 2S H NS6S (alpha) and DeltaU 2S H NS6S G 2S H NS6S (β)；
(4) The structure of the connecting region has abundance higher than medical low molecular heparin, and the types include: deltaU-Gal-Gal-Xyl-Serine, deltaU 2, 1-U-Gal-Gal-Xyl-Serine, deltaU-Gal-Gal-Xyl-CH 2 COOH and DeltaU 4,3,1-U-Gal-Gal-Xyl-CH 2 COOH；
(5) The sulfation modification degree is high: most of the N-position, 6-O-position and 2-O-position of iduronic acid of glucosamine are sulfation modification, the rest of N-position of glucosamine is acetylation modification, but the average value is only 4.9%;
(6) The content of the high sulfated component is more than that of the clinical commercial medical low molecular heparin: in the oligosaccharide component after enzyme selective degradation, including non-reducing end a (trisulfated hexosamine, ANS,3,6S), tetraose Δu4,5,0 and tetraose Δu4,6,0 and larger sulfated oligosaccharide chains, the content is more than that of fine heparin and clinically commercially available medical low molecular heparin; in the oligosaccharide mixture without enzymatic degradation, the high weight average molecular weight components include Δu6,9,0, Δu8,12,0 and Δu10,14,0, in amounts greater than those of clinically commercially available medical low molecular weight heparin;
(7) Ext> Gext> -ext> aext> associatedext> withext> anticoagulantext> activeext> sitesext>,ext> inext> anext> averageext> amountext> ofext> 2.3ext>%ext> -ext> 2.5ext>%ext>;ext>
(8) Iduronic acid is more abundant than glucuronic acid, wherein the iduronic acid/glucuronic acid ratio is on average 4.3.
3. An oligosaccharide mixture as claimed in claim 1 wherein: the oligosaccharide mixture has anticoagulant and antithrombotic effects, and the anti-Xa activity of the oligosaccharide mixture is 80-120U/mg depending on antithrombin III.
4. A process for the preparation of an oligosaccharide mixture as claimed in any one of claims 1 to 3, characterised in that: chemical methods for beta-elimination depolymerization are employed, including: the heparan is derivatized and then used to initiate beta-eliminated depolymerization with alkali.
5. The process for the preparation of an oligosaccharide mixture as claimed in claim 4, wherein: the method comprises the following steps: dissolving heparan in purified water, mixing with an aqueous solution prepared from benzethonium chloride or benzalkonium bromide with the mass of more than or equal to 2.5 times that of the heparan, collecting and separating out white solid insoluble matters, drying, and derivatizing to obtain heparin ammonium salt; dissolving heparin ammonium salt in an organic solvent with the mass of 5 times or more of the heparin, adding benzyl trimethyl ammonium hydroxide with the mass of 0.5 time or more of the heparin, and stirring to depolymerize; adding sodium acetate methanol solution prepared by sodium acetate with mass of 0.5 times or more of that of heparan Caprae Seu Ovis after depolymerization reaction, collecting precipitate, re-dissolving with saline, precipitating with ethanol, grading, collecting component with weight average molecular weight of 4000Da-5000Da, refining, and drying to obtain oligosaccharide mixture.
6. The method of preparing an oligosaccharide mixture according to claim 5, wherein: after adding benzyltrimethylammonium hydroxide, the depolymerization reaction time is 12 hours or longer with stirring.
7. The process for the preparation of an oligosaccharide mixture as claimed in claim 4, wherein: the method comprises the following steps: dissolving heparan in purified water, mixing with an aqueous solution prepared from benzethonium chloride or benzalkonium bromide with the mass of more than or equal to 2.5 times that of the heparan, collecting and separating out white solid insoluble matters, drying, and derivatizing to obtain heparin ammonium salt; dissolving heparin ammonium salt in an organic solvent with the mass of 5 times or more of the heparin, adding benzyl chloride with the mass of 0.5 times or more of the heparin, mixing the mixture with sodium acetate methanol solution prepared from sodium acetate with the mass of 0.5 times or more of the heparin after stirring reaction, collecting precipitated precipitate, drying and derivatizing to obtain heparin benzyl ester; dissolving heparin benzyl ester in water, heating, adding inorganic alkali solution, stirring to depolymerize, precipitating with ethanol, re-dissolving precipitate with saline, precipitating with ethanol, grading, collecting fraction with weight average molecular weight of 4000Da-5000Da, refining, and drying to obtain oligosaccharide mixture.
8. The method of preparing an oligosaccharide mixture according to claim 7, wherein: the inorganic alkali solution adopts sodium hydroxide aqueous solution.
9. The method of preparing an oligosaccharide mixture according to claim 7, wherein: dissolving heparin ammonium salt in an organic solvent, adding benzyl chloride, and stirring to react for more than 12 hours; dissolving heparin benzyl ester in water, heating, adding inorganic alkali solution, stirring, reacting and depolymerizing for more than 1 hour.
10. A pharmaceutical composition comprising an oligosaccharide mixture according to any one of claims 1-3 and a pharmaceutically acceptable carrier.
11. Use of an oligosaccharide mixture according to any of claims 1-3 for the prophylaxis and treatment of thromboembolic disorders.
12. The development of an oligosaccharide mixture according to any one of claims 1-3 as an anticoagulant and antithrombotic agent or a halamic anticoagulant and antithrombotic agent.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310232108.2A CN117064909A (en) | 2023-03-12 | 2023-03-12 | Oligosaccharide mixture derived from heparan caprae seu ovis and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310232108.2A CN117064909A (en) | 2023-03-12 | 2023-03-12 | Oligosaccharide mixture derived from heparan caprae seu ovis and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117064909A true CN117064909A (en) | 2023-11-17 |
Family
ID=88715890
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310232108.2A Pending CN117064909A (en) | 2023-03-12 | 2023-03-12 | Oligosaccharide mixture derived from heparan caprae seu ovis and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117064909A (en) |
-
2023
- 2023-03-12 CN CN202310232108.2A patent/CN117064909A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Linhardt et al. | Production and chemical processing of low molecular weight heparins | |
| Mansour et al. | Primary structure and anticoagulant activity of fucoidan from the sea cucumber Holothuria polii | |
| Wu et al. | Physicochemical characteristics and anticoagulant activities of low molecular weight fractions by free-radical depolymerization of a fucosylated chondroitin sulphate from sea cucumber Thelenata ananas | |
| Mansour et al. | Characterization and anticoagulant activity of a fucosylated chondroitin sulfate with unusually procoagulant effect from sea cucumber | |
| Karamanos et al. | Ion-pair high-performance liquid chromatography for determining disaccharide composition in heparin and heparan sulphate | |
| Yang et al. | Chemical modification, characterization and structure-anticoagulant activity relationships of Chinese lacquer polysaccharides | |
| DK173818B1 (en) | heparin derivative | |
| Togola et al. | Polysaccharides with complement fixing and macrophage stimulation activity from Opilia celtidifolia, isolation and partial characterisation | |
| EP0394971A1 (en) | Oligosaccharide-containing inhibitors of endothelial cell growth and angiogenesis | |
| EP2794665B1 (en) | Non anti-coagulative glycosaminoglycans comprising repeating disaccharide unit and their medical use | |
| HU210925B (en) | Process to prepare polisaccharide mixtures wita low moleculare weight and pharmaceutical compns. contg. them | |
| Liu et al. | Structural analysis and biological activity of a highly regular glycosaminoglycan from Achatina fulica | |
| Maas et al. | Sulfation pattern of citrus pectin and its carboxy-reduced derivatives: Influence on anticoagulant and antithrombotic effects | |
| US10689463B2 (en) | Fuc3S4S substituted oligoglycosaminoglycan and preparation method thereof | |
| Fu et al. | Structure and activity of a new low-molecular-weight heparin produced by enzymatic ultrafiltration | |
| Guan et al. | Comparison of low-molecular-weight heparins prepared from bovine lung heparin and porcine intestine heparin | |
| CN107759712B (en) | Sheep-derived low-molecular-weight heparin and preparation method and application thereof | |
| WO2009105522A1 (en) | Methods of making low molecular weight heparin compositions | |
| EP2985298B1 (en) | Low-molecular-weight fucosylated glycosaminoglycan derivative containing terminal 2,5-anhydrated talose or derivative thereof | |
| Flengsrud et al. | Purification, characterization and in vivo studies of salmon heparin | |
| WO2013139478A1 (en) | Profiling oligosaccharides associated with biological activities of heparin or heparin derivatives | |
| Byankina et al. | Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus | |
| Alban et al. | Gas-liquid chromatography-mass spectrometry analysis of anticoagulant active curdlan sulfates | |
| Baytas et al. | Preparation of low molecular weight heparin from a remodeled bovine intestinal heparin | |
| Tang et al. | A regular Chlorella mannogalactan and its sulfated derivative as a promising anticoagulant: structural characterization and anticoagulant activity |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |