WO2017032276A1 - 一种牛肠粘膜依诺肝素钠及其制备方法与应用 - Google Patents

一种牛肠粘膜依诺肝素钠及其制备方法与应用 Download PDF

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WO2017032276A1
WO2017032276A1 PCT/CN2016/096023 CN2016096023W WO2017032276A1 WO 2017032276 A1 WO2017032276 A1 WO 2017032276A1 CN 2016096023 W CN2016096023 W CN 2016096023W WO 2017032276 A1 WO2017032276 A1 WO 2017032276A1
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bovine intestinal
intestinal mucosal
enoxaparin sodium
heparin
bovine
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PCT/CN2016/096023
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English (en)
French (fr)
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金永生
靳彩娟
姚亦明
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苏州融析生物科技有限公司
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    • 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
    • C08B37/0069—Chondroitin-4-sulfate, i.e. chondroitin sulfate A; Dermatan sulfate, i.e. chondroitin sulfate B or beta-heparin; Chondroitin-6-sulfate, i.e. chondroitin sulfate C; Derivatives thereof
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
    • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
    • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
    • C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
    • C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
    • C08B37/0078—Degradation products
    • 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
    • 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
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • 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
    • 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
    • C08B37/0003—General processes for their isolation or fractionation, e.g. purification or extraction from biomass

Definitions

  • the invention relates to a bovine intestinal mucosal enoxaparin sodium and a preparation method and application thereof, and belongs to the field of medical biotechnology.
  • Heparin is a sulfated acid polysaccharide ester substance produced by the mast cells of connective tissue of animals. It is the most widely used anticoagulant and antithrombotic drug in clinical practice.
  • Enoxaparin Sodium (ES) is a low molecular weight heparin sodium salt that is depolymerized from macromolecular heparin and is one of the most important heparin anticoagulants in the clinic. More than $3 billion.
  • ES Enoxaparin Sodium
  • bovine heparin and heparin including sheep heparin and goat heparin are also used in some areas.
  • the natural heparin from different sources has different degrees of molecular structure, disaccharide composition and physicochemical properties.
  • the low molecular weight heparin prepared from different natural heparins also has the corresponding differences in molecular structure caused by species.
  • bovine heparin and heparin are halal, and there is a strong demand for halal drugs in Muslim countries and regions.
  • Halal is a popular name for Muslims in China.
  • Muslim doctrine has clear requirements for food and medicine.
  • mammals only ruminant products such as cattle, sheep, and goats are allowed to be eaten, and non-reverse animal products such as pigs and dogs are fasted.
  • the global Muslim population exceeded 1.6 billion in 2013, accounting for 23% of the world's 6.9 billion people.
  • countries with a majority of Muslim populations, such as Indonesia, Pakistan, Iran, etc. Muslim medicines that meet Muslim teachings have unparalleled advantages. Therefore, the development of halal cattle, sheep enoxaparin sodium, has an extremely important role.
  • China has abundant livestock product resources.
  • the breeding and slaughtering of pigs, cattle, sheep and goats are among the highest in the world. It is easy to prepare intestinal mucosa or lung enoxaparin sodium of cattle, sheep and goats.
  • the object of the present invention is to provide a method for preparing bovine intestinal mucosal enoxaparin sodium, including bovine intestinal mucosal enoxaparin sodium, an injection, and an anticoagulant antithrombotic and halal medicine.
  • the bovine intestinal mucosal enoxaparin sodium is prepared from hepatic intestinal mucosal heparin.
  • the disaccharide composition of the bovine intestinal mucosa enoxaparin sodium exhibits typical species, organ origin (bovine intestinal mucosa) and regional characteristics, SAX-HPLC analysis after heparinase digestion, ⁇ UA2S-GlcNS ( ⁇ IIIS)
  • the content is 15%-32%, specifically from the bovine intestinal mucosa enoxaparin sodium prepared from the cattle source in mainland China, 15%-24%, from the South American marketed Brazilian bovine intestinal mucosa heparin prepared bovine intestinal mucosal enoxaparin sodium
  • the ⁇ IIIS content is 24%-32%, and ⁇ IIIS is only 5.8%-7.8%, 4.0% in the (intestine mucosa) enoxaparin sodium, the intestinal mucosa enoxaparin sodium and the bovine lung enoxaparin sodium, respectively. 6.0% and 4.5%-5.5%.
  • the biological anticoagulant activity of the bovine intestinal mucosal enoxaparin sodium is analyzed with reference to USP39.
  • the anti-Xa activity is dried between 90-125 units per mg, and the anti-IIa activity is dried at 10-35 units.
  • the ratio of anti-Xa/anti-IIa is between 5-10 per mg.
  • the preparation method of the bovine intestinal mucosal enoxaparin sodium described above is the same as the preparation method claimed by the present inventors in the prior patent application (Application Publication No.: CN 105131153 A), and includes the following steps:
  • pretreatment of raw bovine intestinal mucosa heparin is prepared by dissolving the crude bovine intestinal mucosa heparin sodium in a certain concentration of saline to prepare a solution, decolorizing the bovine intestinal mucosal heparin solution, finely filtering, and then performing alcohol precipitation at room temperature. Refined, collected precipitates, and dried to obtain bovine intestinal mucosal heparin.
  • heparin quaternary ammonium salt of bovine intestinal mucosa is prepared by dissolving the bovine intestinal mucosa heparin sodium obtained in S1 into a bovine intestinal mucosal heparin aqueous solution, mixing with benzethonium chloride aqueous solution, filtering or centrifuging to obtain bovine intestinal mucosa heparin. a quaternary ammonium salt and washed and dried;
  • bovine intestinal mucosal heparin benzyl ester by mixing and esterifying the bovine intestinal mucosal heparin quaternary ammonium salt obtained by drying in S2 with methylene chloride and benzyl chloride in a weight ratio, and the cow intestinal mucosal heparin season after esterification
  • the sodium acetate methanol solution was added dropwise to the ammonium salt to prepare a hepatic intestinal mucosal heparin benzyl ester precipitate, and the bovine intestinal mucosal heparin benzyl ester was precipitated, filtered, washed and dried to obtain a bovine intestinal mucosal heparin benzyl ester;
  • bovine intestinal mucosal enoxaparin sodium finished product is the S3 in the bovine intestinal mucosal heparin benzyl ester alkali depolymerization, decolorization, acid neutralization to neutral, alcohol precipitation, refining, drying, to obtain the bovine Mucosal enoxaparin Sodium finished product.
  • a sodium chloride aqueous solution having a mass concentration of 1% to 3% is used to dissolve the crude heparin sodium of the bovine intestinal mucosa for decolorization, filtration and purification, until the aqueous solution of the heparin sodium of the bovine intestinal mucosa is clarified and the chroma is not deep after the pretreatment.
  • the precipitating agent refined in the alcohol precipitation in S1 is one or a combination of methanol, ethanol, isopropanol or acetone.
  • the weight ratio of benzethonium chloride to bovine intestinal mucosa heparin sodium in S2 is 2-5:1; the esterification temperature in S3 is 30-40 ° C, cow intestinal mucosa heparin quaternary ammonium salt, dichloromethane, benzyl chloride
  • the mass ratio is 1:3-10:1.1
  • washing of the cow intestinal mucosal heparin benzyl ester precipitate in S3 comprises the following steps:
  • S4 is depolymerized by using a sodium hydroxide solution, the depolymerization temperature is between 30 ° C and 70 ° C, and the holding time is more than 0.5 hours; in S4, decoloration with hydrogen peroxide is used, and 0.1-1 times of bovine intestinal mucosal heparin is added at room temperature or below. 30% hydrogen peroxide by weight of benzyl ester, oxidatively decolorized for more than 10 minutes until the color of the reaction solution is as shallow as Y6 and GY6.
  • the detection of the finished product of the bovine intestinal mucosal enoxaparin sodium in S4 is carried out according to the index of enoxaparin sodium in USP39, and the removal of the provenance and the lower anti-IIa activity, the other indicators are in line with the release of USP39. index.
  • the structural analysis of the finished product of the bovine intestinal mucosal enoxaparin sodium in S4 is analyzed by nuclear magnetic resonance spectroscopy ( 1 H-NMR) and nuclear magnetic resonance spectroscopy ( 13 C-NMR) to examine the linkage on the sugar chain. Carbon-hydrogen relationship.
  • the bovine intestinal mucosal enoxaparin sodium and the enoxaparin sodium derived from the porcine intestinal mucosa have the same main structure, but there are also some differences, such as the methyl peak of N-acetyl group at ⁇ 2.04 ppm, the number integral There is less enoxaparin sodium in the bovine intestinal mucosa, indicating that the N-acetyl modification in the enoxaparin sugar chain of the bovine intestinal mucosa is relatively less.
  • HSQC-NMR heteronuclear single quantum relationship-nuclear magnetic resonance
  • the bovine intestinal mucosal enoxaparin sodium is prepared according to the preparation method as described above.
  • the bovine intestinal mucosal enoxaparin sodium is used in the prevention and treatment of diseases related to anticoagulation and antithrombotic, and is developed as a halal anticoagulant antithrombotic drug.
  • a bovine intestinal mucosal enoxaparin sodium injection comprising the above-mentioned bovine intestinal mucosal enoxaparin sodium and water for injection.
  • the method for preparing the bovine intestinal mucosal enoxaparin sodium injection comprises dissolving the bovine intestinal mucosal enoxaparin sodium in water for injection, and after completely dissolving, supplementing the water for injection to a certain concentration, aseptically filtering and filling to Syringes, vials or ampoules.
  • the active concentration of the bovine intestinal mucosal enoxaparin sodium injection is 10,000 anti-Xa units per ml, preferably made into pre-filled needles, the specifications are 4000 anti-Xa units, 6000 anti-Xa units and 10000 anti- Xa units and other specifications.
  • the bovine intestinal mucosal enoxaparin sodium injection is used for anticoagulant, antithrombotic and halal drugs.
  • a bovine intestinal mucosal enoxaparin sodium injection comprising the above-mentioned bovine intestinal mucosal enoxaparin sodium, water for injection and benzyl alcohol.
  • the method for preparing the bovine intestinal mucosal enoxaparin sodium injection comprises dissolving the bovine intestinal mucosal enoxaparin sodium in water for injection, and then adding benzyl alcohol, and after completely dissolving and mixing, supplementing the water for injection to a certain concentration, Sterile filtration, filling to vials, etc.
  • the concentration of benzyl alcohol is between 1.35 mg/ml and 1.65 mg/ml.
  • the above-mentioned bovine intestinal mucosal enoxaparin sodium injection has an active concentration of 10,000 anti-Xa units per ml, preferably potted into a vial, a specification of 30,000 anti-Xa units and other specifications.
  • the bovine intestinal mucosal enoxaparin sodium injection is used for anticoagulant, antithrombotic and halal drugs.
  • the anticoagulant effect of the bovine intestinal mucosal enoxaparin sodium and the bovine intestinal mucosal enoxaparin sodium injection is preferably carried out in vivo in rabbits.
  • rabbit blood is collected before and after administration, and anticoagulation with 3.8% sodium citrate anticoagulant 1:9, the effect of on-machine detection on blood coagulation routine, including but Not limited to APTT, TT and PT, etc., as well as the effects of other clotting factors.
  • the bovine intestinal mucosa enoxaparin sodium and bovine intestinal mucosal enoxaparin sodium injection are resistant in vivo
  • the coagulation test showed an effect similar to or equivalent to the enoxaparin sodium standard.
  • the outstanding effect of the invention is that a bovine intestinal mucosa enoxaparin sodium and an injection thereof are provided, and are prepared by a practical and stable method, except for the molecular structure (disaccharide composition) and antibiotic caused by the characteristics of the provenance.
  • the bovine intestinal mucosal enoxaparin sodium is consistent with the mass release indicators listed in other USP39 enoxaparin sodium.
  • the invention fills the blank of other sources of heparin in the preparation of enoxaparin sodium, and can be developed into a halal drug.
  • the bovine intestinal mucosa heparin sodium the raw materials are simple and easy to obtain, the quality is controllable, which can greatly enrich the source and yield of enoxaparin sodium in the market, and can also promote the effective utilization of cattle breeding and slaughtering waste (intestinal mucosa) with huge economic potential.
  • Fig. 1 is a schematic view showing the comparison of molecular weight distributions of enoxaparin sodium and enoxaparin sodium standards of bovine intestinal mucosa according to Example 2 of the present invention.
  • Example 2 is a schematic diagram showing the disaccharide spectrum and 1,6-anhydride % of the bovine intestinal mucosal enoxaparin sodium according to Example 2 of the present invention.
  • Fig. 3 is a schematic view showing the ratio of sulfonate and carboxylate in the bovine intestinal mucosal enoxaparin sodium according to Example 2 of the present invention.
  • Figure 4 is a schematic diagram showing the 1 H-NMR comparison of the enema heparin sodium and enoxaparin sodium standards of the bovine intestinal mucosa according to Example 2 of the present invention.
  • Fig. 5 is a schematic view showing the comparison of 13 C-NMR of the entorage of enoxaparin sodium and enoxaparin sodium in the bovine intestinal mucosa according to Example 2 of the present invention.
  • Figure 6 is a schematic view showing the comparison of the anti-Xa activity of the bovine intestinal mucosal enoxaparin sodium and its injection sample and the enoxaparin sodium standard in rabbits according to Example 5 of the present invention.
  • the present invention describes a new source of enoxaparin sodium - bovine intestinal mucosal enoxaparin sodium and its injection.
  • the specific experimental examples are taken as an example to illustrate the specific embodiments. It should be understood that the specific implementation described herein. The examples are merely illustrative of the invention and are not intended to limit the invention.
  • the mixture was centrifuged at 6000 rpm for 5 minutes in a high-speed centrifuge, and the pellet was resuspended in 1600 ml of water, and further stirred for 5 minutes, and further centrifuged at 6000 rpm for 5 minutes. repeat.
  • the precipitated bovine intestinal mucosal heparin quaternary ammonium salt was air-dried at 45 ° C for 10 hours, transferred to a vacuum drying oven, and vacuum dried at 60 ° C for 48 hours.
  • the dried cow's intestinal mucosal heparin quaternary ammonium salt had a loss on drying of 0.5% and a weighing of 232.5 g.
  • the solution was dissolved in the above solid, and then precipitated with 1.6 liters of methanol, and the precipitate was harvested by centrifugation at 6000 rpm for 5 minutes.
  • the salt solution was repeated and methanol precipitation was repeated 3 times, and the precipitate was transferred to a vacuum drying oven and vacuum dried at 60 ° C for 50 hours.
  • the obtained bovine intestinal mucosal heparin benzyl ester weighed 60.2 g, the loss on drying was 3.3%, and the degree of esterification was 12.8% after drying.
  • the filtrate was added with 125 g of sodium chloride, stirred to ensure complete dissolution of sodium chloride, adjusted to pH 6.0, and then finely filtered at 0.22 ⁇ m. 3 liters of methanol was added and the precipitate was filtered through a 400 mesh. The precipitate was resuspended in 3 liters of methanol for 30 minutes, and the precipitate was taken up with suction. After the precipitate was dissolved in 120 g of water, it was transferred to a lyophilized bottle and lyophilized under vacuum for 30 hours. Frozen The dry powder is weighed and then bagged and sealed for inspection.
  • enoxaparin sodium was obtained from the bovine intestinal mucosa, and the weight yield was 56.0% based on the initial dose, and the loss on drying was 4.2%.
  • the weight average molecular weight is 4431, the ratio of molecular weight ⁇ 2000 part is 15.7%, the ratio of 2000 ⁇ molecular weight ⁇ 8000 part is 72.8%, the ratio of molecular weight>8000 part is 12.5%; the 1,6-anhydride ring sugar chain accounts for total sugar chain. It is 22.0%.
  • the anti-Xa activity was 95.2 units per mg after drying, the anti-IIa activity was 19.5 units per mg after drying, and the anti-Xa/anti-IIa ratio was 4.9; 1.0 g of the product was dissolved in 10 ml of water, clarified and The chroma is not deeper than the standard color No. 6; 1.0 g of the product is dissolved in 10 ml of water, the pH is 6.76; the sodium content is 12.1% after drying; the nitrogen content is 2.0% after drying; the aqueous solution has the largest wavelength at 232 nm.
  • the characteristic absorption at 231 nm is 16.2; the ratio of sulfonate/carboxylate is 2.8; the content of benzyl alcohol is not more than 0.1% after drying, and the content of benzylammonium is not more than 0.1% after drying; the residual of heavy metal is not more than 30 ppm; methanol in solvent residue is 210 ppm; bacterial endotoxin content, less than 0.01 bacterial endotoxin unit (EU) per anti-Xa unit active enoxaparin sodium.
  • EU bacterial endotoxin unit
  • the bovine intestinal mucosa heparin sodium supplied by Suzhou Terui Pharmaceutical Co., Ltd., extracts and purifies the intestinal mucosa after slaughter of beef cattle in northeast China. Accurately weighed 3.0 kg, which was 156.3 units per milligram after the anti-coagulant activity of whole sheep plasma. According to the method for determining the activity of high-quality heparin sodium anti-Xa and anti-IIa according to USP39, the anti-Xa activity was 139.1 after drying. The unit per mg of anti-IIa activity is 130.0 units per mg, and the anti-Xa/anti-IIa ratio is 1.07.
  • the procedure for preparing the bovine intestinal mucosal enoxaparin sodium was the same as in Example 1, except for the difference in the amount of reagent used. Finally, 1683.1 g of enema heparin sodium was obtained from the bovine intestinal mucosa, and the weight yield was 56.1% based on the initial dose. The weight loss on drying is 3.2%; 1.0 g of the product is dissolved in 10 ml of water, clarified and the color is not deeper than the standard color of No.
  • the molecular weight distribution of the bovine intestinal mucosal enoxaparin sodium was carried out in accordance with the USP 39 method, and the results are shown in Fig. 1 and Table 1.
  • Table 1 Weight average molecular weight and molecular weight distribution of enema heparin sodium samples from bovine intestinal mucosa
  • the bovine intestinal mucosal enoxaparin sodium (RX0025-JCJ-003) in the table was obtained from the product prepared in Example 2.
  • the bovine intestinal mucosal enoxaparin sodium prepared in the second embodiment has a weight average molecular weight and a molecular weight distribution which are very close to the enoxaparin sodium standard derived from the pig intestinal mucosa, and meet the requirements of the USP39 technical index.
  • Table 2 Disaccharide composition ratio and 1,6-anhydride% of enoxaparin sodium and enoxaparin sodium standards in bovine intestinal mucosa
  • the bovine intestinal mucosa enoxaparin sodium enoxaparin sodium prepared in Example 2 exhibited typical characteristics of bovine intestinal mucosal heparin, compared with the disaccharide of enoxaparin sodium standard.
  • the content of the main disaccharide ⁇ IIIS is higher, reaching 17.08%.
  • the disaccharide composition and percentage are not the release indicators of USP39 and EP8.6.
  • the percentage of 1,6-anhydride of the bovine intestinal mucosa enoxaparin sodium prepared by the process is almost the same as that of the standard, which is 19.5%, which is in accordance with the index of 15%-25% required by USP39.
  • the molar ratio of sulfonate to carboxylate of the bovine intestinal mucosal enoxaparin sodium was 2.5, which was slightly higher than that of the usual porcine intestinal mucosal enoxaparin sodium (2.2).
  • This test reflects the degree of sulfonate modification on the sugar chain, indicating that the sulfonate modification of the two is also relatively close.
  • the test results are within the technical standard of USP39 (greater than 1.8), and the ratio of sulfonate and carboxylate in the bovine intestinal mucosal enoxaparin sodium meets the requirements.
  • sample solution to be tested bovine intestinal mucosa enoxaparin sodium (Example 1) and enoxaparin sodium standard, each accurately weigh about 20 mg, and weighed in water (D 2 O) to 20 mg per At a concentration of about ML, 1-2 drops of TSP were added dropwise, and the mixture was shaken and mixed for 0.22 micron filtration. The results are shown in Fig. 4. Among them, ⁇ 3.4 ppm is the residual methyl hydrogen peak of methanol, and ⁇ 4.7 ppm is water hydrogen. peak.
  • the nuclear magnetic carbon spectrum of the bovine intestinal mucosa enoxaparin sodium was analyzed by the 600 MHz NMR spectrometer of the Suzhou University Analytical Testing Center. The method was carried out in accordance with USP39.
  • bovine intestinal mucosal enoxaparin sodium (Example 2) and enoxaparin sodium standard, each accurately weighing 200 mg, adding 0.2 ml of hydrophobic water (D 2 O) and 0.8 ml of pure water to dissolve Then, 50 ⁇ l of deuterated methanol was added dropwise, and the mixture was shaken and mixed for 0.22 ⁇ m filtration. The results are shown in Fig. 5, wherein ⁇ 50 ppm is a methyl carbon peak remaining in methanol.
  • Example 1 The results showed that, consistent with the hydrogen spectrum, the enzymatic heparin sodium carbon skeleton prepared in Example 1 was consistent with the enoxaparin sodium standard, but some specific positions, such as ⁇ 24.9 ppm of nitrogen-acetyl methyl carbon. There is a certain difference in the content. Although the carbon spectrum of the bovine intestinal mucosa enoxaparin sodium is significantly different, the difference is not in the requirements of USP39, so the results are in accordance with USP39.
  • the activity of anti-Xa and anti-IIa of the bovine intestinal mucosal enoxaparin sodium was measured in accordance with USP 39, and the respective activities of the eneviable bovine intestinal mucosal enoxaparin sodium and enoxaparin sodium standards of Example 1 are shown in Table 3 below.
  • the bovine intestinal mucosal enoxaparin sodium sample prepared in Example 2 was inferior to the porcine intestinal mucosal enoxaparin sodium except that the provenance was different and the anti-IIa activity was low.
  • USP39 requires release factors for enoxaparin sodium, compared to the enoxaparin sodium release index required by EP8.6. It is also fully compliant.
  • bovine intestinal mucosal enoxaparin sodium powder (3.2% dry weight loss, 120.2 anti-Xa unit per mg, a total of 0.2 billion anti-Xa units), dissolved in cooled water for injection and The volume is adjusted to 2000 ml, and the two-stage 0.2 micron filter is aseptically filtered into the Class A clean area and potted into a 1 ml glass syringe with a potting machine.
  • the specification is 6000 units (or 0.6 ml), and the loss is removed. 1830 of the finished product of enema heparin sodium injection of bovine intestinal mucosa.
  • bovine intestinal mucosal enoxaparin sodium powder (3.2% dry weight loss, 120.2 anti-Xa unit per mg, a total of 0.3 billion anti-Xa units), dissolved in cooled water for injection.
  • Add 45.0 g of benzyl alcohol stir evenly, and make up to 3000 ml with cooled water for injection.
  • the two-stage 0.2 micron filter is aseptically filtered into the A-grade clean area, and potted into a 5 ml vial with a potting machine.
  • the specification is 30,000 units (or 3.0 ml), and the loss is lost.
  • a total of 870 bottles of enzymatic heparin sodium injection 2 are obtained.
  • Test sample bovine intestinal mucosal enoxaparin sodium sample (described in Example 2, batch number: RX0025-JCJ-003), bovine intestinal mucosal enoxaparin sodium injection 1 (described in Example 3), bovine intestinal mucosa Heparin sodium injection 2 (described in Example 4), enoxaparin sodium standard product is a clinically marketed drug (Kesai, batch number: 24459).
  • Preparation The bovine intestinal mucosal enoxaparin sodium sample was prepared in physiological saline to a concentration of 100 mg per ml, and 0.2 ⁇ m was filtered for use, and other injections were directly used for injection.
  • Experimental method 2-3 kg of Japanese white rabbits were selected and administered subcutaneously at the upper extremities of the anterior and the lower limbs according to their body weight. Injection dose: 2 mg per kg (or 200 units per kg). Blood was collected from 0.5 hour to 1 hour before and after administration, and 2 ml of blood was collected, and anticoagulated with 3.8% sodium citrate anticoagulant 1:9, and detected by machine. Measuring instrument: automatic blood coagulation instrument (Stago Compact) and platelet aggregation instrument (Puli Health LBY-NJ4).
  • Anticoagulant test A routine coagulation set including anti-Xa activity, activated partial thrombin time (APTT) and thrombin time (TT) is determined.
  • Fig. 6 (1) The experimental results are shown in Fig. 6 (1). It can be seen from the figure that compared with the enoxaparin sodium standard, the bovine intestinal mucosal enoxaparin sodium and its injection can significantly prolong the APTT, the bovine intestinal mucosa. Heparin sodium and its injections have similar effects on APTT prolongation, and are slightly weaker than enoxaparin sodium standards; all groups have similar APTT maximum time in rabbits, and the decay time is similar, revealing bovine intestinal mucosal enoxaparin sodium and The injection is consistent with the enoxaparin sodium standard in rabbits.
  • Fig. 6 (3) The experimental results are shown in Fig. 6 (3). It can be seen from the figure that the bovine intestinal mucosal enoxaparin sodium and its injection can significantly prolong TT, but slightly weaker than the enoxaparin sodium standard; At the time of the maximum TT, the bovine intestinal mucosal enoxaparin sodium and its injection were similar to the enoxaparin sodium standard, and the decay time was also comparable.
  • the present invention has various embodiments, and all technical solutions formed by equivalent transformation or equivalent transformation are within the scope of the present invention.

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Abstract

提供了一种牛肠粘膜依诺肝素钠及其制备方法与应用。牛肠粘膜依诺肝素钠由牛肠粘膜肝素制备而来，与USP39和EP8.6等药典规定的由猪肠粘膜肝素来源的依诺肝素钠不同，其呈现出典型的物种和器官来源（牛肠粘膜）特征。还提供了两种牛肠粘膜依诺肝素钠注射剂和制备方法，以及在动物体内抗凝试验的应用。牛肠粘膜依诺肝素钠还具有猪来源依诺肝素钠所不具有的清真性，在广大穆斯林人群、国家和地区，有着巨大的市场。牛肠粘膜依诺肝素钠及其注射剂，可应用于抗凝、抗栓、抗炎、抗癌及清真药物。

Description

一种牛肠粘膜依诺肝素钠及其制备方法与应用 技术领域
本发明涉及一种牛肠粘膜依诺肝素钠及其制备方法与应用，属于医药生物技术领域。
背景技术
肝素(Heparin)是一种硫酸化酸性多糖酯类物质，由动物结缔组织的肥大细胞产生分泌，是目前临床上应用最广泛的抗凝抗栓药物。依诺肝素钠(Enoxaparin Sodium，ES)是一种低分子的肝素钠盐，是由大分子肝素经解聚而来，是临床上最为重要的肝素类抗凝剂之一，全球年销售量超30亿美元以上。目前，医用肝素的主要来源是猪肠粘膜肝素，牛肝素和羊肝素(包括绵羊肝素和山羊肝素)在某些地区也有使用。不同来源的天然肝素，其分子结构、二糖组成和理化性质均有不同程度的差异，由不同天然肝素制备的低分子肝素，也必然存在相应的由种属带来的分子结构等的差异。
此外，不同于猪肝素，牛肝素和羊肝素具有清真性，穆斯林国家和地区对清真药物有着强烈的需求。清真是穆斯林在中国流行的专用名称，穆斯林教义对食品和药品等有着明确的要求，哺乳动物中只允许食用牛、绵羊、山羊等反刍动物产品，禁食猪和狗等不反刍动物产品。全球穆斯林人口2013年突破16亿，占全球69亿人口的23％。在一些由穆斯林人口占多数的国家，如印度尼西亚、巴基斯坦、伊朗等等，符合穆斯林教义的清真药品有着无可比拟的优势。因此，开发清真的牛、羊依诺肝素钠，有着极为重要的作用。中国具有丰富的畜产品资源，猪、牛、绵羊、山羊等养殖和屠宰量均位居世界前列，易于制备牛、绵羊、山羊等的肠粘膜或肺依诺肝素钠。
本发明人在之前的专利申请(申请公布号：CN 105131153 A)中，详细描述了一种羊依诺肝素钠及其制备方法，同时也提供了牛肠粘膜依诺肝素钠和牛肺依诺肝素钠以及他们的制备方法。经不同来源肝素制备而得的依诺肝素钠，都有近似的理化性质、分子结构和生物学抗凝活性，但同时又各有独特的特点。本发明详细介绍了牛肠粘膜依诺肝素钠及其注射剂，包括其制备方法以及具体的理化、生物学特性研究。
发明内容
本发明的目的在于提供一种牛肠粘膜依诺肝素钠，包括牛肠粘膜依诺肝素钠的制备方法、注射剂及在抗凝抗栓和清真药物中的应用。
本发明的目的，将通过以下技术方案得以实现：
牛肠粘膜依诺肝素钠，是以牛的肠粘膜肝素制备得到的。
所述牛肠粘膜依诺肝素钠的二糖组成，呈现典型的物种、器官来源(牛肠粘膜)和地区特征，以肝素酶酶解后的SAX-HPLC分析，ΔUA2S-GlcNS(ΔⅢS)的含量为15％-32％，具体来自中国内地牛源制备的牛肠粘膜依诺肝素钠为15％-24％，来自于南美的市售巴西牛肠粘膜肝素制备的牛肠粘膜依诺肝素钠，其ΔⅢS含量为24％-32％，而ΔⅢS在(猪肠粘膜)依诺肝素钠、羊肠粘膜依诺肝素钠和牛肺依诺肝素钠中仅分别是5.8％～7.8％、4.0％～6.0％和4.5％-5.5％。
所述牛肠粘膜依诺肝素钠的生物学抗凝活性，参照USP39进行分析，抗-Ⅹa活性折干后在90-125单位每毫克之间，抗-Ⅱa活性折干后在10-35单位每毫克之间，抗-Ⅹa/抗-Ⅱa比例在5-10之间。
以上所述的牛肠粘膜依诺肝素钠的制备方法，与本发明人在之前的专利申请(申请公布号：CN 105131153 A)主张的制备方法相同，包括如下步骤：
S1、原料牛肠粘膜肝素的预处理，是将牛肠粘膜肝素钠粗品溶解于一定浓度盐水中配制成溶液，对所述牛肠粘膜肝素溶液进行脱色，精过滤，再在室温下进行醇沉精制，收集沉淀物，干燥获得牛肠粘膜肝素。
S2、牛肠粘膜肝素季铵盐的制备，是将S1中获得的牛肠粘膜肝素钠溶解配制成牛肠粘膜肝素水溶液，并与苄索氯铵水溶液进行混合，过滤或离心获得牛肠粘膜肝素季铵盐，并进行洗涤干燥；
S3、牛肠粘膜肝素苄酯的制备，是将S2中干燥得到的牛肠粘膜肝素季铵盐与二氯甲烷及氯化苄按重量比例混合酯化，在酯化后的牛肠粘膜肝素季铵盐中滴加醋酸钠甲醇溶液，制得牛肠粘膜肝素苄基酯沉淀，将牛肠粘膜肝素苄基酯沉淀进行过滤、洗涤、干燥，制得牛肠粘膜肝素苄基酯；
S4、牛肠粘膜依诺肝素钠成品制得，是将S3中的牛肠粘膜肝素苄基酯进行碱解聚、脱色、以酸中和至中性、醇沉淀，精制、干燥，得到牛肠粘膜依诺肝素 钠成品。
进一步地，S1中采用质量浓度为1％-3％的氯化钠水溶液溶解牛肠粘膜肝素钠粗品进行脱色、过滤和精制，直至预处理后牛肠粘膜肝素钠的水溶液澄清且色度不深于5号标准色；S1中醇沉精制的沉淀剂为甲醇、乙醇、异丙醇或丙酮中的一种或几种的组合。
进一步地，S2中苄索氯铵与牛肠粘膜肝素钠的重量比为2-5:1；S3中酯化温度30-40℃，牛肠粘膜肝素季铵盐、二氯甲烷、氯化苄的质量比为1:3-10:1.1
进一步地，S3中牛肠粘膜肝素苄基酯沉淀的洗涤包括如下步骤：
S31、在加入醋酸钠甲醇溶液的牛肠粘膜肝素季铵盐溶液中加入甲醇静置沉降和分离制得牛肠粘膜肝素苄基酯；
S32、在分离后的牛肠粘膜肝素苄基酯中添加8％-12％的氯化钠水溶液进行复溶，所述氯化钠水溶液与所述牛肠粘膜肝素季铵盐重量比为0.5-2:1；
S33、对S32中获得的溶液以60％-70％的甲醇终浓度进行醇沉结晶；
S34、重复氯化钠水溶液复溶和醇沉结晶2-5次至牛肠粘膜肝素苄基酯复溶不浑浊。
进一步地，S4中采用氢氧化钠溶液解聚，解聚温度在30℃-70℃之间，保温时间在0.5小时以上；S4中采用双氧水脱色，室温或以下加入0.1-1倍牛肠粘膜肝素苄基酯重量的30％双氧水，氧化脱色10分钟以上，直至反应液颜色浅至Y6与GY6以下。
优选地，所述S4中牛肠粘膜依诺肝素钠成品的检测，参照USP39中依诺肝素钠的指标进行，且除去种源和较低的抗-Ⅱa活力外，其余指标均符合USP39的放行指标。
优选地，所述S4中牛肠粘膜依诺肝素钠成品的结构分析，采用核磁共振氢谱(1H-NMR)和核磁共振碳谱(13C-NMR)进行分析，考察糖链上链接的碳-氢关系。所述牛肠粘膜依诺肝素钠与来自猪肠粘膜的依诺肝素钠，主体结构一致，但也存在一定的差异，如在δ2.04ppm处的N-乙酰基的甲基峰，数量积分上牛肠粘膜依诺肝素钠要更少一些，说明牛肠粘膜依诺肝素糖链中N-乙酰基修饰相对要少。所述的核磁共振方法，更优的方案是采用异核单量子关系-核磁共振(HSQC-NMR)等更高级的二维核磁分析，以此可以明确判断一些具体糖链结 构上的差异。
优选地，所述牛肠粘膜依诺肝素钠，按照如以上所述的制备方法制得。
优选地，所述牛肠粘膜依诺肝素钠在防治与抗凝、抗栓有关疾病中的应用，以及开发为清真抗凝抗栓药物。
一种牛肠粘膜依诺肝素钠注射剂，组分包括以上所述牛肠粘膜依诺肝素钠和注射用水。
优选地，所述牛肠粘膜依诺肝素钠注射剂的制备方法，是将牛肠粘膜依诺肝素钠以注射用水溶解，待完全溶解后补注射用水至一定的浓度，无菌过滤，灌装至注射器、西林瓶或安瓿瓶等。
优选地，所述牛肠粘膜依诺肝素钠注射剂的活性浓度在10000抗-Ⅹa单位每毫升，优选制成预灌封针，规格为4000抗-Ⅹa单位、6000抗-Ⅹa单位和10000抗-Ⅹa单位以及其他规格。
优选地，所述牛肠粘膜依诺肝素钠注射剂，在抗凝、抗栓及清真药物中的应用。
一种牛肠粘膜依诺肝素钠注射剂，组分包括以上所述的牛肠粘膜依诺肝素钠、注射用水和苯甲醇。
优选地，所述牛肠粘膜依诺肝素钠注射剂的制备方法，是将牛肠粘膜依诺肝素钠以注射用水溶解，再加入苯甲醇，待完全溶解混匀后补注射用水至一定的浓度，无菌过滤，灌装至西林瓶等。
优选地，所述苯甲醇的浓度在1.35毫克每毫升至1.65毫克每毫升之间。
优选地，以上所述牛肠粘膜依诺肝素钠注射剂的活性浓度在10000抗-Ⅹa单位每毫升，优选灌封成西林瓶，规格为30000抗-Ⅹa单位以及其他规格。
优选地，所述牛肠粘膜依诺肝素钠注射剂，在抗凝、抗栓及清真药物中的应用。
优选地，所述牛肠粘膜依诺肝素钠和牛肠粘膜依诺肝素钠注射剂的抗凝作用，体内试验优选兔进行。优选皮下注射给药后，采集给药前及给药后各时间点的兔血液，用3.8％枸橼酸钠抗凝剂1:9抗凝，上机检测对血凝常规的影响，包括但不限于APTT、TT和PT等，以及其他凝血因子的影响。
优选地，所述牛肠粘膜依诺肝素钠和牛肠粘膜依诺肝素钠注射剂，在体内抗 凝试验，表现出近似或等同于依诺肝素钠标准品的效果。
本发明突出效果为：提供了一种牛肠粘膜依诺肝素钠以及它的注射剂，并采用实用、稳定的方法制得，除了由种源特征性带来的分子结构(二糖组成)和抗凝血活性(较小的抗-Ⅱa活性)外，牛肠粘膜依诺肝素钠均符合其他USP39依诺肝素钠所列的质量放行指标。本发明填补了其他来源肝素在依诺肝素钠制备上的空白，可开发为清真药物。牛肠粘膜肝素钠，原料简便易得，质量可控，可极大丰富市场中依诺肝素钠的来源和产量，还可以促进牛养殖和屠宰废料(肠粘膜)的有效利用，经济潜力巨大。
以下便结合实施例附图，对本发明的具体实施方式作进一步的详述，以使本发明技术方案更易于理解、掌握。
附图说明
图1为本发明实施例2所述牛肠粘膜依诺肝素钠与依诺肝素钠标准品的分子量分布比较示意图。
图2为本发明实施例2所述牛肠粘膜依诺肝素钠的二糖谱及1,6-酐％示意图。
图3为本发明实施例2所述牛肠粘膜依诺肝素钠的磺酸根和羧酸根比例示意图。
图4为本发明实施例2所述牛肠粘膜依诺肝素钠与依诺肝素钠标准品的1H-NMR对比示意图。
图5为本发明实施例2所述牛肠粘膜依诺肝素钠与依诺肝素钠标准品的13C-NMR比较示意图。
图6为本发明实施例5所述牛肠粘膜依诺肝素钠及其注射剂样品与依诺肝素钠标准品在兔体内的抗-Ⅹa活性比较示意图
具体实施方式
本发明实施例描述了一种新来源的依诺肝素钠——牛肠粘膜依诺肝素钠及其注射剂，下面以具体实验案例为例来说明具体实施方式，应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
实施例1
取巴西市售的牛肠粘膜肝素钠，经全绵羊血浆法抗凝活性折干后为156.3单 位每毫克；按USP39规定的精品肝素钠抗-Ⅹa和抗-Ⅱa活性测定方法，抗-Ⅹa活性折干后仅为120.1单位每毫克，抗-Ⅱa活性折干后为93.0单位每毫克，抗-Ⅹa/抗-Ⅱa比例为1.29。
准确称取上述牛肠粘膜肝素钠80克，溶解于800毫升水中；另将200克苄索氯铵，溶解于800毫升水中，配制成澄清的苄索氯铵水溶液；于充分搅拌下，将苄索氯铵水溶液滴加至肝素水溶液中，30分钟内滴加完毕，继续搅拌2小时。用高速离心机6000转/分钟离心5分钟，沉淀用1600毫升水重悬，继续充分搅拌5分钟，再6000转/分钟离心5分钟。重复一次。沉淀的牛肠粘膜肝素季铵盐，45℃鼓风干燥10小时后，转移至真空干燥箱，在60℃下真空干燥48小时。干燥后的牛肠粘膜肝素季铵盐的干燥失重为0.5％，称重为232.5克。
取上述干燥后的牛肠粘膜肝素季铵盐200克于2升反应瓶中，加入1300克二氯甲烷搅拌溶解，升温至38℃，加入220克氯化苄，全程保温30-40℃，反应过夜(＝25小时)，转移反应液至5升瓶。另外，称量160克醋酸钠，溶解于1600毫升的甲醇中，在酯化反应结束后滴加至反应液中，此时产生不溶的牛肠粘膜肝素苄基酯沉淀。再加入3升甲醇，搅拌5分钟，静置过夜。小心地吸去上清液，下层沉降区混合物，用100目滤布抽滤，得到牛肠粘膜肝素苄基酯粗品。粗品再两次以1.6升甲醇重悬，充分搅拌洗涤后抽滤。称取20克氯化钠并溶于160毫升水中，用该氯化钠水
溶液溶解上述固体，再以1.6升的甲醇醇沉，沉淀物以离心机6000转/分钟离心5分钟收获。重复盐水溶再甲醇醇沉3次，沉淀转移至真空干燥箱，60℃真空干燥50小时。所得的牛肠粘膜肝素苄基酯称重60.2克，干燥失重为3.3％，酯化度折干后为12.8％。
取上述牛肠粘膜肝素苄基酯50克，溶于1.25升水中，加热至60℃，保温60±1℃30分钟以上。另外准确称取6.25克50％氢氧化钠溶液，加入上述保温的水溶液中，继续搅拌保温60±2℃，反应90分钟。将反应液冷却至室温，加入25克30％双氧水，氧化脱色30分钟。用稀盐酸调pH至7.0，0.22微米过滤反应液。滤液加125克氯化钠，搅拌确保氯化钠全溶，调pH至6.0，再0.22微米精过滤。加入3升甲醇醇沉，沉淀物经400目过滤。沉淀物再以3升甲醇重悬搅拌30分钟，抽滤取沉淀。沉淀溶于120克水后，转移至冻干瓶，真空冻干30小时。冻 干粉称重后装袋，密封送检。
最终收获牛肠粘膜依诺肝素钠30.4克，按初始投料计重量收率56.0％，干燥失重为4.2％。均重分子量为4431，分子量<2000部分的比例为15.7％，2000<分子量<8000部分的比例为72.8％，分子量>8000部分的比例为12.5％；1,6-酐环糖链占总糖链为22.0％。抗-Ⅹa活性折干后为95.2单位每毫克，抗-Ⅱa活性折干后为19.5单位每毫克，抗-Ⅹa/抗-Ⅱa比例为4.9；1.0克产品溶于10毫升水的溶液，澄清且色度不深于6号标准色；1.0克产品溶于10毫升水的溶液，pH为6.76；钠含量折干后为12.1％；氮含量折干后为2.0％；水溶液在232纳米波长有最大吸收，折干后231纳米特征吸收为16.2；磺酸根/羧酸根比例为2.8；有关物质苄醇含量折干后不大于0.1％，苄铵盐含量折干后不大于0.1％；重金属残留不大于30ppm；溶剂残留中甲醇为210ppm；细菌内毒素含量，每抗-Ⅹa单位活性依诺肝素钠小于0.01细菌内毒素单位(EU)。以上牛肠粘膜依诺肝素钠的所有指标，除了抗-Ⅱa活力偏低外，均符合USP39依诺肝素钠的放行标准。
实施例2
牛肠粘膜肝素钠，由苏州特瑞药业有限公司提供，提取和纯化自中国东北肉牛屠宰后的小肠粘膜。准确称取3.0千克，经全绵羊血浆法抗凝活性折干后为156.3单位每毫克；按USP39规定的精品肝素钠抗-Ⅹa和抗-Ⅱa活性测定方法，抗-Ⅹa活性折干后为139.1单位每毫克，抗-Ⅱa活性折干后为130.0单位每毫克，抗-Ⅹa/抗-Ⅱa比例为1.07
制备牛肠粘膜依诺肝素钠的过程同实施例一，仅试剂使用量上的差异。最终获得牛肠粘膜依诺肝素钠1683.1克，按初始投料计重量收率56.1％。干燥失重为3.2％；1.0克产品溶于10毫升水的溶液，澄清且色度不深于6号标准色；1.0克产品溶于10毫升水的溶液，pH为6.9；钠含量折干后为11.8％；氮含量折干后为2.1％；水溶液在232纳米波长有最大吸收，折干后231纳米特征吸收为16.0；磺酸根/羧酸根比例为2.5；有关物质苄醇含量折干后不大于0.1％，苄铵盐含量折干后不大于0.1％；重金属残留不大于30ppm；溶剂残留中甲醇为240ppm；细菌内毒素含量，每抗-Ⅹa单位活性依诺肝素钠小于0.01细菌内毒素单位。以上所有牛肠粘膜依诺肝素钠的测试结果，均符合USP39依诺肝素钠的放行标准。
牛肠粘膜依诺肝素钠重均分子量及分子量分布分析
牛肠粘膜依诺肝素钠的分子量分布，参照USP39方法进行，结果如附图1和表1所列。
表1：牛肠粘膜依诺肝素钠样品的重均分子量及分子量分布
Figure PCTCN2016096023-appb-000001
表格中的牛肠粘膜依诺肝素钠(RX0025-JCJ-003)来自于实施例二所制备的产品。
结果可以看出，实施例二中制备的牛肠粘膜依诺肝素钠，其均重分子量和分子量分布与来源于猪肠粘膜的依诺肝素钠标准品非常接近，符合USP39技术指标的要求。
牛肠粘膜依诺肝素钠二糖和1,6-酐含量分析
牛肠粘膜依诺肝素钠的二糖组成分析，遵照USP32附录<207>“依诺肝素钠的1,6-酐衍生物检查”进行。样品和标准品的二糖组成和1,6-酐分析结果见图2和表2。
表2：牛肠粘膜依诺肝素钠与依诺肝素钠标准品的二糖组成比例及1,6-酐％
Figure PCTCN2016096023-appb-000002
Figure PCTCN2016096023-appb-000003
从图2和表2可以看出，实施例二制备的牛肠粘膜依诺肝素钠依诺肝素钠呈现出典型的牛肠粘膜肝素特征，与依诺肝素钠标准品的二糖相比，其主要二糖ΔⅢS的含量要更高，达到17.08％，值得注意的是，二糖组成和百分含量并非USP39和EP8.6的放行指标。同时，本工艺制备的牛肠粘膜依诺肝素钠的1,6-酐百分含量与标准品几乎相同，为19.5％，符合USP39要求的15％-25％的指标。
牛肠粘膜依诺肝素钠的磺酸根羧酸根比例分析
牛肠粘膜依诺肝素钠的磺酸根羧酸根比例分析，方法遵照USP39进行，计算磺酸根和羧酸根的摩尔比，结果如图3所示。
从图3可以看出，牛肠粘膜依诺肝素钠的磺酸根和羧酸根的摩尔比是2.5，稍高于通常的猪肠粘膜依诺肝素钠(2.2)一致。该项测试反映糖链上的磺酸根修饰程度，说明两者的磺酸根修饰也较为接近。测试结果都在USP39的技术标准(大于1.8)之内，牛肠粘膜依诺肝素钠的磺酸根和羧酸根比例符合其规定。
牛肠粘膜依诺肝素钠的核磁氢谱(1H-NMR)分析
牛肠粘膜依诺肝素钠的核磁氢谱分析，设备用苏州大学分析测试中心的600MHz核磁共振谱仪，以3-三甲基硅基丙酸钠-d4(TSP)定零点。
待测样品溶液配制：牛肠粘膜依诺肝素钠(实施例一)和依诺肝素钠标准品，各准确各称取20毫克左右，按重以氘水(D2O)溶解成20毫克每毫升左右的浓度，滴加1-2滴TSP，震荡混匀后0.22微米过滤送检，结果如图4所示，其中，δ3.4ppm为甲醇残留的甲基氢峰，δ4.7ppm为水氢峰。
结果显示，实施例一制备的牛肠粘膜依诺肝素钠的氢谱与依诺肝素钠标准品较为一致，但在δ2.04ppm处的氮-乙酰基的甲基峰，牛肠粘膜依诺肝素钠的积分含量要低一些，说明牛肠粘膜依诺肝素钠中，氮-乙酰基修饰更少，相应地，其氮-磺酸基修饰就更多。通常，更多的磺酸基修饰可以带来更高的抗凝活性。 USP39对依诺肝素没有要求氢谱测试。
牛肠粘膜依诺肝素钠核磁碳谱(14C-NMR)分析
牛肠粘膜依诺肝素钠的的核磁碳谱分析，设备用苏州大学分析测试中心的600MHz核磁共振谱仪，方法遵照USP39进行。
待测样品溶液配制：牛肠粘膜依诺肝素钠(实施例二)和依诺肝素钠标准品，各准确各称取200毫克，加入0.2毫升氘水(D2O)和0.8毫升纯水溶清，再滴加50微升氘代甲醇，震荡混匀后0.22微米过滤送检，结果如图5所示，其中，δ50ppm为甲醇残留的甲基碳峰。
结果显示，同氢谱一致，实施例一制备的牛肠粘膜依诺肝素钠碳骨架与依诺肝素钠标准品一致，但一些具体的位置，如δ24.9ppm的氮-乙酰基的甲基碳，含量有一定的差异。该牛肠粘膜依诺肝素钠的碳谱，虽然差异明显，但该差异项不在USP39的要求中，因此结果符合USP39对该项指标的规定。
牛肠粘膜依诺肝素钠抗-Ⅹa、抗-Ⅱa活力和全绵羊血浆法活力对比分析
牛肠粘膜依诺肝素钠的抗-Ⅹa和抗-Ⅱa的活性测定遵照USP39进行，来源于实施例1的牛肠粘膜依诺肝素钠与依诺肝素钠标准品的各活性对比如下表3。
表3：牛肠粘膜依诺肝素钠样品的抗凝活力对比
Figure PCTCN2016096023-appb-000004
结果显示：牛肠粘膜依诺肝素钠和依诺肝素钠标准品相比，全绵羊血浆法的抗凝血活力低了～10％，抗-Ⅹa活力较高，达到了120.2单位每毫克，而抗-Ⅱa活力则非常低，仅15.9单位每毫克，以此得到的抗-Ⅹa/抗-Ⅱa比例在7.6，其抗-Ⅱa活力和比例，偏出了USP39对依诺肝素钠的指标要求。
综合以上所有测试的结果，实施例2制备的牛肠粘膜依诺肝素钠样品与猪肠粘膜依诺肝素钠相比，除了种源不同和抗-Ⅱa活力偏低外，其余的各项都符合USP39对依诺肝素钠的放行指标要求，比对EP8.6要求的依诺肝素钠放行指标， 也是完全符合的。
实施例3
牛肠粘膜依诺肝素钠注射剂1的制备
按活性计算并准确称取牛肠粘膜依诺肝素钠粉末171.9克(干燥失重3.2％，折干后120.2抗-Ⅹa单位每毫克，共0.2亿抗-Ⅹa单位)，以冷却的注射用水溶解并定容至2000毫升，两级0.2微米过滤器无菌过滤进A级洁净区，并以灌封机灌封至1毫升玻璃注射器中，规格为6000单位(或0.6毫升)，去除损耗，共收获牛肠粘膜依诺肝素钠注射剂1成品1830支。
实施例4
牛肠粘膜依诺肝素钠注射剂2的制备
按活性计算并准确称取牛肠粘膜依诺肝素钠粉末286.3克(干燥失重3.2％，折干后120.2抗-Ⅹa单位每毫克，共0.3亿抗-Ⅹa单位)，以冷却的注射用水溶解，加入45.0克苯甲醇，搅拌均匀，再以冷却的注射用水定容至3000毫升，两级0.2微米过滤器无菌过滤进A级洁净区，并以灌封机灌封至5毫升西林瓶中，规格为30000单位(或3.0毫升)，去除损耗，共收获牛肠粘膜依诺肝素钠注射剂2成品870瓶。
实施例5
牛肠粘膜依诺肝素钠与牛肠粘膜依诺肝素钠注射剂的家兔体内抗凝试验
一、实验内容：
供试样品：牛肠粘膜依诺肝素钠样品(实施例二所述，批号：RX0025-JCJ-003)，牛肠粘膜依诺肝素钠注射剂1(实施例三所述)，牛肠粘膜依诺肝素钠注射剂2(实施例四所述)，依诺肝素钠标准品为临床市售药品(克赛，批号：24459)。配制：牛肠粘膜依诺肝素钠样品以生理盐水配制成100毫克每毫升浓度，并0.2微米过滤待用，其他注射剂直接用于注射。
实验方法：选取日本大白兔2-3千克，根据体重分别在前背部近上肢处皮下注射给药。注射剂量：2毫克每千克(或200单位每千克)。于给药前及给药后每0.5小时-1小时分别采血，采血2毫升，用3.8％枸橼酸钠抗凝剂1：9抗凝，上机检测。测定仪器：全自动血凝仪(Stago Compact)和血小板聚集仪(普利 生LBY-NJ4)。抗凝血试验：测定包括抗-Ⅹa活性、活化部分凝血酶时间(APTT)和凝血酶时间(TT)等常规凝血全套。
二、实验结果与分析：
1)APTT：
实验结果如附图6(1)所示，从图中可以看出：与依诺肝素钠标准品相比，牛肠粘膜依诺肝素钠及其注射剂均能显著延长APTT，牛肠粘膜依诺肝素钠及其注射剂对APTT延长作用相当，均比依诺肝素钠标准品稍弱一些；所有组别在兔体内达到APTT最大值时间相近，衰减时间也相近，揭示牛肠粘膜依诺肝素钠及其注射剂在兔体内与依诺肝素钠标准品是一致的。
2)PT：
实验结果如附图6(2)所示，从图中可以看出：各组样品在兔体内均对PT影响较小，无显著延长作用。
3)TT：
实验结果如附图6(3)所示，从图中可以看出：牛肠粘膜依诺肝素钠及其注射剂均能显著延长TT，但比依诺肝素钠标准品稍弱；在兔体内达到TT最大值的时间，牛肠粘膜依诺肝素钠及其注射剂与依诺肝素钠标准品相近，衰减时间也相当。
4)抗-Xa活性：
实验结果如附图6(4)所示，从图中可以看出：各组皮下注射后，兔血浆中肝素的抗-Ⅹa活性，其吸收和代谢(或衰减)曲线均一致，都是在大约2小时至4小时达到吸收峰值，在8小时几乎全部衰减。
所有以上数据揭示，牛肠粘膜依诺肝素钠及其注射剂，在兔体内有着良好的抗凝效果，且与依诺肝素钠标准品相当。
本发明尚有多种实施方式，凡采用等同变换或者等效变换而形成的所有技术方案，均落在本发明的保护范围之内。

Claims (17)

  1. 一种牛肠粘膜依诺肝素钠，其特征在于，以牛的肠粘膜肝素制备得到。
  2. 根据权利要求1所述的牛肠粘膜依诺肝素钠，其特征在于，二糖ΔUA2S-GlcNS(ΔⅢS)的含量为15％-32％。
  3. 根据权利要求2所述的牛肠粘膜依诺肝素钠，其特征在于，来自中国内地牛源制备的牛肠粘膜依诺肝素钠二糖ΔUA2S-GlcNS(ΔⅢS)的含量为15％-24％，来自南美巴西牛源制备的牛肠粘膜依诺肝素钠二糖ΔUA2S-GlcNS(ΔⅢS)的含量为24％-32％。
  4. 根据权利要求1所述的牛肠粘膜依诺肝素钠，其特征在于，抗-Ⅹa活性折干后为90-125单位每毫克，抗-Ⅱa活性折干后为10-35单位每毫克，抗-Ⅹa/抗-Ⅱa的比例为5-10。
  5. 一种权利要求1-4任一项所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，包括如下步骤：
    S1、原料牛肠粘膜肝素的预处理：将牛肠粘膜肝素钠粗品溶解后进行溶液脱色，精过滤，再在室温下进行醇沉精制，收集沉淀物，干燥获得牛肠粘膜肝素；
    S2、牛肠粘膜肝素季铵盐的制备：将S1中获得的牛肠粘膜肝素溶解配制成牛肠粘膜肝素水溶液，并与苄索氯铵水溶液进行混合，过滤或离心获得牛肠粘膜肝素季铵盐，并进行洗涤干燥，制得牛肠粘膜肝素季铵盐；
    S3、牛肠粘膜肝素苄酯的制备：将S2中干燥得到的牛肠粘膜肝素季铵盐与二氯甲烷及氯化苄按重量比例混合酯化，在酯化后的牛肠粘膜肝素季铵盐中滴加醋酸钠甲醇溶液，制得牛肠粘膜肝素苄基酯沉淀，将牛肠粘膜肝素苄基酯沉淀进行过滤、洗涤、干燥，制得牛肠粘膜肝素苄基酯；
    S4、牛肠粘膜依诺肝素钠成品制得，是将S3中的牛肠粘膜肝素苄基酯进行碱解聚、脱色、以酸中和至中性、醇沉淀，精制、干燥，得到牛肠粘膜依诺肝素钠成品。
  6. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S1中采用质量浓度为1％-3％的氯化钠水溶液溶解牛肠粘膜肝素钠粗品进行脱色、过滤和精制，直至预处理后牛肠粘膜肝素钠的水溶液澄清且色度不深于5号标准色。
  7. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S1中醇沉精制的沉淀剂为甲醇、乙醇、异丙醇或丙酮中的一种或几种的组合。
  8. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S2中苄索氯铵与牛肠粘膜肝素钠的重量比为2-5:1。
  9. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S3中酯化温 度30-40℃，牛肠粘膜肝素季铵盐、二氯甲烷、氯化苄的质量比为1:3-10:1.1。
  10. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S3中牛肠粘膜肝素苄基酯沉淀的洗涤包括如下步骤：
    S31、在加入醋酸钠甲醇溶液的牛肠粘膜肝素季铵盐溶液中加入甲醇静置沉降和分离制得牛肠粘膜肝素苄基酯；
    S32、在分离后的牛肠粘膜肝素苄基酯中添加8％-12％的氯化钠水溶液进行复溶，所述氯化钠水溶液与所述牛肠粘膜肝素季铵盐重量比为0.5-2:1；
    S33、对S32中获得的溶液以60％-70％的甲醇终浓度进行醇沉结晶；
    S34、重复氯化钠水溶液复溶和醇沉结晶2-5次至牛肠粘膜肝素苄基酯复溶不浑浊。
  11. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S4中采用氢氧化钠溶液解聚，解聚温度在30℃-70℃之间，保温时间在0.5小时以上。
  12. 根据权利要求5所述的牛肠粘膜依诺肝素钠的制备方法，其特征在于，S4中采用双氧水脱色，室温或以下加入0.1-1倍牛肠粘膜肝素苄基酯重量的30％双氧水，氧化脱色10分钟以上，直至反应液颜色浅至Y6与GY6以下。
  13. 权利要求1-4任一项所述的牛肠粘膜依诺肝素钠在抗凝、抗栓及清真药物中的应用。
  14. 一种牛肠粘膜依诺肝素钠注射剂，其特征在于，组分包括权利要求1-4任一项所述的牛肠粘膜依诺肝素钠和注射用水。
  15. 权利要求14所述的牛肠粘膜依诺肝素钠注射剂在抗凝、抗栓及清真药物中的应用。
  16. 根据权利要求14所述的牛肠粘膜依诺肝素钠注射剂，其特征在于，组分还包括苯甲醇。
  17. 权利要求16所述的牛肠粘膜依诺肝素钠注射剂在抗凝、抗栓及清真药物中的应用。
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