An ultra-low molecular weight heparin, its preparation method and application

Abstract

The present invention introduces a super low molecular weight heparin and its preparation method and application, belonging to the field of biomedical technology. The weight-average molecular weight of the super low molecular weight heparin is 3000 Da - 4000 Da, with an average of about 3500 Da, and the percentage of sugar chain components greater than 6000 Da is below 10%. It has an extremely high anti-Xa / anti-IIa titer ratio (10–14), meeting the performance (quality) requirements for second-generation low molecular weight heparin in the biomedical field. The preparation method of the super low molecular weight heparin uses porcine intestinal mucosa heparin as the raw material, undergoes salification and esterification derivatives, and then uses alkali to initiate and control β-depolymerization at high temperature, and is prepared by organic solvent fractional precipitation and other refining methods. The super low molecular weight heparin has unique properties, and the preparation method is stable and efficient, suitable for industrial production, and is developed to prepare drugs for preventing and treating thromboembolism.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to an ultra-low molecular weight heparin, a preparation method thereof, and an application thereof. Background Art

[0002] Heparin is a highly sulfated glycosaminoglycan extracted from mammalian tissues, and has physiological functions of anticoagulation and lipid regulation. Clinically, it is widely used for preventing and treating various thromboembolic diseases. Clinical heparin drugs are generally divided into unfractionated heparin (UFH) and low molecular weight heparin (LMWH) according to the difference in molecular weight. The molecular weight of UFH is generally 3000 Da - 30000 Da. Before use, it is necessary to monitor the activated partial thromboplastin time (aPTT) to prevent spontaneous bleeding caused by excessive medication. It is natural heparin. The molecular weight of LMWH is generally 3000 Da - 8000 Da. Compared with natural heparin, it has a smaller relative molecular weight, stronger selectivity, and does not require monitoring of the activated partial thromboplastin time during use. Clinically, side effects such as potential bleeding, allergic reactions, and thrombocytopenia are significantly reduced. In addition, there is a class of ultra-low molecular weight heparin (ULMWH), whose molecular weight is generally less than 4000 Da, and it is regarded as the second-generation low molecular weight heparin. The ratio of anti-Xa / anti-IIa factor potency of ULMWH is larger than that of the first-generation low molecular weight heparin (the low molecular weight heparin is generally about 2 - 4, and the ultra-low molecular weight heparin is above 5). It has less anti-IIa factor potency and has less risk in inducing hemorrhage, allergic reactions, and heparin-induced thrombocytopenia.

[0003] Currently, the main production methods of LMWH and ULMWH include chemical depolymerization method, biodegradation method, and synthetic preparation method, etc. Existing technologies show that LMWH or ULMWH obtained by different preparation methods have different molecular weights, physicochemical properties, and biological potencies, and there are also differences in pharmacokinetic characteristics and anticoagulant effects. Therefore, they cannot be substituted for each other during clinical use.

[0004] Low molecular weight heparins such as dalteparin sodium, nadroparin sodium, enoxaparin sodium, and parnaparin sodium are the mainstream products of heparin drugs in the international market at present. However, as an upgraded variety of the first-generation low molecular weight heparins, there are only a few ultra-low molecular weight heparin products under the premise of better efficacy and lower adverse reactions. One of the important reasons is the deficiency in the industrial preparation method. Therefore, it is of great significance to study an ultra-low molecular weight heparin suitable for industrial production and its preparation process. Summary of the Invention

[0005] The object of the present invention is to provide an ultra-low molecular weight heparin, its preparation method and application.

[0006] The object of the present invention is achieved by the following technical solutions:

[0007] An ultra-low molecular weight heparin, having the following characteristics:

[0008] (1) The weight-average molecular weight is 3000 Da - 4000 Da, with an average of about 3500 Da, and the percentage of sugar chain components (M 6000 ) greater than 6000 Da is below 10%;

[0009] (2) The ratio of anti-Xa / anti-IIa factor potency is between 10 and 14.

[0010] The said ultra-low molecular weight heparin is prepared by the general chemical depolymerization method. First, heparin is derivatized into a salt and heparin ester is prepared, and then the depolymerization is initiated by the action of an alkali to prepare an ultra-low molecular weight heparin, which is then refined.

[0011] The core characteristics of the said ultra-low molecular weight heparin are ensured by the key process steps and parameter control during its preparation: (1) When the depolymerization reaction is initiated by the action of an alkali, the reactant ratio is controlled and the depolymerization is carried out at a high temperature (defined: in this article, high temperature refers to the reaction solution temperature of 60°C or above); (2) When the product is refined, it is fractionated and precipitated with an organic solvent to control the molecular weight and molecular weight distribution.

[0012] As is well known, the above chemical depolymerization reaction initiated by the action of alkali is the β-depolymerization method. Its advantages include fast reaction speed, no damage to the anticoagulant active sites of heparin, and uniform product molecular weight distribution. Among existing low-molecular-weight heparin drugs, enoxaparin and bemiparin are both prepared using this principle. The average molecular weight of enoxaparin is about 4500 Da, and the anti-Xa / anti-IIa factor potency ratio is around 3 - 5. The average molecular weight of bemiparin is about 3600 Da, and the anti-Xa / anti-IIa factor potency ratio is around 7 - 9. Above all, the reaction temperature is extremely important for the β-depolymerization reaction and the size of the product molecular weight. When the temperature is too low, the product has a large molecular weight and a large number of macromolecular components. When the temperature is too high, the product is extremely fragmented and has a small molecular weight. Through a large number of experimental screenings, the inventor found that high temperature (60°C or above) is more conducive to the production of ultra-low-molecular-weight heparin, which can ensure that the molecular weight of the product is below 4000 Da and is easy to control the number of macromolecular components.

[0013] In addition, as is well known to those skilled in the art, organic solvent fractional precipitation is one of the important methods for controlling the molecular weight and molecular weight distribution of heparin drugs. The advantages of this method are simple operation, and the solvents used are often the same organic solvents used in the established process, without introducing other solvent impurities, and it is very efficient. Through a large number of experimental screenings, the inventor determined a molecular weight fractional refining method suitable for the ultra-low-molecular-weight heparin. In addition, the inventor also introduced size exclusion-high performance liquid chromatography (SEC-HPLC) to control the refining process of this step, which can ensure the molecular weight and molecular weight distribution of the product.

[0014] Regarding how to ensure that the ultra-low-molecular-weight heparin has an ultra-high anti-Xa / anti-IIa factor potency ratio (10 - 14), the inventor manages it in combination with the above molecular weight control, which is specifically described as follows.

[0015] As is well known, the anti-Xa (regarded as anticoagulation) and anti-IIa (regarded as antithrombosis) activities of heparin molecules rely on binding to antithrombin III to inhibit the activities of these coagulation factors (factor Xa and factor IIa). Among them, the anti-Xa activity only requires the heparin molecule to bind to antithrombin III with the heparin pentasaccharide center (molecular weight about 1725 Da) to form a complex to inhibit the activity of factor Xa. The anti-IIa activity not only requires the heparin molecule to bind to antithrombin III but also requires the heparin molecule to have a longer sugar chain length to simultaneously bind to factor IIa. That is to say, it is necessary to form a ternary complex of "heparin + antithrombin III + factor IIa". According to existing literature reports, this heparin chain length is considered to require 16 sugar units or more, that is, the molecular weight needs to be above 5600 Da. For the convenience of control, the present invention examines the sugar chain components with a molecular weight greater than 6000 Da (M 6000)by percentage. For example, in the prior art bemiparin which is relatively close to the present invention, the sugar chain component with a molecular weight greater than 6000 Da in the quality standard is less than 15%, with an average of about 12%, and the ratio of anti-Xa / anti-IIa factor titer is between 7 and 9. In combination with the above molecular weight control measures, specifically, in Examples 1 - 6, the sugar chain component (M 6000 )percentage of the ultra-low molecular weight heparin is less than 10%, and the average value is only about 6 - 7%. Correspondingly, these products have the characteristic of a lower anti-IIa factor titer, and the ratio of anti-Xa / anti-IIa factor titer is about 11 - 13.

[0016] The preparation method of the ultra-low molecular weight heparin is schematically shown in the following synthesis route:

[0017] .

[0018] Combined with the above synthesis route diagram, the preparation method of the ultra-low molecular weight heparin is characterized in that it consists of the following four steps:

[0019] S1. Preparation of heparin ammonium salt: Take heparin sodium, dissolve it in 8 - 12 times of water, mix it with an aqueous solution prepared from 2.5 - 3.5 times of organic ammonium salt (heparin sodium), collect the precipitated white solid insoluble matter, and dry it;

[0020] S2. Preparation of heparin ester: Dissolve heparin ammonium salt in 4 - 12 times of organic solvent, add 0.9 - 1.3 times of benzyl chloride (heparin ammonium salt), stir and react at 28 - 38°C for 12 hours or more, prepare a sodium acetate methanol solution with 0.5 - 1.0 times of sodium acetate (heparin ammonium salt), mix it with the reaction solution, collect the precipitated sediment, and dry it;

[0021] S3. Depolymerization preparation of crude ultra-low molecular weight heparin: Dissolve heparin benzyl ester in 15 - 25 times of water, heat it to 60°C - 70°C and keep it, add 0.08 - 0.12 times of base (previously prepared into a 20% - 35% concentrated solution with water) of heparin benzyl ester, stir and react for depolymerization for 30 - 60 minutes, add sodium chloride with a weight of 0.02 - 0.12 times the volume of the feed liquid, stir to dissolve clearly, add ethanol with a volume of 1.0 - 2.0 times the volume of the feed liquid or methanol with a volume of 1.5 - 3.5 times, stir for not less than 10 minutes, and centrifuge or filter to collect the precipitate;

[0022] S4. Grading and refining of low molecular weight heparin: The precipitate is redissolved in 3% - 7% brine at 4 - 6 times the volume, decolorized with hydrogen peroxide at a final concentration of 1% - 3% and / or activated carbon at a concentration of 0.1% - 1.0%, filtered, fractionally precipitated with an organic solvent ethanol or methanol, and the precipitate obtained by ethanol fractionation between 0.3 times and 2.0 times the solution volume or the precipitate obtained by methanol fractionation between 0.5 - 3.5 times the solution volume is taken for in - process control, or continue with organic solvent fractional precipitation until the molecular weight of the target substance is between 3000 Da and 4000 Da, and the sugar chain component with a molecular weight greater than 6000 Da is below 10%, and then dried.

[0023] Preferably, the heparin sodium in step S1 is used as the starting material, and its source is porcine intestinal mucosa.

[0024] Preferably, the organic ammonium salt in step S1 is selected from benzethonium chloride and / or benzalkonium bromide.

[0025] Preferably, the drying method in step S1 includes hot - air drying, vacuum drying, fluid - bed drying and / or freeze - drying.

[0026] Preferably, the organic solvent in step S2 is selected from dichloromethane or N,N - dimethylformamide.

[0027] Preferably, the heparin ester in step S2 can be further purified by the common method of redissolving in brine and then reprecipitating.

[0028] Preferably, the base in step S3 is selected from sodium hydroxide and / or potassium hydroxide.

[0029] The reaction temperature in step S3 uses high - temperature depolymerization at 60℃ - 70℃ to make the molecular weight of the resulting low molecular weight heparin smaller and the distribution more concentrated. As mentioned above, temperature is extremely important for the β - depolymerization reaction of heparin initiated by base. When the temperature is too low, the molecular weight of the product is large and there are many macromolecular components, while when the temperature is too high, the product is extremely fragmented and the molecular weight is on the small side. In a set of examples but not limited to this example, the temperature conditions for this depolymerization were investigated.

[0030] The reaction time in step S3 is 30 - 60 minutes. As mentioned above, the principle of this step reaction is the β - depolymerization of heparin polysaccharide initiated by the action of base. This chemical reaction responds quickly and can be completed in a short time. In addition, the color of the solution under high - temperature and alkaline conditions is prone to deepen with the extension of time. Therefore, controlling the reaction time can reduce side reactions such as color deepening.

[0031] Preferably, for the alcohol precipitation in step S3, when using ethanol, the dosage is 1.3 - 1.7 times the volume of the solution, or when using methanol, the dosage is 2.0 - 3.0 times the volume of the solution. Ethanol and methanol are common organic solvent precipitants in the art. The principle lies in the change of the relative dielectric constant of the aqueous solution during the precipitation process, which causes the fractional precipitation of polysaccharides with different properties.

[0032] Preferably, the decolorization with hydrogen peroxide in step S4 means decolorizing for 1 hour or more at room temperature until the color of the solution becomes light yellow.

[0033] Preferably, the decolorization with activated carbon in step S4 means stirring and adsorbing at room temperature for 15 minutes or more, and then removing the adsorbed activated carbon particles by filtration or other means.

[0034] Preferably, the filtration in step S4, in addition to the filtration after using the above-mentioned activated carbon, also includes filtration with 0.45 μm and / or 0.22 μm, aiming to remove particulate matter and microbial contamination.

[0035] Preferably, for the fractional precipitation with the organic solvents ethanol or methanol in step S4, when using ethanol, collect the part precipitated by 0.5 to 1.5 times the volume of ethanol of the solution, or when using methanol, collect the part precipitated by 0.8 to 3.0 times the volume of methanol of the solution.

[0036] The above-mentioned fractional precipitation with organic solvents is beneficial to removing and controlling the large molecular weight components and small molecular weight components therein, and ensuring the weight average molecular weight and the distribution of molecular weight size components of the product. Preferably, the process of fractional alcohol precipitation is investigated for the molecular weight distribution by size exclusion-high performance liquid chromatography (SEC-HPLC).

[0037] As described in step S4, the fractional precipitation with organic solvents can be repeated or carried out multiple times until the molecular weight of the target substance is controlled within 3000 Da - 4000 Da by SEC-HPLC molecular weight distribution, and the sugar chain components with a molecular weight greater than 6000 Da are less than 10%.

[0038] Preferably, the drying in step S4 includes hot air drying, vacuum drying, fluidized bed drying and / or freeze drying.

[0039] More preferably, the drying in step S4 adopts the freeze drying method. Specifically, it is reconstituted with water for injection, preferably at a concentration between 5% - 25%, filtered with 0.22 μm, loaded into a stainless steel freeze drying tray, and freeze dried.

[0040] The low molecular weight heparin has anticoagulant and antithrombotic activity, and its anti-Xa factor titer is about 100 IU / mg.

[0041] The low molecular weight heparin can be used as a raw material for preparing drugs for preventing and treating thromboembolism.

[0042] A pharmaceutical composition comprises: a pharmaceutically acceptable carrier and a low molecular weight heparin described in this solution. The pharmaceutically acceptable carrier includes: pH buffer, isotonic regulator, preservative, excipient, etc., which are specified in conventional pharmaceutical preparations. This pharmaceutical composition can be used as a drug for preventing and treating thromboembolism.

[0043] The prominent effect of the present invention is: to provide a low molecular weight heparin and its preparation method. The weight-average molecular weight of the low molecular weight heparin is 3000 Da - 4000 Da, with an average of about 3500 Da. The percentage of sugar chain components with a molecular weight greater than 6000 Da is below 10%. The ratio of anti-Xa / anti-IIa factor potency is between 10 and 14, meeting the performance (quality) requirements for second-generation low molecular weight heparin in the field. Its preparation method is to utilize the β-depolymerization initiated by the action of alkali on heparin ester, and high-temperature depolymerization to produce heparin derivatives with ultra-low molecular weight and molecular weight distribution, and use organic solvent fractional precipitation to control the weight-average molecular weight and the percentage of sugar chain components with a molecular weight greater than 6000 Da, so as to obtain the low molecular weight heparin with the above-mentioned molecular weight and potency ratio characteristics. This preparation method is stable and efficient, suitable for industrial production, and used to develop and prepare drugs for preventing and treating thromboembolism. Description of the Drawings

[0044] Figure 1 Schematic diagram comparing the weight-average molecular weight and molecular weight distribution of low molecular weight heparin prepared by different high-temperature depolymerization methods.

[0045] Figure 2 Schematic diagram comparing the weight-average molecular weight and molecular weight distribution of low molecular weight heparin. Detailed Embodiments

[0046] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described in detail below in conjunction with specific embodiments, but it is not used to limit the protection scope of the present invention.

[0047] Example 1: Preparation 1 of Low Molecular Weight Heparin

[0048] This example is the small-scale preparation of low molecular weight heparin, and the steps and experimental results are as follows.

[0049] S1. Preparation of ammonium heparinate:

[0050] Take 100 g of commercially available heparin sodium (Chinese Pharmacopoeia grade) in a 3 L beaker, add 1000 mL of purified water and stir until dissolved and clear; weigh another 250 g of benzethonium chloride and dissolve it in 1.5 L of purified water; under sufficient stirring, mix the two solutions, at this time a large amount of white precipitate is produced, collect it by centrifugation, evenly spread the precipitate on a stainless steel freeze-drying tray, and freeze-dry (procedure: pre-freeze at -40°C for 3 hours, start the cold trap to cool down and the vacuum pump (50 Pa), raise the partition to 25°C for 2 hours, maintain at 25°C for 12 hours, vacuum 20 ± 5 Pa; raise the partition to 40°C for 2 hours, maintain at 40°C for 2 hours, ultimate vacuum; after completion, release the vacuum and take out of the box), to obtain 285 g of heparin ammonium salt.

[0051] S2. Preparation of heparin ester:

[0052] Take 200 g of the heparin ammonium salt prepared in S1 above, add 1 L of N,N-dimethylformamide, and stir until completely dissolved; add 180 g of benzyl chloride and stir and react at 35°C for 12 hours; add a solution prepared with 100 g of sodium acetate and 1.0 L of methanol to terminate the reaction, stir well, collect the precipitate by centrifugation, and vacuum dry in an oven at 60°C to obtain 68 g of heparin ester.

[0053] S3. Depolymerization preparation of crude low molecular weight heparin:

[0054] Take 50 g of the heparin ester prepared in S2 above in a 1 L three-necked flask, add 750 mL of purified water to dissolve, heat and maintain the temperature at 65°C; weigh 6.0 g of potassium hydroxide, add purified water to prepare a 20% alkali solution; add the alkali solution and carry out the depolymerization reaction for 45 minutes, then quickly cool down to room temperature in an ice-water bath; add 25 g of solid sodium chloride and stir until dissolved and clear; transfer the reaction solution to a beaker, add 1.5 L of ethanol, stir well, collect the precipitate by centrifugation to obtain crude low molecular weight heparin.

[0055] S4. Fractionation and refining of low molecular weight heparin to obtain:

[0056] Dissolve the crude ultralow molecular weight heparin in about 220 mL of 5% sodium chloride solution in portions, transfer all to a beaker, add about 28 mL of 30% hydrogen peroxide, the total volume of the material liquid is about 300 mL, continuously adjust the pH to the range of 9 - 10 with dilute sodium hydroxide solution, and decolorize at room temperature for 1.5 hours; adjust the pH to neutral with dilute hydrochloric acid, and filter through a 0.45 μm filter; add 150 mL of methanol, stir well, centrifuge, and transfer the supernatant to a clean beaker; continue to add 750 mL of methanol, stir well, centrifuge, and collect the precipitate; take the sample for SEC - HPLC molecular weight inspection of the in - process control, showing that the molecular weight of the target product is about 3800 Da, and the sugar chain component with a molecular weight greater than 6000 Da is about 6%, meeting the expected indicators; continue to add 200 mL of methanol to dehydrate the precipitate, centrifuge, transfer to an oven, and dry at 60°C for about 8 hours to obtain 21.3 g of the ultralow molecular weight heparin finished product.

[0057] The product test results are as follows: loss on drying is 6.3%, weight - average molecular weight is 3661 Da, the percentage of the component with a molecular weight greater than 6000 Da is 6.7%, the anti - Xa factor titer calculated based on the dried product is 97.5 IU / mg, and the anti - Xa / anti - Ⅱa titer ratio is 11.4.

[0058] Example 2: Preparation of ultralow molecular weight heparin 2

[0059] This example is the small - scale preparation of ultralow molecular weight heparin, and the preparation steps and experimental results are as follows.

[0060] S1. Preparation of heparin ammonium salt:

[0061] Dissolve 100 g of heparin sodium (pharmaceutical grade) in 1200 mL of purified water; dissolve 350 g of benzalkonium bromide in 2.5 L of purified water until clear; as in Example 1, mix the two solutions, centrifuge to collect the precipitated white solid, and dry by vacuum drying at 70°C for 11 hours to obtain 304 g of heparin ammonium salt.

[0062] S2. Preparation of heparin ester:

[0063] Take about 200 g of the above - mentioned heparin ammonium salt, add 2.0 L of dichloromethane, stir until completely dissolved; add 260 g of benzyl chloride, stir and react at 30°C for 24 hours, add the solution prepared with 200 g of sodium acetate and 2.0 L of methanol to terminate the reaction, stir for 15 minutes, then centrifuge to collect the precipitate; redissolve with 0.3 L of 10% sodium chloride solution, add 0.9 L of ethanol, centrifuge to collect the precipitate; redissolve again with 0.3 L of 10% sodium chloride solution, add 0.9 L of ethanol, centrifuge to collect the precipitate; vacuum - dry in an oven at 60°C for about 9 hours to obtain 66 g of heparin ester.

[0064] S3. Depolymerization preparation of crude ultralow molecular weight heparin

[0065] Take 50 g of the above heparin ester, dissolve it in 1.0 L of purified water, heat it and keep the temperature at 68 °C, add a 30% alkaline solution prepared with 5.0 g of sodium hydroxide for depolymerization. After adding the alkaline solution and carrying out the depolymerization reaction for 30 minutes, quickly cool it to room temperature in an ice-water bath; add 100 g of solid sodium chloride, stir until dissolved clearly, then add 2.0 L of 95% ethanol, stir for 15 minutes, centrifuge to collect the precipitate, and obtain crude low molecular weight heparin.

[0066] S4. Preparation of fractionation and refinement of low molecular weight heparin

[0067] Take the above crude low molecular weight heparin, dissolve it in 300 mL of 7% sodium chloride solution, and transfer it to a beaker; add 10 mL of 30% hydrogen peroxide, control the pH at about 9, and carry out decolorization for 1 hour; then add about 1 g of activated carbon, stir and adsorb at room temperature for 0.5 hour, centrifuge, and filter the solution through 0.45 μm and 0.22 μm filter membranes respectively; the rest is the same as in Example 1, only dry it in a 70 °C oven under vacuum for 13 hours to obtain 22.6 g of the finished product of low molecular weight heparin.

[0068] The product test results are as follows: loss on drying is 3.2%, weight average molecular weight is 3282 Da, the percentage of components greater than 6000 Da is 6.5%, the anti-Xa factor titer calculated based on the dried product is 101.3 IU / mg, and the anti-Xa / anti-IIa titer ratio is 11.6.

[0069] Example 3: Investigation on the molecular weight distribution of low molecular weight heparin products by different high-temperature depolymerization

[0070] This example investigates different reaction high temperatures during the depolymerization of heparin ester to illustrate the influence of high temperature on the molecular weight distribution of low molecular weight heparin products. The process and results are as follows.

[0071] The preparation of ammonium heparinate is the same as S1 in Example 1. Using 200 g of heparin sodium (pharmaceutical grade) as the raw material, 613 g of ammonium heparinate is obtained; the preparation of heparin ester is the same as S2 in Example 2, only the ammonium salt raw material comes from the above S1 prepared with benzethonium chloride, and 196 g of heparin ester is obtained.

[0072] Preparation of crude ultra-low molecular weight heparin by depolymerization (S3): Take 90 g of the above heparin ester, add 1.5 L of purified water, stir to dissolve, then add water to make up the reaction liquid volume to about 1.8 L, and divide it into three portions, each 600 mL, and put them into different 1 L reaction bottles; heat each reaction bottle in a water bath and keep it warm at different target high temperatures ±1°C (60°C, 65°C and 70°C); weigh 3 g of sodium hydroxide and dissolve it in 10 mL of water to form a concentrated solution, and after the temperature of the feed liquid stabilizes, add it to the three groups of reaction liquids respectively; the reaction time is 60 minutes, and after the end, it is quickly transferred and cooled to room temperature in a cold water bath; adjust the pH to neutral, add 60 g of sodium chloride solid respectively, stir until dissolved, and then add 500 mL of ethanol to precipitate each, stir thoroughly and collect the precipitates by centrifugation to obtain 3 portions of crude ultra-low molecular weight heparin wet products, each about 15 g.

[0073] The ultra-low molecular weight heparin was prepared by classification and purification (S4): 85 mL of 5% sodium chloride solution was added to the above three portions of crude ultra-low molecular weight heparin, and the total volume of each portion was about 100 mL, and stirred until dissolved; 6.7 mL of 30% hydrogen peroxide was added, and the pH was adjusted to about 8.5, and decolorized for 1 hour; after decolorization, the pH was adjusted to neutral with dilute hydrochloric acid, and filtered at 0.45 μm; the rest was the same as in Example 1, and the solid was dried in an oven at 60°C for 13 hours, and the weight average molecular weight and molecular weight distribution of the solid were checked by SEC-HPLC.

[0074] Experimental results:

[0075] The weight average molecular weight and molecular weight distribution results of ultra-low molecular weight heparin samples depolymerized at different high temperatures (60℃, 65℃ and 70℃) are shown in Tables 1 and Figure 1 .

[0076] Table 1 Molecular weight distribution of ultra-low molecular weight heparin samples depolymerized at different high temperatures

[0077] Temperature Mw Mn <![CDATA[M 6000 > <![CDATA[M 2000~6000 > <![CDATA[M 2000 > Pd 60℃ 3911 Da 2577 Da 7.6% 63.3% 29.1% 1.46 65℃ 3672 Da 2398 Da 6.5% 62.5% 31.0% 1.47 70℃ 3244 Da 2109 Da 5.1% 63.5% 31.4% 1.45

[0078] Note: Mw is the weight average molecular weight, Mn is the number average molecular weight, 6000 is the percentage of components with molecular weight greater than 6000 Da, M 2000-6000 is the percentage of components with molecular weight between 2000 Da and 6000 Da, M 2000 is the percentage of components with molecular weight less than 2000 Da, and Pd is the dispersion = Mw / Mn.

[0079] The above results show that high temperature depolymerization has an important influence on the molecular weight of ultra-low molecular weight heparin products. The higher the temperature, the lower the weight average molecular weight of the product. 6000 The percentage is also smaller.

[0080] Example 4: Investigation on the molecular weight distribution of ultra-low molecular weight heparin products by fractional precipitation

[0081] Take the remaining heparin ester prepared in Example 3, a total of 100 g. The depolymerization preparation (S3) of the crude ultra-low molecular weight heparin is the same as the method in Example 3, and the depolymerization temperature is 65 °C. Finally, about 130 g of wet crude ultra-low molecular weight heparin is obtained; add 600 mL of 5% sodium chloride solution and stir until dissolved and clear, add 30 mL of 30% hydrogen peroxide, adjust the pH to about 9, decolorize for 2 hours, then adjust the pH to 7.2 with dilute hydrochloric acid, filter through a 0.45 μm filter, take the filtrate, divide it into two equal parts, each about 350 mL.

[0082] (1) Methanol fractional alcohol precipitation: Label the ultra-low molecular weight heparin solution 1, add 280 mL of methanol, stir magnetically for 15 minutes, centrifuge, and transfer the supernatant to a new beaker; continue to add 770 mL of methanol, stir for 15 minutes, centrifuge, and collect the precipitate; add 200 mL of methanol and shake to dehydrate, centrifuge, and transfer the solid to a 70 °C oven for vacuum drying for 12 hours to obtain the fractionated ultra-low molecular weight heparin sample 1.

[0083] (2) Ethanol fractional alcohol precipitation: Label the ultra-low molecular weight heparin solution 2, add 175 mL of ethanol, stir magnetically for 15 minutes, centrifuge, and transfer the supernatant to a new beaker; continue to add 350 mL of ethanol, stir for 15 minutes, centrifuge, and collect the precipitate; add 100 mL of ethanol and shake to dehydrate, centrifuge, and transfer the solid to a 70 °C oven for vacuum drying for 12 hours to obtain the fractionated ultra-low molecular weight heparin sample 2.

[0084] Experimental results:

[0085] The weight-average molecular weight and molecular weight distribution results of the ultra-low molecular weight heparin samples 1 and 2 by fractional alcohol precipitation with organic solvents (methanol and ethanol) are shown in Table 2.

[0086] Table 2 Molecular weight distribution of ultra-low molecular weight heparin samples by fractional alcohol precipitation with different organic solvents

[0087] Solvent Mw Mn <![CDATA[M 6000 > <![CDATA[M 2000~6000 > <![CDATA[M 2000 > Pd Methanol (0.8 - 3.0 times) 3672 Da 2298 Da 6.8% 63.4% 29.7% 1.46 Ethanol (0.5 - 1.5 times) 3544 Da 2263 Da 6.4% 63.5% 30.1% 1.45

[0088] Note: Mw is the weight-average molecular weight, Mn is the number-average molecular weight, M 6000 is the percentage of the component with a molecular weight greater than 6000 Da, M 2000-6000 is the percentage of the component with a molecular weight between 2000 Da and 6000 Da, M 2000 is the percentage of the component with a molecular weight less than 2000 Da, and Pd is the dispersity = Mw / Mn.

[0089] Example 5: Kilogram-scale preparation of ultra-low molecular weight heparin

[0090] The kilogram-scale preparation of low molecular weight heparin is consistent with the methods of Examples 1-4 described above, except for differences in batch size, some excipients, and process parameters, which are as follows.

[0091] Preparation of ammonium heparinate (S1): Similar to Example 1, take 3.0 Kg of heparin sodium (pharmaceutical grade), add 30.0 L of water and stir until dissolved and clear; separately weigh 9.0 Kg of benzalkonium bromide and dissolve it in 60 L of purified water; under sufficient stirring, mix the two solutions and continue stirring for 30 minutes; centrifuge with a plate centrifuge to collect the resulting white precipitate, transfer and evenly spread it on a stainless steel freeze-drying tray, and freeze-dry (procedure: pre-freeze at -30°C for 4 hours, start cooling the cold trap and the vacuum pump (50 Pa), raise the baffle to 40°C for 2 hours, maintain at 40°C for 36 hours, vacuum at 20 ± 5 Pa, then pull the ultimate vacuum for 2 hours; after completion, release the vacuum and take out of the chamber), to obtain 9.1 Kg of ammonium heparinate.

[0092] Preparation of heparin ester (S2): Take the above ammonium heparinate, add 60 L of dichloromethane and stir until completely dissolved; add 10 Kg of benzyl chloride, stir and react at 35°C for 26 hours, add a solution prepared from 8.0 Kg of sodium acetate and 80 L of methanol to terminate the reaction, stir well and then let it stand, centrifuge to collect the precipitate; redissolve with 40 L of 5% sodium chloride solution, add 60 L of ethanol, stir for 20 minutes, let it stand for 6 hours, discard the supernatant, and collect the precipitate; add 45 L of ethanol, centrifuge to collect the precipitate; spread the precipitate evenly on a stainless steel tray and dry it under vacuum at 65°C for 14 hours to obtain 2.89 Kg of heparin ester.

[0093] Depolymerization preparation of crude low molecular weight heparin (S3): Take the above heparin ester, add 58 L of purified water, turn on the stirrer and heater until the material is dissolved and clear, heat and maintain the temperature of the liquid material at 65 ± 2°C; take 289 g of solid sodium hydroxide, prepare a concentrated solution of about 20% with about 1.5 L of water, add it all to the reaction kettle, stir and react for 45 minutes; quickly cool the jacket of the reaction kettle with cold water bath, adjust the pH to about neutral 7; add 3.0 Kg of solid sodium chloride, stir for 5 minutes until the liquid material is dissolved and clear; add 150 L of methanol, stir for 15 minutes, then let it stand for 12 hours, remove the supernatant, centrifuge the lower slurry, and collect the wet precipitate.

[0094] Fractionation and refinement of low molecular weight heparin (S4): Transfer the above precipitate and dissolve it in 20 L of 5% sodium chloride solution, stir until clear; add 0.7 L of 30% hydrogen peroxide, adjust the pH of the liquid material to 9.5, stir evenly and then let it stand for decolorization for 2 hours. At this time, the reaction solution is light yellow; filter the reaction solution, adjust the pH to 7.1, add 300 g of activated carbon, and stir for 30 minutes; filter with a 5 μm metal titanium rod and an air pump to remove activated carbon particles, and continue to filter the filtrate with a 0.45 μm filter. The total volume of the liquid material is about 20 L; slowly add 16 L of methanol, stir well and then let it stand for 4 hours, transfer the upper clear liquid (the solid in the lower slurry is recovered by another method), continue to add 44 L of methanol, stir well and then let it stand overnight, remove the supernatant, centrifuge the lower slurry, and collect the precipitate; resuspend the precipitate with 6 L of methanol, filter by suction and collect; take a sample and check the molecular weight distribution by SEC-HPLC. At this time, the molecular weight of the target substance is about 3800 Da, and the sugar chain component with a molecular weight greater than 6000 Da is about 6%, meeting the established goal; therefore, transfer the solid and dry it in a 60 °C oven under vacuum for 15 hours; collect the powder, dissolve it with 8 L of injection water, filter with 0.22 μm, load it into a stainless steel freeze-drying tray, about 1.5 – 2.0 L per tray, and freeze-dry (procedure: pre-freeze at -40 °C for 4 hours, start the cold trap cooling and the vacuum pump (50 Pa), raise the partition to 0 °C for 2 hours, maintain at 0 °C for 12 hours, vacuum 20 ± 5 Pa; raise the partition to 40 °C for 2 hours, maintain at 40 °C for 2 hours, ultimate vacuum; after completion, release the vacuum and take out of the box), and a total of 1.64 Kg of low molecular weight heparin is obtained, with a total yield of about 54.7%.

[0095] The product test results are as follows: loss on drying is 2.3%; weight average molecular weight is 3550 Da, and the percentage of components with a molecular weight greater than 6000 Da is 6.7%; the anti-Xa titer after drying is 101 U / mg; the ratio of anti-Xa / anti-IIa is 12.7; the pH of the solution prepared by dissolving 1.0 g of the product in 10 mL of water is 6.8; the sodium content after drying is 12.0%; the aqueous solution has a maximum absorption at a wavelength of 232 nm; the ratio of sulfonate / carboxylate is 2.0; the residual solvent of methanol is 178 ppm.

[0096] Example 6: Weight average molecular weight and molecular weight distribution of low molecular weight heparin

[0097] This example is an experiment on the analysis of the weight average molecular weight and molecular weight distribution of the low molecular weight heparin sample prepared in Example 5, and the SEC-HPLC method is used to determine its molecular weight distribution characteristics.

[0098] Solution preparation: Weigh accurately the test sample and the molecular weight calculation standard product respectively, and dissolve them with the mobile phase to prepare a 10 mg / mL solution.

[0099] Chromatographic column: Hydrophilic modified silica gel as filler (TSK pre-column, 6 mm × 40 mm; TSK GEL G2000SWxl, 7.8 mm × 300 mm; TSK GEL G3000SWxl, 7.8 mm × 300 mm, used in series);

[0100] Mobile phase: 0.5 mol / L lithium nitrate solution, pH 6.6;

[0101] Flow rate: 0.6 mL / min;

[0102] Column temperature: 30 °C;

[0103] Detector: Differential refractive index detector

[0104] Injection volume: 20 μL

[0105] Running time: 50 min.

[0106] Experimental results:

[0107] The weight-average molecular weight and molecular weight of low-molecular-weight heparin are shown in Table 3 and Figure 2 as follows.

[0108] Table 3 Molecular weight distribution of low-molecular-weight heparin samples

[0109] Sample Mw Mn <![CDATA[M 6000 > <![CDATA[M 2000~6000 > <![CDATA[M 2000 > Pd Ultra-low molecular weight heparin 3550 Da 2301 Da 6.7% 64.6% 28.7% 1.42

[0110] Note: Mw is the weight-average molecular weight, Mn is the number-average molecular weight, M 6000 is the percentage of components with a molecular weight greater than 6000 Da, M 2000-6000 is the percentage of components with a molecular weight between 2000 Da and 6000 Da, M 2000 is the percentage of components with a molecular weight less than 2000 Da, Pd is the dispersity = Mw / Mn.

[0111] The above embodiments introduce a low molecular weight heparin and its preparation method. The weight average molecular weight of the low molecular weight heparin is 3000 Da - 4000 Da, with an average of approximately 3500 Da. The percentage of sugar chains with a molecular weight greater than 6000 Da is below 10%. The anti-Xa factor titer is on average approximately 100 IU / mg, and it has an extremely high anti-Xa / anti-IIa titer ratio (between 10 - 14). Therefore, its antithrombotic titer (anti-Xa) is strong while its anticoagulant titer (anti-IIa) is low, reducing the side effects caused by the high anticoagulant titer of heparin drugs. The preparation method of this low molecular weight heparin uses porcine intestinal mucosa heparin as the raw material, undergoes salification and esterification derivatization, then uses the action of alkali to initiate and control β-depolymerization at high temperature, and uses organic solvent fractional precipitation and other refining methods to obtain the low molecular weight heparin with the characteristics of the above-mentioned molecular weight and titer ratio. This preparation method is stable and efficient, suitable for industrial production, and is used to develop and prepare drugs for preventing and treating thromboembolism.

[0112] There are still various implementation manners of the present invention. All technical solutions formed by using equivalent transformation or equivalent substitution fall within the protection scope of the present invention.

Claims

1. An ultra-low molecular weight heparin, characterized in that, The weight-average molecular weight of the ultra-low molecular weight heparin is 3000 Da - 4000 Da, the percentage of sugar chain components with a molecular weight greater than 6000 Da is below 10%, and the anti-Xa / anti-IIa factor titer ratio is between 10 and 14; the preparation method of the ultra-low molecular weight heparin includes the following steps: S1. Preparation of ammonium heparinate: Take sodium heparin, dissolve it in 8 - 12 times of water, mix it with an aqueous solution prepared from 2.5 - 3.5 times of the organic ammonium salt of sodium heparin, collect the precipitated white solid insoluble matter, and dry it; among them, sodium heparin is used as the starting material, and the source is porcine intestinal mucosa; S2. Preparation of heparin ester: Dissolve ammonium heparinate in 4 - 12 times of organic solvent, add benzyl chloride of 0.9 - 1.3 times of ammonium heparinate, stir and react at 28 - 38 °C for 12 hours or more, prepare a sodium acetate methanol solution with 0.5 - 1.0 times of ammonium heparinate of sodium acetate, mix it with the reaction solution, collect the precipitated sediment, and dry it; S3. Depolymerization preparation of crude ultra-low molecular weight heparin: Dissolve heparin benzyl ester in 15 - 25 times of water, heat it to 60 °C - 70 °C and keep it, add alkali of 0.08 - 0.12 times of heparin benzyl ester, stir and react for depolymerization for 30 - 60 minutes, add sodium chloride with a weight of 0.02 - 0.12 times of the volume of the feed liquid, stir until clear, add ethanol with a volume of 1.0 - 2.0 times of the volume of the feed liquid or methanol with a volume of 1.5 - 3.5 times, stir for not less than 10 minutes, and centrifuge or filter to collect the precipitate; among them, the alkali is a concentrated solution prepared in advance with water at a concentration of 20% - 35%; S4. Grading and refining of ultra-low molecular weight heparin: Redissolve the precipitate in 4 - 6 times of 3% - 7% brine, decolorize it with hydrogen peroxide with a final concentration of 1% - 3% and / or activated carbon with a concentration of 0.1% - 1.0%, filter it, fractionate and precipitate it with an organic solvent ethanol or methanol, take the precipitate fractionated by ethanol with a volume between 0.3 times and 2.0 times of the solution volume or take the precipitate fractionated by methanol with a volume between 0.5 - 3.5 times of the solution volume, perform in-process control, or continue fractionation and precipitation with an organic solvent until the molecular weight of the target substance is between 3000 Da and 4000 Da, and the sugar chain components with a molecular weight greater than 6000 Da are below 10%, and dry it.

2. An ultra-low molecular weight heparin as claimed in claim 1, characterized in that, The organic ammonium salt in step S1 is selected from benzethonium chloride and / or benzalkonium bromide.

3. An ultra-low molecular weight heparin as claimed in claim 1, wherein, The drying method in step S1 includes hot air drying, vacuum drying, fluidized bed drying and / or freeze drying.

4. An ultra-low molecular weight heparin as claimed in claim 1, characterized in that, The organic solvent in step S2 is selected from dichloromethane or N,N-dimethylformamide.

5. An ultra-low molecular weight heparin as claimed in claim 1, wherein, The alkali in step S3 is selected from sodium hydroxide and / or potassium hydroxide.

6. An ultra-low molecular weight heparin as claimed in claim 1, characterized in that, In step S3, when using ethanol, the dosage is 1.3 - 1.7 times of the solution volume, or when using methanol, the dosage is 2.0 - 3.0 times of the solution volume.

7. An ultra-low molecular weight heparin as claimed in claim 1, characterized in that, For the fractionation and precipitation with an organic solvent ethanol or methanol in step S4, when using ethanol, collect the part of the ethanol precipitate with a volume between 0.5 times and 1.5 times of the solution volume, or when using methanol, collect the part of the methanol precipitate with a volume between 0.8 times and 3.0 times of the solution volume.

8. An ultra-low molecular weight heparin as claimed in claim 1, characterized in that, The drying in step S4 is carried out by methods including: hot air drying, vacuum drying, fluidized bed drying, and / or freeze drying.

9. A pharmaceutical composition, characterized in that, Comprising: A pharmaceutically acceptable carrier and the low molecular weight heparin described in claim 1.

10. Use of the low molecular weight heparin described in claim 1 or the pharmaceutical composition claimed in claim 9 in the preparation of a medicament for preventing and treating thromboembolism.

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