A method for preparing ovine enoxaparin sodium

Abstract

The application relates to the field of medicine biotechnology, and discloses a preparation method of sheep-derived enoxaparin sodium, which comprises the following steps: after sheep-derived heparin sodium is subjected to salification and esterification, benzyl heparin ester is obtained; the benzyl heparin ester is degraded twice; then, the degradation products of the two times are collected to obtain an intermediate product meeting the quality standard of enoxaparin sodium; and finally, the intermediate product is subjected to oxidation and drying to obtain fine enoxaparin sodium. Through the technical scheme, the problem of low yield of enoxaparin sodium in the prior art is solved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine biotechnology, in particular, to a preparation method of sheep-derived enoxaparin sodium. BACKGROUND

[0002] Enoxaparin sodium is developed by Sanofi, France, which is a low molecular heparin product prepared by alkaline hydrolysis of benzyl ester derivatives of heparin. It is mainly used for treating antithrombotic, anticoagulant, acute myocardial infarction and unstable angina pectoris and other cardiovascular diseases, and is one of the main therapeutic drugs for anticoagulation and antithrombosis.

[0003] The main source of heparin is pig intestinal mucosa. In 2008, the pig heparin chondroitin sulfate (OSCS) pollution event and the recent "swine fever" event made countries realize the risk of single heparin source and encouraged the diversification of heparin sources.

[0004] Due to the low potency of bovine heparin (only 2 / 3 of the potency of pig-derived heparin and sheep-derived heparin), it cannot be used to prepare low molecular heparin. Meanwhile, the diet habit in China is mainly eating mutton, and most of the sheep resources are wasted except for a small part being consumed and used for medical treatment. Therefore, sheep-derived heparin is the best choice.

[0005] The patent with the authorization announcement number CN103554305B degrades heparin benzyl ester in a high-temperature water bath by sodium hydroxide, adds ethanol with a volume of 2 times of the drug solution for fractional precipitation, and collects the precipitate to obtain enoxaparin sodium crude product. The patent with the authorization announcement number CN102603925B adds sodium hydroxide to the heparin benzyl ester solution for degradation at high temperature, adjusts the pH to neutral, adds 2-3 times of ethanol for precipitation and fractionation, separates the precipitate to obtain enoxaparin sodium crude product, and then adds 2 times of ethanol for fractional precipitation after oxidation. The patent with the authorization announcement number CN104086674B degrades heparin benzyl ester through an anion exchange chromatography column, first washes out the excess low molecular oligosaccharide fragments by using a low-concentration sodium chloride solution, and then elutes and collects enoxaparin sodium by using a high-concentration sodium chloride solution.

[0006] The above disclosed preparation processes of enoxaparin sodium all adopt a degradation method to meet the molecular weight and distribution of enoxaparin sodium, and then collect the target heparin fragments. Whether it is ethanol fractional precipitation, washing and elution collection after column chromatography, or the use of ultrafiltration of the degradation solution to filter out the over-degraded low molecular fragments, there is a problem of low yield. Therefore, it is of great significance to study a preparation method of sheep-derived enoxaparin sodium with high yield. SUMMARY

[0007] The present application provides a preparation method of sheep-derived enoxaparin sodium, which solves the problem of low yield of enoxaparin sodium in the related art.

[0008] The technical solution of the present invention is as follows:

[0009] This invention proposes a method for preparing sheep-derived enoxaparin sodium, comprising the following steps:

[0010] S1. Preparation of heparin benzyl chloride: Dissolve heparin sodium in water, add benzyl chloride, mix, filter, and dry to obtain heparin benzyl chloride.

[0011] S2. Preparation of heparin benzyl ester: The heparin benzyl chloride ammonium salt is dissolved in the first solvent, benzyl chloride is added, the reaction is carried out, sodium acetate ethanol solution is added after the reaction, filtered, and dried to obtain heparin benzyl ester;

[0012] S3. Stepwise Degradation: The heparin benzyl ester is dissolved in water, and after the first degradation with the addition of the degrading agent, the mixture is cooled, the pH is adjusted to neutral, and the reaction is terminated to obtain the first degradation solution. A second solvent is added until the first degradation solution is 40%-50% complete. The mixture is allowed to stand and separate into layers. The supernatant and precipitate are separated. The second solvent is added to the supernatant until the supernatant is ≥80% complete. The low molecular weight heparin is collected for the first time to obtain intermediate m1. The precipitate is dissolved in water, and after the second degradation with the addition of the degrading agent, the mixture is cooled, the pH is adjusted to neutral, and the reaction is terminated to obtain the second degradation solution. The second solvent is added until the second degradation solution is ≥80% complete. The low molecular weight heparin is collected for the second time to obtain intermediate m2.

[0013] S4. Oxidation: After mixing the intermediates m1 and m2, dissolve them in water, add an oxidizing agent to oxidize them, and obtain an oxidized mixture. Add a third solvent to the oxidized mixture until the volume fraction of the oxidized mixture is ≥80%, then collect it all to obtain enoxaparin sodium.

[0014] S5. Drying: Dissolve the enoxaparin sodium in water, filter, and dry to obtain the refined enoxaparin sodium.

[0015] As a further technical solution, in step S1, the heparin sodium can be porcine mucosal heparin or ovine mucosal heparin.

[0016] As a further technical solution, in step S1, the mass ratio of heparin sodium to benzyl chloride is 1:2.5-3.

[0017] As a further technical solution, in step S2, the mass-to-volume ratio of heparin benzyl chloride to benzyl chloride is 1g:0.1-2mL. Preferably, the first solvent is N,N-dimethylformamide or dichloromethane.

[0018] As a further technical solution, in step S2, the reaction is carried out at a temperature of 30-40℃ for a time of 20-48h.

[0019] As a further technical solution, in step S2, the mass fraction of the sodium acetate ethanol solution is 8%-10%.

[0020] As a further technical solution, in step S3, the degradation agent is one or more of sodium hydroxide, sodium carbonate, and sodium bicarbonate;

[0021] Preferably, during the first degradation, the mass ratio of the degrading agent to heparin benzyl ester is 2-8:100, and during the second degradation, the mass ratio of the degrading agent to the precipitate is 1-4:100.

[0022] As a further technical solution, in step S3, the temperature during the first degradation is 56-65℃ and the time is 30-90 min, and the temperature during the second degradation is 50-60℃ and the time is 60-120 min.

[0023] As a further technical solution, in steps S3 and S4, the second solvent and the third solvent are each independently selected from one or more of ethanol, methanol, and acetone.

[0024] As a further technical solution, in step S3, the weight-average molecular weight of intermediate m1 is 2500-4000, and the weight-average molecular weight of intermediate m2 is 4500-6000.

[0025] As a further technical solution, in step S4, the oxidant is hydrogen peroxide or peracetic acid;

[0026] Preferably, the mass-to-volume ratio of heparin benzyl ester to oxidant is 100g:9-16mL.

[0027] As a further technical solution, in step S4, the oxidation process is carried out at a temperature of 20-35℃, a pH value of 9.0-11.0, and a time of 6-24h.

[0028] The working principle and beneficial effects of this invention are as follows:

[0029] 1. In this invention, during the preparation of enoxaparin sodium, the heparin benzyl ester is degraded twice and collected completely. By controlling the weight-average molecular weight range of the intermediates obtained from the two degradations, the degradation of different components of heparin benzyl ester is controlled, enabling the complete conversion of high-molecular-weight heparin sodium into the target low-molecular-weight enoxaparin sodium. The resulting enoxaparin sodium meets the CP / EP / USP pharmacopoeia standards and its quality is higher than the current pharmacopoeia standards. Simultaneously, this invention changes the traditional preparation process where excessive degradation is followed by fractional precipitation, column chromatography, or ultrafiltration to remove some of the over-degraded low-molecular-weight fragments. This avoids the loss of low-molecular-weight enoxaparin sodium caused by directly discarding the supernatant containing low-molecular-weight enoxaparin sodium in the traditional preparation process, achieving full utilization of raw materials and significantly improving the yield of enoxaparin sodium, with a weight yield exceeding 78%. Attached Figure Description

[0030] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments.

[0031] Figure 1 This is a flowchart of the preparation method of enoxaparin sodium according to the present invention. Detailed Implementation

[0032] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0033] Unless otherwise specified, in the following examples and comparative examples, sheep heparin sodium was prepared by salting out, refining and oxidizing sheep intestinal mucosa. The absorbance of sheep heparin sodium was tested to be ≤0.2 at A260nm and ≤0.2 at A280nm, and the anti-IIa factor titer was not less than 180 IU / mg; the purity of benzyl chloride was ≥98%; and the purity of benzyl chloride was ≥99%.

[0034] Example 1

[0035] A method for preparing sheep-derived enoxaparin sodium includes the following steps:

[0036] S1. Preparation of heparin benzyl chloride: Dissolve 100g of sheep-derived sodium heparin in 1000mL of purified water by stirring. Dissolve 280g of benzyl chloride in 1500mL of purified water by stirring. Add the sodium heparin solution to the benzyl chloride solution and stir for 1 hour. Filter and dry to obtain heparin benzyl chloride.

[0037] S2. Preparation of heparin benzyl ester: Heparin benzyl chloride was dissolved in N,N-dimethylformamide by stirring, and benzyl chloride was added (the mass-volume ratio of heparin benzyl chloride to benzyl chloride was 1 g: 1 mL). The mixture was stirred at 35 °C for 34 h. After the reaction, a 9% sodium acetate ethanol solution was added to precipitate the product. The product was then filtered and dried to obtain heparin benzyl ester.

[0038] S3. Stepwise Degradation: Heparin benzyl ester was dissolved in water, and a degradation agent (composed of sodium hydroxide and sodium carbonate in a mass ratio of 4:1, with a mass ratio of degradation agent to heparin benzyl ester of 5:100) was added. After degradation at 60℃ for 60 min, the solution was cooled, the pH was adjusted to neutral, and the reaction was terminated to obtain the first degradation solution. Ethanol was added until the first degradation solution volume was 45%, and the solution was allowed to stand and separate into layers. The supernatant and precipitate were separated. Ethanol was added to the supernatant until the supernatant volume was 80%. The low molecular weight heparin was collected for the first time to obtain intermediate m1. The precipitate was dissolved in water, and the above degradation agent (mass ratio of degradation agent to precipitate of 2.5:100) was added. After degradation at 55℃ for 90 min, the solution was cooled, the pH was adjusted to neutral, and the reaction was terminated to obtain the second degradation solution. Ethanol was added until the second degradation solution volume was 80%, and the low molecular weight heparin was collected for the second time to obtain intermediate m2.

[0039] S4. Oxidation: Intermediate m1 and intermediate m2 are mixed and dissolved in water. Hydrogen peroxide (mass-volume ratio of heparin benzyl ester to oxidant 100g:13mL) is added. The mixture is oxidized at 25℃ and pH 10.0 for 15h to obtain an oxidized mixture. Ethanol is added to the oxidized mixture until the volume fraction of the oxidized mixture is 80%. The mixture is then collected to obtain enoxaparin sodium.

[0040] S5. Drying: Dissolve enoxaparin sodium in water, filter, and dry to obtain purified enoxaparin sodium with a weight yield of 78.5%.

[0041] The flowchart of the preparation method of enoxaparin sodium is as follows: Figure 1 As shown.

[0042] Example 2

[0043] A method for preparing sheep-derived enoxaparin sodium includes the following steps:

[0044] S1. Preparation of heparin benzyl chloride: Dissolve 100g of sheep-derived sodium heparin in 1000mL of purified water by stirring. Dissolve 280g of benzyl chloride in 1500mL of purified water by stirring. Add the sodium heparin solution to the benzyl chloride solution and stir for 1 hour. Filter and dry to obtain heparin benzyl chloride.

[0045] S2. Preparation of heparin benzyl ester: Heparin benzyl chloride was dissolved in N,N-dimethylformamide by stirring, and benzyl chloride was added (the mass-volume ratio of heparin benzyl chloride to benzyl chloride was 1 g: 2 mL). The mixture was stirred at 35 °C for 34 h. After the reaction, a 9% sodium acetate ethanol solution was added to precipitate the product. The product was then filtered and dried to obtain heparin benzyl ester.

[0046] S3. Stepwise Degradation: Heparin benzyl ester was dissolved in water, sodium hydroxide was added (the mass ratio of sodium hydroxide to heparin benzyl ester was 2:100), and the degradation was carried out at 56℃ for 90 min. After cooling, the pH was adjusted to neutral, and the reaction was terminated to obtain the first degradation solution. Ethanol was added until the first degradation solution volume was 49%, and the mixture was allowed to stand and separate into layers. The supernatant and precipitate were separated. Ethanol was added to the supernatant until the supernatant volume was 80%. The low molecular weight heparin was collected for the first time to obtain intermediate m1. The precipitate was dissolved in water, sodium hydroxide was added (the mass ratio of sodium hydroxide to precipitate was 4:100), and the degradation was carried out at 50℃ for 120 min. After cooling, the pH was adjusted to neutral, and the reaction was terminated to obtain the second degradation solution. Ethanol was added until the second degradation solution volume was 80%, and the low molecular weight heparin was collected for the second time to obtain intermediate m2.

[0047] S4. Oxidation: Intermediate m1 and intermediate m2 are mixed and dissolved in water. Hydrogen peroxide (mass-volume ratio of heparin benzyl ester to oxidant 100g:13mL) is added. The mixture is oxidized at 25℃ and pH 10.0 for 15h to obtain an oxidized mixture. Ethanol is added to the oxidized mixture until the volume fraction of the oxidized mixture is 80%. The mixture is then collected to obtain enoxaparin sodium.

[0048] S5. Drying: Dissolve enoxaparin sodium in water, filter, and dry to obtain purified enoxaparin sodium with a weight yield of 78.1%.

[0049] Example 3

[0050] A method for preparing sheep-derived enoxaparin sodium includes the following steps:

[0051] S1. Preparation of heparin benzyl chloride: Dissolve 100g of sheep-derived sodium heparin in 1000mL of purified water by stirring. Dissolve 280g of benzyl chloride in 1500mL of purified water by stirring. Add the sodium heparin solution to the benzyl chloride solution and stir for 1 hour. Filter and dry to obtain heparin benzyl chloride.

[0052] S2. Preparation of heparin benzyl ester: Heparin benzyl chloride was dissolved in N,N-dimethylformamide by stirring, and benzyl chloride was added (the mass-volume ratio of heparin benzyl chloride to benzyl chloride was 1 g: 0.5 mL). The mixture was stirred at 35 °C for 34 h. After the reaction, a 9% sodium acetate ethanol solution was added to precipitate the product. The product was then filtered and dried to obtain heparin benzyl ester.

[0053] S3. Stepwise Degradation: Heparin benzyl ester was dissolved in water, sodium carbonate was added (the mass ratio of sodium carbonate to heparin benzyl ester was 8:100), and degradation was carried out at 65℃ for 30 min. After cooling, the pH was adjusted to neutral, and the reaction was terminated to obtain the first degradation solution. Ethanol was added until the first degradation solution volume was 40%, and the mixture was allowed to stand to separate the layers. The supernatant and precipitate were separated. Ethanol was added to the supernatant until the supernatant volume was 80%. The low molecular weight heparin was collected for the first time to obtain intermediate m1. The precipitate was dissolved in water, sodium carbonate was added (the mass ratio of sodium carbonate to precipitate was 1:100), and degradation was carried out at 60℃ for 60 min. After cooling, the pH was adjusted to neutral, and the reaction was terminated to obtain the second degradation solution. Ethanol was added until the second degradation solution volume was 80%, and the low molecular weight heparin was collected for the second time to obtain intermediate m2.

[0054] S4. Oxidation: Intermediate m1 and intermediate m2 are mixed and dissolved in water. Hydrogen peroxide (mass-volume ratio of heparin benzyl ester to oxidant 100g:13mL) is added. The mixture is oxidized at 25℃ and pH 10.0 for 15h to obtain an oxidized mixture. Ethanol is added to the oxidized mixture until the volume fraction of the oxidized mixture is 80%. The mixture is then collected to obtain enoxaparin sodium.

[0055] S5. Drying: Dissolve enoxaparin sodium in water, filter, and dry to obtain purified enoxaparin sodium with a weight yield of 79.8%.

[0056] Comparative Example 1

[0057] A method for preparing sheep-derived enoxaparin sodium includes the following steps:

[0058] S1. Preparation of heparin benzyl chloride: Dissolve 100g of sheep-derived sodium heparin in 1000mL of purified water by stirring. Dissolve 280g of benzyl chloride in 1500mL of purified water by stirring. Add the sodium heparin solution to the benzyl chloride solution and stir for 1 hour. Filter and dry to obtain heparin benzyl chloride.

[0059] S2. Preparation of heparin benzyl ester: Heparin benzyl chloride was dissolved in N,N-dimethylformamide by stirring, and benzyl chloride was added (the mass-volume ratio of heparin benzyl chloride to benzyl chloride was 1 g: 1 mL). The mixture was stirred at 35 °C for 34 h. After the reaction, a 9% sodium acetate ethanol solution was added to precipitate the product. The product was then filtered and dried to obtain heparin benzyl ester.

[0060] S3. Degradation: Heparin benzyl ester was dissolved in water, and a degradation agent (composed of sodium hydroxide and sodium carbonate in a mass ratio of 4:1, with a mass ratio of degradation agent to heparin benzyl ester of 10:100) was added. After degradation at 60℃ for 60 min, the solution was cooled, the pH was adjusted to neutral, and the reaction was terminated to obtain the degradation solution. Ethanol was added until the degradation liquid volume was 58%, and the solution was fractionated and precipitated. The supernatant was discarded to obtain enoxaparin sodium intermediate.

[0061] S4. Oxidation: Dissolve the intermediate in water, add hydrogen peroxide (heparin benzyl ester to oxidant mass-volume ratio 100g:13mL), oxidize for 15h at 25℃ and pH 10.0 to obtain an oxidized solution, add ethanol to the oxidized solution until the oxidized mixture is 80% complete, collect it all to obtain enoxaparin sodium.

[0062] S5. Drying: Dissolve enoxaparin sodium in water, filter, and dry to obtain purified enoxaparin sodium with a weight yield of 62%.

[0063] The molecular weight and distribution of intermediates m1 and m2 in Examples 1-3 were determined according to European Pharmacopoeia EP10.5, and the test results are shown in Table 1 below.

[0064] Table 1. Molecular weight and distribution of intermediates

[0065]

[0066] The molecular weight, fractionation, potency, 1,6-dehydrated derivatives, and weight yield of enoxaparin sodium prepared in Examples 1-3 and Comparative Example 1 were determined. The methods for detecting molecular weight, fractionation, potency, and 1,6-dehydrated derivatives were performed according to European Pharmacopoeia EP10.5. The results are shown in Table 2 below.

[0067] Table 2. Results of Enoxaparin Sodium Detection

[0068]

[0069] The data in the table show that the enoxaparin sodium prepared by the method of this invention meets the CP / EP / USP pharmacopoeia standards for enoxaparin sodium and its quality is higher than the current pharmacopoeia standards, with a weight yield of over 78%. A comparison between Example 1 and Comparative Example 1 shows that two degradation and collection processes of heparin benzyl ester help improve the yield of enoxaparin sodium.

[0070] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing sheep-derived enoxaparin sodium, characterized in that, Includes the following steps: S1. Preparation of heparin benzyl chloride: Dissolve heparin sodium in water, add benzyl chloride, mix, filter, and dry to obtain heparin benzyl chloride. S2. Preparation of heparin benzyl ester: The heparin benzyl chloride ammonium salt is dissolved in the first solvent, benzyl chloride is added, the reaction is carried out, sodium acetate ethanol solution is added after the reaction, filtered, and dried to obtain heparin benzyl ester; S3. Stepwise Degradation: The heparin benzyl ester is dissolved in water, and after the first degradation with the addition of the degrading agent, the mixture is cooled, the pH is adjusted to neutral, and the reaction is terminated to obtain the first degradation solution. A second solvent is added until the first degradation solution is 40%-50% complete. The mixture is allowed to stand and separate into layers. The supernatant and precipitate are separated. The second solvent is added to the supernatant until the supernatant is ≥80% complete. The low molecular weight heparin is collected for the first time to obtain intermediate m1. The precipitate is dissolved in water, and after the second degradation with the addition of the degrading agent, the mixture is cooled, the pH is adjusted to neutral, and the reaction is terminated to obtain the second degradation solution. The second solvent is added until the second degradation solution is ≥80% complete. The low molecular weight heparin is collected for the second time to obtain intermediate m2. S4. Oxidation: After mixing the intermediates m1 and m2, dissolve them in water, add an oxidizing agent to oxidize them, and obtain an oxidized mixture. Add a third solvent to the oxidized mixture until the volume fraction of the oxidized mixture is ≥80%, then collect it all to obtain enoxaparin sodium. S5. Drying: Dissolve the enoxaparin sodium in water, filter, and dry to obtain the refined enoxaparin sodium. In step S3, the degrading agent is one or more of sodium hydroxide, sodium carbonate, and sodium bicarbonate. During the first degradation, the mass ratio of the degrading agent to heparin benzyl ester is 2-8:

100. During the second degradation, the mass ratio of the degrading agent to the precipitate is 1-4:

100. During the first degradation, the temperature is 56-65℃ and the time is 30-90 min. During the second degradation, the temperature is 50-60℃ and the time is 60-120 min. The weight-average molecular weight of intermediate m1 is 2500-4000, and the weight-average molecular weight of intermediate m2 is 4500-6000. In steps S3 and S4, the second solvent and the third solvent are each independently selected from one or more of ethanol, methanol, and acetone.

2. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, In step S1, the mass ratio of heparin sodium to benzyl chloride is 1:2.5-3.

3. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, In step S2, the mass-to-volume ratio of heparin benzyl chloride to benzyl chloride is 1g:0.1-2mL.

4. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, The first solvent is N,N-dimethylformamide or dichloromethane.

5. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, In step S2, the reaction is carried out at a temperature of 30-40°C for a time of 20-48 hours.

6. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, In step S4, the oxidant is hydrogen peroxide or peracetic acid.

7. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, The mass-to-volume ratio of heparin benzyl ester to oxidant is 100g:9-16mL.

8. The method for preparing sheep-derived enoxaparin sodium according to claim 1, characterized in that, In step S4, the oxidation process is carried out at a temperature of 20-35°C, a pH value of 9.0-11.0, and a time of 6-24 hours.

Images