Combined method for converting nadroparin calcium into calcium and adjusting molecular weight

Abstract

The invention discloses a combined method for converting nadroparin calcium into calcium and adjusting molecular weight. The method comprises the following steps: degrading common heparin sodium by using a sodium nitrite solution, then reducing by using sodium borohydride, and finally carrying out alcohol precipitation, adding the obtained precipitate into purified water, stirring and dissolving, and carrying out ultraviolet radiation; adding into an ion exchange resin column, washing by using a 100-500mmol / L calcium chloride solution, eluting by using a 1-5mol / L calcium chloride solution, detecting the molecular weight and distribution, and collecting the eluent; adding hydrogen peroxide into the obtained feed liquid for oxidation, and then carrying out alcohol precipitation; and adding purified water into the obtained precipitate, dissolving, and freeze-drying. Calcium conversion and molecular weight adjustment can be achieved at the same time through only one procedure, the calcium content of the obtained nadroparin calcium meets the standard, and the molecular weight is basically consistent with the reference. The method is simple in process operation, greatly shortens the working procedure time, reduces the solvent, labor and power cost of multi-step reaction, and has remarkable economic benefits.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and specifically relates to a combined method of nadroparin calcium conversion and molecular weight adjustment. Background Art

[0002] Heparin Sodium is a mucopolysaccharide sulfate anticoagulant. Heparin Sodium is the sodium salt of aminoglucan sulfate extracted from the intestinal mucosa of pigs or cattle, and belongs to the mucopolysaccharide class. It has anticoagulant effects both in vivo and in vitro, and is currently the mainstream anticoagulant drug. However, ordinary heparin has low bioavailability and large side effects. Later, people found that the sodium salt of aminoglucan sulfate fragments obtained by the cleavage of heparin sodium has an average molecular weight of 4000-6000 Daltons, which is called low molecular weight heparin sodium. Low molecular weight heparin sodium injection, like heparin sodium injection, is an antithrombin III (ATⅢ)-dependent thrombin inhibitor. However, compared with heparin sodium injection, it has a longer half-life, good antithrombotic effect, weaker bleeding tendency, and convenient administration, but it is more expensive. Ordinary heparin products are all sodium salt type. Compared with calcium salt type, the latter has the advantages of reduced local hematoma and significantly improved pain conditions during subcutaneous injection. Therefore, heparin products are currently more inclined to be made into calcium salt type. Nadroparin calcium has the function of preventing and treating venous thromboembolic diseases, especially preventing thromboembolic diseases in general surgery or orthopedic surgery, and preventing blood clot formation in extracorporeal circulation during hemodialysis.

[0003] Nadroparin calcium is a type of low molecular weight heparin in the form of calcium salt. It was developed by GlaxoSmithKline Pharmaceuticals Co., Ltd. and had global sales of 229 million pounds in 2009. It is made from heparin sodium by nitrous acid lysis, with an average molecular weight of 3600 to 5000. The portion with a molecular weight of less than 2000 shall not exceed 15% of the total amount, the portion with a molecular weight of 2000 to 4000 shall account for 35% to 55% of the total amount, and the portion with a molecular weight of 2000 to 8000 shall account for 75% to 95% of the total amount.

[0004] Patent CN103275246A uses crude heparin sodium as raw material, obtains fine heparin sodium through enzymatic purification, degrades, and then obtains crude nadroparin calcium through reduction, ultraviolet irradiation, oxidation, and calcium replacement. After refining it through nanofiltration and ultrafiltration, it is freeze-dried to obtain fine nadroparin calcium.

[0005] Patent CN103382232A reduces the raw material of sodium heparin with sodium nitrite under the acidic conditions of acetic acid, reduces it with sodium borohydride, and recrystallizes it with ethanol to obtain low molecular weight heparin sodium, converts it into calcium chloride, recrystallizes it with ethanol, performs exchange column chromatography, precipitates it with ethanol of the washing solution, and filters and freeze-dries it to obtain nadroparin calcium.

[0006] Patent CN103408676A obtains low molecular weight heparin sodium by dissolving heparin sodium raw material in purified water, adding sodium nitrite for degradation under certain temperature and pH conditions, reducing with sodium borohydride, and precipitating with ethanol. The low molecular weight heparin sodium is then oxidized with hydrogen peroxide, filtered, converted to calcium with an anion exchange column, and the eluent is precipitated with ethanol. The precipitate is oxidized with hydrogen peroxide, and the oxidation reaction liquid is precipitated with ethanol to obtain crude nadroparin calcium. The crude nadroparin calcium is ultrafiltered with a 1000D ultrafiltration membrane and sterilized and filtered to obtain fine nadroparin calcium.

[0007] Patent CN104072639 uses heparin sodium as a raw material and obtains nadroparin calcium by degradation, reduction, ethanol precipitation, ion exchange resin calcium conversion, oxidation, filtration precipitation and dehydration.

[0008] Patents CN103275246A, CN103382232A and 103408676A all obtain low molecular weight heparin sodium through degradation and reduction methods, among which patent CN103275246A needs to go through calcium transfer, nanofiltration, ultrafiltration and other steps, and the process is cumbersome and complicated. Patent CN103382232A separates calcium transfer and exchange column chromatography, which increases the complexity of operation. Patent 103408676A undergoes two hydrogen peroxide oxidations, two filtrations, anion exchange column calcium transfer, and 1000D ultrafiltration membrane ultrafiltration, which is also complex in process and complicated in operation. Patent CN104072639 does not adjust the molecular weight, and the final nadroparin calcium product is quite different from the reference. Summary of the invention

[0009] The purpose of the present invention is to fill the gap in the existing technical solutions and propose a new method for combining nadroparin calcium conversion with molecular weight adjustment, which has the advantages of short process, simple operation, good reproducibility, etc., and meets the needs of laboratory research and large-scale chemical production in the pharmaceutical and chemical industries.

[0010] The combined method of nadroparin calcium conversion and molecular weight adjustment is: (1) Degrading ordinary heparin sodium with sodium nitrite solution, then reducing it with sodium borohydride, and finally precipitating it with alcohol to obtain a precipitate; (2) adding purified water to the precipitate obtained in step (1), stirring and dissolving the precipitate, and subjecting the precipitate to ultraviolet irradiation; (3) The slurry obtained by ultraviolet irradiation in step (2) is added to an ion exchange resin column, first washed with a 100-500 mmol / L calcium chloride solution, then eluted with a 1-5 mol / L calcium chloride solution, and the molecular weight and distribution are detected at the same time, and the eluate that meets the requirements is collected and combined; (4) adding 30% hydrogen peroxide to the liquid obtained in step (3) for oxidation, and then performing alcohol precipitation; (5) Add purified water to the precipitate obtained in step (4), dissolve it, and then freeze-dry it.

[0011] The ion exchange resin used in step (3) is a cation exchange resin.

[0012] The present invention can simultaneously realize calcium conversion and molecular weight adjustment in only one step, and the calcium content of the obtained nadroparin calcium meets the standard, and the molecular weight is basically consistent with the reference. The present invention is simple to operate, greatly shortens the process time, reduces the solvent, labor and power costs of multi-step reactions, and has significant economic benefits. DETAILED DESCRIPTION Example 1

[0013] (1) Degradation and reduction: Weigh 200 g of heparin sodium and add it to a reactor. Add 2000 ml of purified water and stir to dissolve. Adjust the pH. Add 60 ml of 10% sodium nitrite solution for degradation. After degradation, adjust the pH of the solution to below 12 with 5 mol / L sodium hydroxide solution. Add 0.2 g of sodium borohydride for reduction. After reduction, adjust the pH to acidic. Stir for 10 min. Adjust the pH of the solution to 7.2 with 5 mol / L sodium hydroxide solution. The volume of the solution is 3 L. Add 6 L of ethanol to the solution under stirring. Allow to settle for 12 h. Discard the supernatant and save the precipitate.

[0014] (2) Ultraviolet irradiation: The precipitate obtained in (1) is added into purified water, stirred and dissolved, and subjected to ultraviolet irradiation to obtain a sample with qualified N-NO groups.

[0015] (3) Combined calcium transfer and molecular weight adjustment: Add the above-mentioned ultraviolet irradiation solution to the prepared cation exchange resin column, first wash with 100mmol / L calcium chloride solution, then elute with 1mol / L calcium chloride solution, and collect the eluate that meets the requirements. Combine the eluates. Oxidation: Add 30% hydrogen peroxide to the solution obtained in (3) for oxidation, and then precipitate with alcohol; (4) Freeze-drying: Add purified water to the precipitate obtained by oxidation, dissolve it, and then freeze-dry it to obtain nadroparin calcium (sample 1). Example 2

[0016] (1) Degradation and reduction: Weigh 200 g of heparin sodium and add it to a reactor. Add 2000 ml of purified water and stir to dissolve. After dissolution, adjust the pH and stir for 10 min. Add 60 ml of 10% sodium nitrite solution to degrade. After degradation, adjust the pH of the solution to below 12 with 5 mol / L sodium hydroxide solution. Add 0.2 g of sodium borohydride to reduce. After reduction, adjust the pH to acidic with 4 mol / L hydrochloric acid and stir for 10 min. Then adjust the pH of the solution to 7.2 with 5 mol / L sodium hydroxide solution. The volume of the solution is 3 L. Add 6 L of ethanol to the solution under stirring. Let it stand for 12 h and discard the supernatant to save the precipitate.

[0017] (2) Ultraviolet irradiation: The precipitate obtained in (1) is added into purified water, stirred and dissolved, and subjected to ultraviolet irradiation to obtain a sample with qualified N-NO groups.

[0018] (3) Combined calcium transfer and molecular weight adjustment: Add the above-mentioned ultraviolet irradiation solution to the prepared cation exchange resin column, first wash with 500mmol / L calcium chloride solution, then elute with 5mol / L calcium chloride solution, and collect the eluate that meets the requirements. Combine the eluates. Oxidation: Add 30% hydrogen peroxide to the solution obtained in (3) for oxidation, and then precipitate with alcohol; (4) Freeze-drying: Add purified water to the precipitate obtained by oxidation, dissolve it, and then freeze-dry it to obtain nadroparin calcium (sample 2).

[0019]

Claims

1. A combined method of nadroparin calcium conversion and molecular weight adjustment, characterized in that: The specific steps of the method are: The ordinary heparin sodium is degraded by using a sodium nitrite solution, then reduced by using sodium borohydride, and finally precipitated by alcohol to obtain a precipitate; The precipitate obtained in step (1) is added into purified water, stirred and dissolved, and subjected to ultraviolet irradiation; The feed solution obtained by ultraviolet irradiation in step (2) is added to an ion exchange resin column, first washed with a 100-500 mmol / L calcium chloride solution, then eluted with a 1-5 mol / L calcium chloride solution, and the molecular weight and distribution are detected at the same time, and the eluate that meets the requirements is collected and combined; The feed solution obtained in step (3) is oxidized by adding 30% hydrogen peroxide, and then precipitated with alcohol; Purified water is added to the precipitate obtained in step (4), and the precipitate is dissolved and then freeze-dried.

2. The method according to claim 1, characterized in that The ion exchange resin used in step (3) is a cation exchange resin.

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