CN102952202A

CN102952202A - Preparation method and use of ultralow-molecular weight heparin sodium

Info

Publication number: CN102952202A
Application number: CN 201110245021
Authority: CN
Inventors: 王芃
Original assignee: SUZHOU WISMED PHARMACEUTICAL CO Ltd
Current assignee: SUZHOU WISMED PHARMACEUTICAL CO Ltd
Priority date: 2011-08-25
Filing date: 2011-08-25
Publication date: 2013-03-06

Abstract

The invention provides a preparation method of ultralow-molecular weight heparin sodium. The preparation method comprises that common heparin is subjected to beta-elimination degradation in an organic solvent to produce the ultralow-molecular weight heparin sodium and the ultralow-molecular weight heparin sodium is refined and purified to form the medical ultralow-molecular weight heparin sodium. The preparation method is feasible, allows mild degradation conditions and guarantees end product anticoagulant activity. The ultralow-molecular weight heparin sodium obtained by the preparation method has anti-FXa titer of 140 to 200IU/mg (wherein a typical value is in a range of 160 to 180IU/mg), has anti-FIIa titer of 0.5 to 2IU/mg (wherein a typical value is in a range of 0.5 to 1IU/mg), has average molecular weight of 1600 to 3000Da (wherein a typical value is in a range of 1800 to 2400Da), and has heparin hexaose fragment content of 40 to 50%. The ultralow-molecular weight heparin sodium obtained by the preparation method is suitable for anticoagulant and antithrombotic clinical application, is applied by subcutaneous injection, and has a daily administration single dosage of 20 to 100mg for adults, wherein the ultralow-molecular weight heparin sodium is applied once a day.

Description

A kind of preparation method of ultra-low molecular heparin sodium and application thereof

Technical field

The invention belongs to biomedicine field, be specifically related to the preparation method of a kind of ultra-low molecular heparin sodium (ULMWH) and the medicinal application of described ultra-low molecular heparin sodium.

Background technology

Heparin is a kind of sulfated polysaccharides that comes from animal, often is used as main anti-freezing clinically, antithrombotic reagent uses.But owing to using heparin that higher hemorrhage risk is arranged, its use need to be carried out the medical matters observation, and in time adjusts dosage, so its clinical use has significant limitation.

Low molecular heparin is the small molecules heparin product of degrading and obtaining by diverse ways from unfractionated heparin, has the advantages such as bioavailability is high, plasma half-life is long, and particularly its hemorrhage side effect that causes significantly reduces.But the anti-FIIa value of Low molecular heparin is still higher, and about 30, the ratio of the anti-FIIa of anti-FXa/ is 3.5～5.5 such as the anti-FIIa value of the Low molecular heparin for preparing by patent EP0040144 method.Use this class Low molecular heparin, although than the security of unfractionated heparin significantly raising is arranged, still have certain hemorrhage risk.Equally, reported a kind of synthetic method of very low molecular weight heparin in US Patent No. 6384021, although the molecular weight of the very low molecular weight heparin that obtains is lower, its anti-FXa value is no more than 120IU/mg, and the anti-FIIa ratio of anti-FXa/ is between 15～50.

Ultra low molecular weight heparin is than low molecular sodium heparin molecular-weight average less (being usually less than 3000 dalton), the heparitin derivative that anti-FIIa value is lower.Compare with Low molecular heparin, ultra low molecular weight heparin tool anticoagulation safety, its anti thrombotic action is strong, subcutaneous injection easily absorbs, be mainly used in the prevention of thrombotic disease, such as nephrotic syndrome, the prevention of formation, old phlebothrombosis and the cardiovascular and cerebrovascular diseases of operation posterior vein thrombus and anti-curing hyperlipemia etc.Chinese patent CN200810020765 has reported a kind of preparation method of ultra low molecular weight heparin, but the anti-FXa activity of the ultra low molecular weight heparin that utilizes this preparation method to obtain is low, is difficult to satisfy clinical requirement, and the feasibility of suitability for industrialized production is not high.

Summary of the invention

Technical problem to be solved by this invention provides a kind of preparation method of ultra-low molecular heparin sodium, this preparation method's degradation condition gentleness can reduce sulfonic group coming off from the heparin, ultra-low molecular heparin sodium molecular-weight average by the method preparation has high resistance Fxa biological activity less than 3000Da.

For solving above technical problem, the preparation method of a kind of ultra-low molecular heparin sodium of the present invention comprises the steps:

(1) get the elaboration heparin and be made into 10% the aqueous solution, add while stirring quaternary ammonium salt such as benzethonium chloride under the room temperature, stop after 1～2 hour stirring, sedimentation, topple over, washing precipitate, filter at last, drying, obtain white heparin quaternary ammonium salt solid;

(2) the heparin quaternary ammonium salt dissolution of solid in (1) is made into 5%～10% solution in organic solvent (anhydrous methylene chloride), add the Benzyl Chloride with heparin quaternary ammonium salt equivalent, under 30 ℃～35 ℃, stirring reaction is after 24 hours, be chilled to room temperature, 10% sodium-acetate of 2 times of volumes of adding/methanol solution stirs after 30 minutes quiescent setting, filter, methanol wash, vacuum-drying under the room temperature obtains the carboxylate of heparin;

(3) heparin ester that (2) is obtained is dissolved in wiring solution-forming in 10 times the water, the benzethonium chloride of 2.3～2.5 times carboxylate amounts is dissolved in 10 times the water, after the dissolving, under room temperature, the vigorous stirring this solution is slowly joined in the aqueous solution of heparin ester fully; After interpolation is finished, continue to stir 1-2 hour, then stop to stir; After the white solid sedimentation, incline the upper strata stillness of night, add water washing, to filter, again washing, obtains the quaternary ammonium salt of heparin ester at then dry (not being higher than 50 ℃) under the vacuum;

The quaternary ammonium salt of the heparin ester that (4) (3) is obtained is dissolved in 10 times the methylene dichloride of drying, after dissolving, add the organic bases of 0.05～0.5 volumetric molar concentration such as benzyltrimethylammonium hydroxide, be that 30 ℃～35 ℃ lower reactions 4～24 hours (preferred 8～12 hours) are carried out β-eliminations and degraded in temperature;

(5) with the degradation product after (4) reaction end, at room temperature add the methanol solution of 10% sodium-acetate of two volumes, stir after 1～2 hour, in 0 ℃～4 ℃ lower sedimentations, filtration, methanol wash, 25 ℃～35 ℃ lower vacuum-dryings, obtain the crude product of ultra-low molecular heparin sodium;

(6) the ultra-low molecular heparin sodium crude product in above-mentioned (5) is made into 10% solution, and 0 ℃～4 ℃ lower sodium hydroxide solutions that add are transferred pH 9～10, and reacted 3 hours under this temperature; Then with the neutralization of 1M hydrochloric acid, adding sodium-chlor to salinity is 8～10%, with 5 times methanol extraction, and throw out methanol wash, vacuum-drying;

(7) the low molecular sodium heparin crude product in above-mentioned (6) is made into 10% solution, the 1M sodium hydroxide solution, adjust pH is 9～10, the hydrogen peroxide at room temperature of adding 30% stirred after 6 hours, drip the hydrochloric acid of 1M to neutral, then add 8～10% sodium-chlor, use again 5 times methanol extraction ultra-low molecular heparin sodium; Filter, with the dry elaboration that makes low molecular sodium heparin after the methanol wash;

(8) product that above-mentioned (7) is obtained is water-soluble be made into 10% solution after, adding sodium-chlor to concentration is 10%, with the methanol extraction of 5 times of volumes, filtration, methanol wash, vacuum-drying obtains the ultra-low molecular heparin sodium elaboration;

(9) the elaboration ultra-low molecular heparin sodium that above-mentioned (8) is obtained is dissolved in deionized water and is made into 10% solution, behind 0.2 micron membrane filtration, lyophilize, finally obtain medical ultra-low molecular heparin sodium, its anti-FXa tires, and (representative value is 160～180IU/mg) at 140～200IU/mg, anti-FIIa tires, and (representative value is 0.5～1IU/mg) at 0.5～2IU/mg, (representative value is 1800～2400Da) to molecular-weight average, and wherein heparin six sugar account for the ratio of total ultra low molecular weight heparin 40～50% at 1600～3000Da.

The present invention also provides the application of medical ultra-low molecular heparin sodium in preparation anti-freezing, antithrombotic reagent, as anti-freezing, antithrombotic reagent, the elaboration heparin that uses in described preparation method's step (1) comes from chitterlings, and the dermatan sulfate that wherein contains≤1%; The medical ultra low molecular weight heparin of the present invention's preparation can be made into for hypodermic injection through regular injection agent preparation technologies such as preparation, filtration, embedding, sterilization, quality inspection, packaging sterilizing, and dosage is 20～100mg of per day for adults.

Compared with prior art, the beneficial effect that has of the present invention is:

1, the present invention provides a method for preparing the esterified unfractionated heparin into a quaternary ammonium salt in an organic solvent in the β-elimination of the degradation, reducing the sulfonic acid group detached from heparin, preparation method is feasible, and the degradation conditions moderate, thus ensuring a high anticoagulant activity of the final product.

2, tire according to the anti-FXa of the ultra-low molecular heparin sodium of present method preparation and tire at 0.5～2IU/mg at 140～200IU/mg, anti-FIIa, molecular-weight average is at 1600～3000Da, and wherein the content of heparin six bglii fragments is 40～50%.

Embodiment

Embodiment 1

The refined heparin sodium of getting 100 grams is dissolved in 1 premium on currency, then under rapid stirring, add 600 milliliters of the aqueous solution containing 270 gram benzethonium chlorides, react after 2 hours, add 2 premium on currency, quiescent settling, topple over the top stillness of night, add again water washing, then filter water washing, drying approximately obtains the 270 heparin quaternary ammonium salts that restrain;

Get in the methylene dichloride that heparin quaternary ammonium salt 100 grams that above-mentioned reaction obtains are dissolved in 500 milliliters, the Benzyl Chloride that adds 100 milliliters under the room temperature, after reacting 24 hours under 30-35 ℃, the 10% sodium-acetate methanol solution that adds 1.2 liters, stir after 20 minutes, sedimentation, filtration, methanol wash, drying obtain heparin ester 44 grams;

10 gram heparin esters are dissolved in wiring solution-forming in 200 ml waters, add 100 milliliters the aqueous solution that contains 23 gram benzethonium chlorides under room temperature, the rapid stirring, react after 2 hours, sedimentation, wash with water, 45-50 ℃ of lower vacuum-drying, obtain 22 the gram heparin esters quaternary ammonium salt;

The quaternary ammonium salts of 10 gram heparin esters are dissolved in the methylene dichloride of 100 milliliters of dryings, add 17 milliliters benzyltrimethylammonium hydroxide under the room temperature, 35 ℃ of lower stirring reactions 24 hours.After reaction finished, cool to room temperature added 200 milliliters 10% sodium-acetate methanol solution, continues to stir 1 hour, and quiescent settling filters, methanol wash, and room temperature vacuum-drying obtains 3.6 and restrains the ultra low molecular weight heparin crude products;

Crude product ultra-low molecular heparin sodium 3 grams that obtain are dissolved in 30 ml waters, the sodium hydroxide of 0 ℃ of lower 1M of adding is transferred pH to 9.5, behind the stirring reaction 3～4 hours, hydrogen peroxide to the concentration of adding 30% is 0.5-1%, stir under the room temperature after 6 hours, transfer solution to neutral with the hydrochloric acid of 1M, add sodium-chlor, so that the concentration of sodium-chlor is 8%, with 100 milliliters methanol extraction.Filter, methanol wash, 45-50 ℃ of lower vacuum-drying obtains half purified product, 2.6 grams;

Half elaboration ultra-low molecular heparin sodium, 1 gram is dissolved in 10 milliliters is made into 10% solution, after the adding 10 gram sodium-chlor dissolvings, add 50 ml methanol precipitation, 4 ℃ of static placements were filtered after 4 hours, methanol wash, vacuum-drying under the room temperature obtains 0.8 gram ultra-low molecular heparin sodium elaboration;

0.8 gram ultra-low molecular heparin sodium is dissolved in deionized water is made into 10% solution, with 0.2 micron membrane filtration, lyophilize, finally obtain medical ultra-low molecular heparin sodium.

The molecular-weight average of above-mentioned ultra-low molecular heparin sodium is 2400Da, and heparin six bglii fragments account for 45% of total amount, and (it is 165IU/mg that anticoagulin a) is worth to anti-FXa, and anti-FIIa (zymoplasm) value is 0.6IU/mg.

Embodiment 2

100 milligrams of ultra low molecular weight heparins are dissolved in 2.5 milliliter 0.5% the physiological saline, behind 0.2 micron the membrane filtration, sample carries out rat, and (body weight is 140～160g) subcutaneous injections, injected dose is 2 milligrams/, respectively at injection rear 0.5,1,1.5,2,3,4, each point was taked 0.5 milliliter of serum in 6,8,12 hours, carry out anti-FXa and analyze peak time T _MaxBe about 2 hours, medicine disappears in mouse blood substantially after 12 hours.

Claims

1. the preparation method of a ultra-low molecular heparin sodium is characterized in that comprising the steps:

(1) gets the aqueous solution of elaboration heparin, add quaternary ammonium salt, obtain white heparin quaternary ammonium salt solid;

(2) the heparin quaternary ammonium salt dissolution of solid in above-mentioned (1) adds Benzyl Chloride and obtains heparin ester in organic solvent;

(3) heparin ester in above-mentioned (2) is water-soluble, adds quaternary ammonium salt, obtains white heparin ester quaternary ammonium salt solid;

(4) quaternary ammonium salt of the heparin ester in above-mentioned (3) is dissolved in the organic solvent, carries out β-elimination degraded under the organic bases effect;

(5) degradation product in above-mentioned (4) is converted into sodium salt under the sodium hydroxide effect, obtains the crude product of elaboration ultra-low molecular heparin sodium;

(6) ultra-low molecular heparin sodium that obtains in above-mentioned (5) obtains medical ultra low molecular weight heparin bulk drug through precipitation, secondary filter, lyophilize.

2. according to the preparation method of the described a kind of ultra-low molecular heparin sodium of claim 1, it is characterized in that: quaternary ammonium salt is benzethonium chloride described in the preparation process (1).

3. according to the preparation method of the described a kind of ultra-low molecular heparin sodium of claim 1, it is characterized in that: organic solvent is anhydrous methylene chloride described in the preparation process (4).

4. according to the preparation method of the described a kind of ultra-low molecular heparin sodium of claim 1, it is characterized in that: organic bases is benzyltrimethylammonium hydroxide described in the preparation process (4).

5. according to the preparation method of the described a kind of ultra-low molecular heparin sodium of claim 1, it is characterized in that: the ultra-low molecular heparin sodium that makes according to this preparation method is that anti-FXa tires and tires at 0.5～2IU/mg at 140～200IU/mg, anti-FIIa, molecular-weight average is at 1600～3000Da, and the content of heparin six bglii fragments is 40～50%.

6. according to the preparation method of the described a kind of ultra-low molecular heparin sodium of claim 5, it is characterized in that: the ultra-low molecular heparin sodium that makes according to this preparation method is that anti-FXa tires and tires at 0.5～1IU/mg at 160～180IU/mg, anti-FIIa, molecular-weight average is at 1800～2400Da, and the content of heparin six bglii fragments is 40～50%.

7. according to the preparation method of the described a kind of ultra-low molecular heparin sodium of claim 5, it is characterized in that: the dermatan sulfate that contains in the described elaboration heparin of step (1)≤1%.

8. the application of the medical ultra-low molecular heparin sodium of claim 7 preparation in preparation anti-freezing, antithrombotic reagent.