CN101711771B - A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition - Google Patents
A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition Download PDFInfo
- Publication number
- CN101711771B CN101711771B CN200810200817.8A CN200810200817A CN101711771B CN 101711771 B CN101711771 B CN 101711771B CN 200810200817 A CN200810200817 A CN 200810200817A CN 101711771 B CN101711771 B CN 101711771B
- Authority
- CN
- China
- Prior art keywords
- heparin
- mixture
- polysaccharide
- anticoagulin
- activity
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
- A61K31/726—Glycosaminoglycans, i.e. mucopolysaccharides
- A61K31/727—Heparin; Heparan
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
- C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
- C08B37/0063—Glycosaminoglycans or mucopolysaccharides, e.g. keratan sulfate; Derivatives thereof, e.g. fucoidan
- C08B37/0075—Heparin; Heparan sulfate; Derivatives thereof, e.g. heparosan; Purification or extraction methods thereof
- C08B37/0078—Degradation products
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
- C08L5/00—Compositions of polysaccharides or of their derivatives not provided for in groups C08L1/00 or C08L3/00
- C08L5/10—Heparin; Derivatives thereof
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Epidemiology (AREA)
- Polymers & Plastics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- General Chemical & Material Sciences (AREA)
- Heart & Thoracic Surgery (AREA)
- Cardiology (AREA)
- Materials Engineering (AREA)
- Dermatology (AREA)
- Biochemistry (AREA)
- Diabetes (AREA)
- Hematology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Polysaccharides And Polysaccharide Derivatives (AREA)
Abstract
The present invention relates to a kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition.Particularly, the polysaccharide mixture of heparin derivative of the present invention is the catabolite of heparin or heparin ester or heparin ester salt, and has following characteristic: weight average molecular weight is 3100 ~ 4500 dalton; The activity of anticoagulin Xa is high and activity that is anticoagulin IIa is low.The antithrombotic property of the polysaccharide mixture of heparin derivative of the present invention is excellent, and side effect is low, does not have the residual of the unfavorable composition such as organic base and dermatan sulfate.
Description
Technical field
The present invention relates to by the polysaccharide mixture of heparin derivative, and preparation method thereof with the pharmaceutical composition containing described polysaccharide mixture.
Background technology
Heparin is the sulfated polysaccharide mixture that animal (pig or cattle) is originated, because they have good antithrombus formation character, thus be widely applied, such as, prepare anti-thrombosis drug, the medicine of Cardiovarscular and the medicine etc. for the treatment of cerebrovascular disease.
But Natural heparin has many defects, the application of these drawbacks limit heparin.A major defect is that heparin medication too much can cause hematostaxis, therefore should measure clotting time before per injection.In addition, heparin also can cause allergic reaction, thrombocytopenia, osteoporosis and spontaneous fracture.
The danger of heparin sodium hematostaxis is very large, must monitor clotting time in Clinical practice, and this all brings very big inconvenience to doctor and patient.Meanwhile, the untoward reaction such as thrombocytopenia and osteoporosis also allows doctor in use have certain scruples.Therefore, numerous medical personal wish to find a kind of optimization method, have both retained the Effective Anti thrombosis of heparin sodium, can reduce the untoward reaction such as it is hemorrhage again.
Scientific research shows, the highly blood coagulation resistant activity (anticoagulin IIa is active) of heparin may cause bleeding, and reduces anti-IIa activity and can reduce bleeding risk.
The highly blood coagulation resistant factor IIa activity of heparin is relevant with the length of its strand and molecular size.Therefore, by the chemical degradation to heparin, can Low molecular heparin being obtained, as carried out alkaline bleach liquor degradation (or depolymerization) to heparin ester, obtaining Enoxaparin Sodium.Clinical trial shows, these Low molecular heparin reducing anticoagulin IIa activity greatly reduce the defect such as hemorrhage of heparin.But it is active that these products also have comparatively highly blood coagulation resistant factor IIa, therefore, still has certain hemorrhage equivalent risk.
Disclose a kind of low molecular weight heparin mixture in Chinese patent CN01814187, its mean molecule quantity is 1500 ~ 3000 dalton.But the mixture of the document contains the component of (at least about 35%) molecular weight in a large number too low (as being less than 1700 dalton) thus nonreactive thrombin xa activity.In addition, the document discloses a kind of preparation method, which includes the step of degrading with benzethonium chloride secondary salify and carrying out with guanidine class organic base.But secondary salify can increase cost, and guanidine class organic base easily residues in finished product, works the mischief.
Therefore, this area had both had excellent antithrombus formation character in the urgent need to exploitation, and the heparin product of side effect little (as bleeding risk is low).
Summary of the invention
Object of the present invention is just to provide one and has excellent antithrombus formation character, and the heparin product of side effect little (as bleeding risk is low).
In a first aspect of the present invention, provide a kind of polysaccharide mixture of heparin derivative, described mixture is the catabolite of heparin or heparin ester or heparin ester salt, and described mixture has following characteristic:
-weight average molecular weight is 3100 ~ 4500 dalton;
The activity of-anticoagulin Xa is 100-150IU/ milligram, and the activity of anticoagulin IIa is 0.001-10IU/ milligram, and ratio >=10 of anticoagulin xa activity and anticoagulin IIa activity;
In-formation polysaccharide in described mixture, there are in its one end undersaturated 4,5-glucuronic acid 2-0-sulfuric ester construction units; With
-described polysaccharide mixture is alkali metal salt or alkaline earth metal salt.
In another preference, the anticoagulin xa activity of described polysaccharide mixture is 125-150IU/ milligram.
In another preference, weight average molecular weight is 3200 ~ 4000 dalton.
In another preference, described mixture is sodium salt, potassium salt, calcium salt or magnesium salt form.
In another preference, the anticoagulin IIa activity of described mixture is 0.01-5IU/ milligram.
In another preference, the anticoagulin xa activity of described mixture and ratio >=20 of anticoagulin IIa activity, preferably >=25, more preferably >=30.
In another preference, by following formula, content Y≤20% of the polysaccharide of the daltonian heparin derivative in described mixture middle-molecular-weihydroxyethyl≤1700:
Preferably, described content Y≤15%; More preferably, described content Y≤10%.
In another preference, described heparin is the heparin deriving from cattle, pig, namely Hepar Sus domestica element or Hepar Bovis seu Bubali plain.
In a second aspect of the present invention, provide the preparation method of the polysaccharide mixture of the above-mentioned heparin derivative of the present invention, comprise step:
(a) in aqueous solvent, under pH10-14 (more preferably 11-13) and alkali metal hydroxide exist, degraded heparin benzyl ester alkali metal salt or alkali salt, thus the polysaccharide forming the heparin derivative of degraded; With
B () isolated or purified from reaction system goes out the polysaccharide mixture of heparin derivative of the present invention.
In another preference, described heparin benzyl ester alkali metal salt or the esterification degree of alkali salt are 60-99%, are more preferably 70 ~ 95%.
In another preference, described alkali metal hydroxide is potassium hydroxide or sodium hydroxide.
In another preference, in degradation step (a), the weight ratio of described alkali metal hydroxide and heparin benzyl ester alkali metal salt or alkali salt is 0.06 ~ 0.2, is more preferably 0.08 ~ 0.15.
In step (a), range of reaction temperature, at 60 DEG C ~ 90 DEG C, is more preferably 70 DEG C ~ 85 DEG C; Reaction time range, at 30 minutes ~ 3 hours, is more preferably 1 ~ 3 hour.
In another preference, also comprise in step (b) and desolventing technology is carried out to the polysaccharide mixture sodium borohydride of the heparin derivative described in obtained.
In a third aspect of the present invention, provide the purposes of the polysaccharide mixture of heparin derivative of the present invention, they are used to (a) and prepare anti-thrombosis drug; B () prepares the medicine of Cardiovarscular; Or the medicine of (c) preparation treatment cerebrovascular disease.
In a fourth aspect of the present invention, provide a kind of pharmaceutical composition, it contains polysaccharide mixture as the above-mentioned heparin derivative of the present invention of active component and pharmaceutically acceptable carrier.
Detailed description of the invention
The present inventor is through extensive and deep research, by improving the preparation method of heparin derivative polysaccharide mixture, adopt and degrade with inorganic base under given conditions, and with methanol, the polysaccharide derivative be in specific weight average molecular weight range is separated out, thus prepared one first there is excellent antithrombus formation character, and the heparin product of side effect little (as bleeding risk is low).The polysaccharide mixture of heparin derivative of the present invention has following major advantage: the content of (1) active substance is low; (2) activity of anticoagulin Xa high (being generally 100-150IU/ milligram) and the activity of anticoagulin IIa very low (being generally 0.001-10IU/ milligram), therefore the ratio of anticoagulin xa activity and anticoagulin IIa activity is much larger than 10 (as 20-200 or higher).
As used herein, term " mixture of the present invention " and " polysaccharide mixture of heparin derivative of the present invention " are used interchangeably.
The polysaccharide mixture of heparin derivative of the present invention can be adopted and prepare with the following method: in aqueous solvent (as the mixed solvent that water or water and alcohol (as methanol, ethanol) are formed), by sodium hydroxide, the heparin benzyl ester sodium salt of high esterification degree is degraded, thus form the heparin derivative polysaccharide of degraded, then carry out being separated and/or purification.
In a preference of the present invention, described preparation method comprises step:
1, heparin and quaternary ammonium salt (be good with Benzethonium salt, such as benzethonium chloride) reaction in atent solvent (as water) is formed salt, thus obtain heparin benzyl ethoxy ammonium salt.
The weight ratio of heparin and quaternary ammonium salt (as benzethonium chloride) is about 1:4, is good with 1:2.5 ~ 2.8.
Wherein, reaction temperature and time are not particularly limited, and usual reaction temperature can be 4 DEG C-60 DEG C, are preferably 10-50 DEG C; Response time is generally 5 minutes-24 hours.
2, in atent solvent (as dichloromethane), heparin ammonium salt (as heparin benzyl ethoxy ammonium salt) and benzyl chloride are reacted and forms heparin ester.The esterification degree of the heparin ester usually formed is 60% ~ 99%, preferably 75% ~ 95%.After formation heparin ester, can react with the methanol solution of acetic acid alkali metal salt (as sodium acetate) further, form heparin ester alkali metal salt.
Wherein, the temperature and time of esterification is not particularly limited, and usual reaction temperature can be 15-60 DEG C; Response time is generally 30 minutes-72 hours.A kind of particularly preferred heparin ester is heparin benzyl ester.
The reaction temperature and the time that form heparin ester alkali metal salt are not particularly limited, and usual reaction temperature can be 15-60 DEG C; Response time is generally 15 minutes-24 hours.A kind of particularly preferred heparin ester alkali metal salt is heparin benzyl ester sodium.
In one embodiment, for obtaining the esterification degree that scope is 60% ~ 99%, in the chlorine solvent of 3-5 part volume, about particularly advantageous with the benzyl chloride of 1 ~ 1.5 part of volume with every 1 part of weight heparin benzyl ethoxy ammonium salt, at temperature is 25 DEG C ~ 45 DEG C, be good with 30 DEG C ~ 40 DEG C, the response time continuing 24 ~ 48 hours carries out.After the completion of reaction, add 10% sodium acetate methanol solution of reaction volume 1 ~ 1.5 times of volume, obtain heparin benzyl ester sodium salt.
3, in aqueous solvent, under pH10-14 (more preferably pH11-13), with alkali metal hydroxide, heparin ester alkali metal salt is degraded, form the heparin derivative polysaccharide of degraded.
As used herein, term " aqueous solvent " comprises the mixed solvent that water and water and alcohol (as methanol, ethanol) are formed.Such as, alcohol content can be considered aqueous solvent lower than the aqueous solution of 5% (v/v).
A kind of preferred inorganic base is sodium hydroxide.
Usually, the weight ratio of alkali and ester is 0.06 ~ 0.2, is more preferably 0.08 ~ 0.15.
Temperature in degradation reaction is generally 50 DEG C ~ 90 DEG C, better with 60 DEG C ~ 85 DEG C, and reaction time range, at 30 minutes ~ 3 hours, was good with 1 ~ 3 hour.
In degradation reaction, water/ester weight ratio is generally 5 ~ 50:1, is preferably 10 ~ 30:1.
4, for obtained heparin derivative polysaccharide, abstraction and purification can be carried out by conventional method.
Polysaccharide mixture by heparin derivative of the present invention, they are compared with Natural heparin, have higher antithrombotic acitivity and lower side effect.In other words, the activity of anticoagulin X (factor Xa) is very high, and the activity of anticoagulin IIa (factor IIa) is very low.Such as, anticoagulin xa activity/anticoagulin IIa specific activity >=10 of inventive mixture, preferably >=20, more preferably >=25.
More specifically, polysaccharide mixture of the present invention has following characteristic:
Their weight average molecular weight is 3100-4500 dalton, and anticoagulin xa activity is 100-150IU/ milligram, and anticoagulin IIa activity is 0.001-10IU/ milligram, and anticoagulin xa activity/anticoagulin IIa specific activity is higher than 10;
In the formation polysaccharide of these mixture, there are in its one end undersaturated 4,5-glucuronic acid 2-0-sulfuric ester construction units;
These polysaccharide mixtures are alkali metal salt or alkaline earth metal salt.
In the present invention, as alkali metal salt or alkali salt, preferably sodium, potassium, calcium and magnesium salt.
Pharmaceutical composition and application process
The polysaccharide mixture of heparin derivative of the present invention can be used as antithrombotic agent, prevention venous thromboembolic disease (prevention intravenous thrombosis), particularly relevant with orthopaedics or general surgical operation thrombosis.Treat established venae profunda conducted, companion or without pulmonary infarction.Treatment unstable angina and non-Q ripple heart infarction, in hemodialysis extracorporeal circulation, prevent thrombosis.
In addition, the polysaccharide mixture of heparin derivative of the present invention also can be used for treatment or adjuvant therapy of cardiovascular disease and cerebrovascular disease.
Therefore, the present invention also provides a kind of pharmaceutical composition, and it contains polysaccharide mixture as the heparin derivative of the present invention of active component and pharmaceutically acceptable carrier.Usually, in pharmaceutical composition, the content of the polysaccharide mixture of heparin derivative of the present invention is 0.01-50wt%, is more preferably 0.1-30wt%.A kind of preferred medicine composition dosage form is injection (comprising solution and lyophilized preparation).
Pharmaceutical composition using conventional procedures of the present invention is prepared and is used.A kind of preferred application process is subcutaneous or intravenous administration.
Major advantage of the present invention is:
(1) component content without antithrombotic acitivity low (usually can be less than 10%).
(2) activity of anticoagulin Xa high (being generally 100-150IU/ milligram) and the activity of anticoagulin IIa very low (being generally 0.001-10IU/ milligram), the ratio of anticoagulin xa activity and anticoagulin IIa activity is much larger than 10 (as 20-200 or higher), therefore antithrombotic property is excellent, and side effect is low.
(3) preparation method of the present invention is without secondary salt-forming steps, and adopts inorganic base, therefore cost is low, can not produce organic base and remain.
(4) prepare in the process of Low molecular heparin mixture of the present invention, dermatan sulfate (more than molecular weight about 20000 dalton) can not degrade, therefore be easy in follow-up isolated or purified step with weight average molecular weight only the daltonian heparin derivative polysaccharide of 3100-4500 separate, therefore in mixture of the present invention not containing or be substantially free of the non-heparin compositions such as dermatan sulfate.
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually conveniently condition (condition as described in " European Pharmacopoeia "), or according to the condition that manufacturer advises.
General assay method
(1) degree of esterification of heparin ester or heparin ester salt can measure with the HPLC of routine, namely determines degree of esterification by being determined at the benzyl alcohol amount produced by the saponification of ester at 0 DEG C.
(2) molecular weight and molecualr weight distribution and anti-Xa and anti-IIa tire and all adopt the method for Low molecular heparin quality standard conventional in European Pharmacopoeia to carry out.
Embodiment 1
The preparation of heparin benzyl ethoxy ammonium salt
To in the aqueous solution (1250ml) of benzethonium chloride (270g), add water (1000ml) solution (note: the heparin in pig source) containing commercially available heparin sodium (100g).Stir lower placement 30 minutes, form heparin benzyl ethoxy ammonium salt precipitation.Then, reactant mixture is at room temperature filtered, obtain filter cake, wash final vacuum drying with water, obtain heparin benzyl ethoxy ammonium salt (about 310 grams).
Embodiment 2
The preparation of the polysaccharide mixture of heparin derivative
1, the preparation of heparin benzyl ester sodium salt
Benzyl chloride (15ml) is joined in heparin benzyl ethoxy ammonium salt (15g) dichloromethane (75ml) solution.Solution is heated to 35 DEG C, maintains 36 hours.Then add the sodium acetate methanol solution (90ml) of 10%, separate out heparin benzyl ester sodium salt precipitation.Reactant mixture is filtered, obtains filter cake, also dry with washed with methanol filter cake.Obtain the heparin benzyl ester (7.1g) (referred to as " heparin benzyl ester sodium salt ") existed with sodium-salt form thus.After measured, degree of esterification is 91%.
2, the polysaccharide mixture of heparin derivative
By obtained above with in the heparin benzyl ester (5g) water-soluble (125ml) of sodium-salt form existence, be heated to 75 DEG C.In this solution, add sodium hydroxide (0.45g), and holding temperature is at 75 DEG C, 1.5 hours, thus makes depolymerized heparin.Then reactant mixture is cooled to about 20 DEG C, adds hydrochloric acid and neutralize.Adding sodium chloride again makes the sodium chloride concentration in reactant mixture be 10% (w/v).In reactant mixture, add methanol (375ml), separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, obtains the polysaccharide mixture (light yellow, 3.9g) of heparin derivative.
3, purification
The polysaccharide mixture of heparin derivative previous step prepared is soluble in water, forms the aqueous solution of concentration about 10%.PH to 8.4 ± 0.2 is regulated with 1MNaOH.Sodium borohydride (polysaccharide mixture by 15g/kg heparin derivative) is joined in this solution, leave standstill 1 hour, then with 3M hydrochloric acid by pH regulator to 4.0-4.4 to eliminate too much boron hydride.Leave standstill 30 minutes, and regulate pH to 7.0 with 2MNaOH.Then add the methanol of liquor capacity 3 times of volumes, separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, thus the polysaccharide mixture (white, 3.7g) obtaining the heparin derivative of purification.
After measured, the character of the polysaccharide mixture of described heparin derivative is as follows:
| Weight average molecular weight | 3,800 dalton |
| Molecular weight is less than 1700 daltonian constituent contents (Y) | 9％ |
| Anticoagulin xa activity | 146IU/ milligram |
| Anticoagulin IIa is active | 9.6IU/ milligram |
| Anticoagulin xa activity/anticoagulin IIa specific activity | 15.2 |
In addition, in described polysaccharide mixture, the residual quantity of methanol and dichloromethane does not measure, and shows that the safety of this polysaccharide mixture is good.
Measure through conventional method, in the formation polysaccharide in described mixture, there are in its one end undersaturated 4,5-glucuronic acid 2-0-sulfuric ester construction units.
Embodiment 3
The preparation of the polysaccharide mixture of heparin derivative
1, the preparation of heparin benzyl ester sodium salt
Benzyl chloride (15ml) is joined in heparin benzyl ethoxy ammonium salt (15g) dichloromethane (75ml) solution.Solution is heated to 35 DEG C, maintains 48 hours.Then add the sodium acetate methanol solution (90ml) of 10%, separate out heparin benzyl ester sodium salt precipitation.Reactant mixture is filtered, obtains filter cake, also dry with washed with methanol filter cake.Obtain the heparin benzyl ester (7.4g) existed with sodium-salt form thus.After measured, degree of esterification is 97%.
2, the polysaccharide mixture of heparin derivative
By obtained above with in the heparin benzyl ester (5g) water-soluble (125ml) of sodium-salt form existence, be heated to 75 DEG C.In this solution, add sodium hydroxide (0.5g), and holding temperature is at 75 DEG C, 1.5 hours, thus makes depolymerized heparin.Then reactant mixture is cooled to about 20 DEG C, adds hydrochloric acid and neutralize.Adding sodium chloride again makes the sodium chloride concentration in reactant mixture be 10% (w/v).In reactant mixture, add methanol (375ml), separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, obtains the polysaccharide mixture (light yellow, 4.0g) of heparin derivative.
3, purification
The polysaccharide mixture of heparin derivative previous step prepared is soluble in water, forms the aqueous solution of concentration about 10%.PH to 8.4 ± 0.2 is regulated with 1MNaOH.Sodium borohydride (polysaccharide mixture by 10g/kg heparin derivative) is joined in this solution, leave standstill 1 hour, then with 3M hydrochloric acid by pH regulator to 4.0-4.4 to eliminate too much boron hydride.Leave standstill 30 minutes, and regulate pH to 7.0 with 2MNaOH.Then add the methanol of liquor capacity 3 times of volumes, separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, thus the polysaccharide mixture (white, 3.8g) obtaining the heparin derivative of purification.
After measured, the character of the polysaccharide mixture of described heparin derivative is as follows:
| Weight average molecular weight | 3,100 dalton |
| Molecular weight is less than 1700 daltonian constituent contents (Y) | 18％ |
| Anticoagulin xa activity | 106IU/ milligram |
| Anticoagulin IIa is active | 2.9IU/ milligram |
| Anticoagulin xa activity/anticoagulin IIa specific activity | 36.6 |
Measure through conventional method, in the formation polysaccharide in described mixture, there are in its one end undersaturated 4,5-glucuronic acid 2-0-sulfuric ester construction units.
Embodiment 4
The preparation of the polysaccharide mixture of heparin derivative
1, the preparation of heparin benzyl ester sodium salt
Benzyl chloride (15ml) is joined in heparin benzyl ethoxy ammonium salt (15g) dichloromethane (75ml) solution.Solution is heated to 37 DEG C, maintains 36 hours.Then add the sodium acetate methanol solution (90ml) of 10%, separate out heparin benzyl ester sodium salt precipitation.Reactant mixture is filtered, obtains filter cake, also dry with washed with methanol filter cake.Obtain the heparin benzyl ester (7.2g) existed with sodium-salt form thus.After measured, degree of esterification is 93%.
2, the polysaccharide mixture of heparin derivative
By obtained above with in the heparin benzyl ester (5g) water-soluble (125ml) of sodium-salt form existence, be heated to 75 DEG C.In this solution, add sodium hydroxide (0.45g), and holding temperature is at 75 DEG C, 1.5 hours, thus makes depolymerized heparin.Then reactant mixture is cooled to about 20 DEG C, adds hydrochloric acid and neutralize.Adding sodium chloride again makes the sodium chloride concentration in reactant mixture be 10% (w/v).In reactant mixture, add methanol (750ml), separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, obtains the polysaccharide mixture (light yellow, 3.9g) of heparin derivative.
3, purification
The polysaccharide mixture of heparin derivative previous step prepared is soluble in water, forms the aqueous solution of concentration about 10%.PH to 8.4 ± 0.2 is regulated with 1MNaOH.Sodium borohydride (polysaccharide mixture by 10g/kg heparin derivative) is joined in this solution, leave standstill 1 hour, then with 3M hydrochloric acid by pH regulator to 4.0-4.4 to eliminate too much boron hydride.Leave standstill 30 minutes, and regulate pH to 7.0 with 2MNaOH.Then add the methanol of liquor capacity 3 times of volumes, separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, thus the polysaccharide mixture (white, 3.6g) obtaining the heparin derivative of purification.
After measured, the character of the polysaccharide mixture of described heparin derivative is as follows:
| Weight average molecular weight | 3,600 dalton |
| Molecular weight is less than 1700 daltonian constituent contents (Y) | 13％ |
| Anticoagulin xa activity | 126IU/ milligram |
| Anticoagulin IIa is active | 5.3IU/ milligram |
| Anticoagulin xa activity/anticoagulin IIa specific activity | 23.8 |
Measure through hydrogen spectrometry, in the formation polysaccharide in described mixture, there are in its one end undersaturated 4,5-glucuronic acid 2-0-sulfuric ester construction units.
Embodiment 5
The preparation of the polysaccharide mixture of heparin derivative
1, the preparation of heparin benzyl ester sodium salt
Benzyl chloride (15ml) is joined in heparin benzyl ethoxy ammonium salt (15g) dichloromethane (75ml) solution.Solution is heated to 40 DEG C, maintains 24 hours.Then add the sodium acetate methanol solution (90ml) of 10%, separate out heparin benzyl ester sodium salt precipitation.Reactant mixture is filtered, obtains filter cake, also dry with washed with methanol filter cake.Obtain the heparin benzyl ester (7.3g) existed with sodium-salt form thus.After measured, degree of esterification is 93%.
2, the polysaccharide mixture of heparin derivative
By obtained above with in the heparin benzyl ester (5g) water-soluble (125ml) of sodium-salt form existence, be heated to 80 DEG C.In this solution, add sodium hydroxide (0.45g), and holding temperature is at 75 DEG C, 1.5 hours, thus makes depolymerized heparin.Then reactant mixture is cooled to about 20 DEG C, adds hydrochloric acid and neutralize.Adding sodium chloride again makes the sodium chloride concentration in reactant mixture be 10% (w/v).In reactant mixture, add methanol (750ml), separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, obtains the polysaccharide mixture (light yellow, 3.9g) of heparin derivative.
3, purification
The polysaccharide mixture of heparin derivative previous step prepared is soluble in water, forms the aqueous solution of concentration about 10%.PH to 8.4 ± 0.1 is regulated with 1MNaOH.Sodium borohydride (polysaccharide mixture by 12g/kg heparin derivative) is joined in this solution, leave standstill 1 hour, then with 3M hydrochloric acid by pH regulator to 4.0-4.4 to eliminate too much boron hydride.Leave standstill 30 minutes, and regulate pH to 7.0 with 2MNaOH.Then add the methanol of liquor capacity 3 times of volumes, separate out precipitation, filter and obtain filter cake, after methanol wash, vacuum drying, thus the polysaccharide mixture (white, 3.7g) obtaining the heparin derivative of purification.
After measured, the character of the polysaccharide mixture of described heparin derivative is as follows:
| Weight average molecular weight | 3,300 dalton |
| Molecular weight is less than 1700 daltonian constituent contents (Y) | 16％ |
| Anticoagulin xa activity | 116IU/ milligram |
| Anticoagulin IIa is active | 3.8IU/ milligram |
| Anticoagulin xa activity/anticoagulin IIa specific activity | 30.5 |
Embodiment 2-5 result shows, the polysaccharide mixture of heparin derivative of the present invention has excellent antithrombotic property, and side effect is low, and there is no the residual of the unfavorable composition such as organic base and dermatan sulfate.
Embodiment 6
Pharmaceutical composition
The preparation of injection (be equivalent to the polysaccharide mixture of 50mg heparin derivative /)
1. prescription
2. technique
The degradation product of 2.1 recipe quantities is dissolved in the water for injection of a certain amount of (about 400 milliliters);
2.2 are settled to 500ml with water for injection;
2.3 aseptic filtrations, fill,
2.4 quality inspection.
Embodiment 7
Pharmaceutical composition
The preparation of injection (be equivalent to the polysaccharide mixture of 30mg heparin derivative /)
1. prescription
2. technique
The degradation product of 2.1 recipe quantities is dissolved in the water for injection of a certain amount of (about 400 milliliters);
2.2 are settled to 500ml with water for injection;
2.3 aseptic filtrations, fill,
2.4 quality inspection.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (12)
1. a polysaccharide mixture for heparin derivative, described mixture is the catabolite of heparin or heparin ester or heparin ester salt, it is characterized in that, described mixture has following characteristic:
-weight average molecular weight is 3100 ~ 4500 dalton;
The activity of-anticoagulin Xa is 100-150IU/ milligram, and the activity of anticoagulin IIa is 0.001-10IU/ milligram, and ratio >=10 of anticoagulin xa activity and anticoagulin IIa activity;
In-formation polysaccharide in described mixture, there are in its one end undersaturated 4,5-glucuronic acid 2-O-sulfuric ester construction units; With
-described polysaccharide mixture is alkali metal salt;
And described polysaccharide mixture adopts following methods to prepare:
(a) in aqueous solvent, under pH10-14 and alkali metal hydroxide exist, degraded heparin benzyl ester alkali metal salt, thus form the polysaccharide of the heparin derivative of degraded; With
B () isolated or purified from reaction system goes out the polysaccharide mixture of described heparin derivative,
Wherein, in step (a), the weight ratio of described alkali metal hydroxide and heparin benzyl ester alkali metal salt is 0.06 ~ 0.2; Reaction temperature is 60 DEG C ~ 90 DEG C; Response time is 30 minutes ~ 3 hours; The esterification degree of described heparin benzyl ester alkali metal salt is 60-99%;
Wherein said aqueous solvent is the mixed solvent that water or water and alcohol are formed, and wherein alcohol content is lower than 5% (v/v), and described alcohol is methanol or ethanol;
And also comprise in step (b): desolventing technology is carried out to the polysaccharide mixture sodium borohydride of the heparin derivative described in obtained.
2. polysaccharide mixture as claimed in claim 1, it is characterized in that, the anticoagulin xa activity of described polysaccharide mixture is 125-150IU/ milligram, and/or weight average molecular weight is 3200 ~ 4000 dalton.
3. polysaccharide mixture as claimed in claim 1, it is characterized in that, described mixture is sodium salt or potassium salt form.
4. polysaccharide mixture as claimed in claim 1, it is characterized in that, the anticoagulin IIa activity of described mixture is 0.01-5IU/ milligram.
5. polysaccharide mixture as claimed in claim 1, is characterized in that, the anticoagulin xa activity of described mixture and ratio >=20 of anticoagulin IIa activity.
6. polysaccharide mixture as claimed in claim 1, is characterized in that, the anticoagulin xa activity of described mixture and ratio >=25 of anticoagulin IIa activity.
7. polysaccharide mixture as claimed in claim 1, is characterized in that, the anticoagulin xa activity of described mixture and ratio >=30 of anticoagulin IIa activity.
8. polysaccharide mixture as claimed in claim 1, is characterized in that, by following formula, and content Y≤20% of the polysaccharide of the daltonian heparin derivative in described mixture middle-molecular-weihydroxyethyl≤1700:
9. a preparation method for the polysaccharide mixture of heparin derivative according to claim 1, is characterized in that, comprises step:
(a) in aqueous solvent, under pH10-14 and alkali metal hydroxide exist, degraded heparin benzyl ester alkali metal salt, thus form the polysaccharide of the heparin derivative of degraded; With
B () isolated or purified from reaction system goes out the polysaccharide mixture of heparin derivative according to claim 1,
Wherein, in step (a), the weight ratio of described alkali metal hydroxide and heparin benzyl ester alkali metal salt is 0.06 ~ 0.2; Reaction temperature is 60 DEG C ~ 90 DEG C; Response time is 30 minutes ~ 3 hours; The esterification degree of described heparin benzyl ester alkali metal salt is 60-99%;
Wherein said aqueous solvent is the mixed solvent that water or water and alcohol are formed, and wherein alcohol content is lower than 5% (v/v), and described alcohol is methanol or ethanol;
And also comprise in step (b): desolventing technology is carried out to the polysaccharide mixture sodium borohydride of the heparin derivative described in obtained.
10. method as claimed in claim 9, it is characterized in that, the esterification degree of described heparin benzyl ester alkali metal salt is 70 ~ 95%.
The purposes of the polysaccharide mixture of 11. heparin derivatives as claimed in claim 1, is characterized in that, prepare anti-thrombosis drug for (a); B () prepares the medicine of Cardiovarscular; Or the medicine of (c) preparation treatment cerebrovascular disease.
12. 1 kinds of pharmaceutical compositions, is characterized in that, it contains polysaccharide mixture as the heparin derivative according to claim 1 of active component and pharmaceutically acceptable carrier.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810200817.8A CN101711771B (en) | 2008-10-07 | 2008-10-07 | A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition |
| PCT/CN2009/074330 WO2010040317A1 (en) | 2008-10-07 | 2009-09-30 | Mixture of oligosaccharides of heparin derivatives and its preparation and medicament |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200810200817.8A CN101711771B (en) | 2008-10-07 | 2008-10-07 | A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101711771A CN101711771A (en) | 2010-05-26 |
| CN101711771B true CN101711771B (en) | 2015-12-09 |
Family
ID=42100221
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200810200817.8A Active CN101711771B (en) | 2008-10-07 | 2008-10-07 | A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition |
Country Status (2)
| Country | Link |
|---|---|
| CN (1) | CN101711771B (en) |
| WO (1) | WO2010040317A1 (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399379B (en) * | 2010-09-09 | 2016-08-31 | 上海喜恩医药科技发展有限公司 | Polysaccharide mixture of a kind of heparin derivative and preparation method thereof and pharmaceutical composition |
| CN103012620B (en) * | 2011-09-26 | 2017-09-19 | 徐美英 | A kind of High-purity heparin benzyl ester salt and its preparation method and application |
| CN103012621B (en) * | 2011-09-26 | 2017-09-19 | 徐美英 | A kind of High-purity heparin benzyl ester salt and its preparation method and application |
| CN103175925B (en) * | 2013-03-20 | 2014-12-03 | 山东辰中生物制药有限公司 | Method for detecting esterification rate of heparin benzyl ester in production process of enoxaparin sodium |
| CN108503721A (en) * | 2018-03-28 | 2018-09-07 | 美瑞多生物科技（天津）有限公司 | With anticoagulant functions active polysaccharide and its preparation and application |
| CN109762079B (en) * | 2019-01-15 | 2021-04-27 | 湖北亿诺瑞生物制药有限公司 | Method for separating and purifying sulodexide bulk drug from heparin by-product |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1070503A1 (en) * | 1999-07-23 | 2001-01-24 | Laboratorios Farmaceuticos Rovi, S.A. | Compositions comprising very low molecular weight Heparin |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2548672A1 (en) * | 1983-07-04 | 1985-01-11 | Pharmuka Lab | SULPHATE OLIGOSACCHARIDES AND THEIR USE AS MEDICAMENTS |
| FR2857971B1 (en) * | 2003-07-24 | 2005-08-26 | Aventis Pharma Sa | MIXTURES OF HEPARIN DERIVED OLIGOSACCHARIDES, THEIR PREPARATION AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THEM |
| JP2009538386A (en) * | 2006-05-25 | 2009-11-05 | モメンタ ファーマシューティカルズ インコーポレイテッド | Low molecular weight heparin compositions and uses thereof |
-
2008
- 2008-10-07 CN CN200810200817.8A patent/CN101711771B/en active Active
-
2009
- 2009-09-30 WO PCT/CN2009/074330 patent/WO2010040317A1/en active Application Filing
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1070503A1 (en) * | 1999-07-23 | 2001-01-24 | Laboratorios Farmaceuticos Rovi, S.A. | Compositions comprising very low molecular weight Heparin |
Non-Patent Citations (2)
| Title |
|---|
| 《基层医院如何使用低分子肝素钙》;张淑敏;《中国社区医师》;20080310;第24卷(第7期);第20页 * |
| 《肝素降解和低分子量肝素的制备》;张万忠等;《中国生化药物杂志Chinese Journal of Biochemical Pharmaceutics》;20011231;第22卷(第1期);第2.1.2节 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101711771A (en) | 2010-05-26 |
| WO2010040317A1 (en) | 2010-04-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101711771B (en) | A kind of polysaccharide mixture of heparin derivative and method for making thereof and pharmaceutical composition | |
| US7812007B2 (en) | Compositions of polysaccharides derived from heparin, their preparation and pharmaceutical compositions containing them | |
| CA1327968C (en) | Low molecular weight heparins, with regular structure, preparation thereof and biological applications thereof | |
| EP2794666B1 (en) | Use of chemically modified heparin derivates in sickle cell disease | |
| EP1070503B1 (en) | Compositions comprising low molecular weight Heparin | |
| AU2012354229B2 (en) | Non anti-coagulative glycosaminoglycans comprising repeating disaccharide unit and their medical use | |
| JP2006291028A (en) | Low-molecular heparin or salt thereof, and manufacturing method thereof | |
| CN102711463B (en) | Hypersulfated disaccharide formulations | |
| EP3010940B1 (en) | New processes for the production of chemically-modified heparins | |
| CN102399306B (en) | A kind of preparation method of polysaccharide mixture of heparin derivative | |
| CN102952202A (en) | Preparation method and use of ultralow-molecular weight heparin sodium | |
| EP3569608A1 (en) | Oligosaccharide compound for inhibiting endogenous coagulation factor x-enzyme complex, and preparation method therefor and uses thereof | |
| CN102399379B (en) | Polysaccharide mixture of a kind of heparin derivative and preparation method thereof and pharmaceutical composition | |
| CN103012620B (en) | A kind of High-purity heparin benzyl ester salt and its preparation method and application | |
| CN103012621B (en) | A kind of High-purity heparin benzyl ester salt and its preparation method and application | |
| WO2013044793A1 (en) | High-purity heparin benzyl ester salt, preparation method therefor and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Free format text: FORMER OWNER: SHANGHAI INSTITUTE OF PHARMACEUTICAL INDUSTRY Effective date: 20130909 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20130909 Address after: 200040 room 703, building 1320, Beijing West Road, Shanghai, China Applicant after: Shanghai Seanpharm Co., Ltd. Address before: 200040 room 703, building 1320, Beijing West Road, Shanghai, China Applicant before: Shanghai Seanpharm Co., Ltd. Applicant before: Shanghai Institute of pharmaceutical industry |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |