RU2295538C2 - Method for preparing heparins of low molecular mass - Google Patents

Abstract

FIELD: medicine, biochemical technology.

SUBSTANCE: invention proposes a method for preparing low-molecular heparins using enzymatic cleavage. For enzymatic depolymerization the method involves using papain, chymotrypsin or complexes of hydrolases as components of preparations "Protease C" and celloviridine immobilized on silochrome or polyvinyl alcohol. Method provides preparing low-molecular heparins by effect of immobilized hydrolases with the yield of the end product about 70-80%.

EFFECT: improved preparing method.

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Description

The invention relates to medicine and can be used to create an effective means of prevention and treatment of thrombotic conditions (heart attacks, strokes of various localization).

In the last decade, low molecular weight heparins (LMWH) with a narrower molecular weight distribution of 4-6 kDa have begun to displace unfractionated heparin as a drug. The main advantage of LMWH follows from their pharmacokinetic properties: a 2-4 times longer half-life from the body, noticeably better bioavailability with subcutaneous administration and a more stable dose response (the dose can be calculated only based on the patient’s weight, without additional laboratory tests). A smaller toxic effect is associated with thrombocytopenia caused by unfractionated heparin, which in turn is due to a lesser interaction of LMWH with platelets.

There are various methods for producing LMWH with a low yield by fractionation of a natural preparation or with a high yield by controlled depolymerization [1]. It is possible to obtain LMWH by partial depolymerization of heparin α-glycosidic bonds using heparinase. The enzyme used in this process is produced by Flavobacterium heparinum [2]. Obtaining LMWH in an enzymatic manner that minimally affects the structure of the heparin molecule, Logiparin is obtained on the basis of this process [1].

We investigated the possibility of obtaining NMH under the influence of hydrolytic enzymes that have never been used for this purpose. The purpose of the invention is the obtaining of NMH by the action of immobilized hydrolases.

This goal is achieved by the fact that for hydrolysis, papain, chymotrypsin, "prosthesis C" or celloviridine are used.

The hydrolysis of such a polysaccharide as chitosan has been described by the action of various hydrolases: hemicellulases, lysozyme, papain, lipase, glucanase, protease, and other enzymes [3, 4]. We hypothesized that heparin might also be exposed to the nonspecific effects of hydrolytic enzymes. We have chosen proteolytic enzyme preparations such as Protease C, papain and chymotrypsin, as well as the enzyme preparation celoviridin — an industrial enzyme preparation of hydrolases based on the Trichoderma viride strain. Celloviridin G20X contains cellulases, xylonases, β-glucanases and hemicellulases, an acidic protease, etc. The drug is active in the pH range from 3 to 6 at temperatures of 30-55 ° C and is used in veterinary medicine as a feed additive. The drug "Protease C" is a mixture of exoproteinases. The composition of the drug includes: serine proteinase I, serine proteinase II, neutral proteinase, arboxypeptidase, aminopeptidase. The drug is active in the pH range from 5 to 10.5 at temperatures of 30-50 ° C and is a medical substance based on the Acremonium chrysogenum strain, intended for the manufacture on its basis of drugs used in various fields of medicine, as well as in the food industry. Papain is a thiol cysteine endoprotease from subclass KF 3.4.22, which has a cistern in the active center of the SH group. By the nature of the enzymatic action, it is called "plant pepsin." But unlike pepsin, papain is active not only in acidic, but also in neutral and alkaline media (pH range 3-12, optimum pH 5). It retains activity over a wide temperature range. Papain has a wide specificity and remains active in a wide temperature range. To exhibit maximum activity during its use, preliminary activation with mild reducing agents is necessary, for example, the addition of 5 mM L-cysteine and 2 mM sodium ethylene diamine tetraacetate. The pathway of the papain-catalyzed reaction is similar to that for chymotrypsin, but in the case of papain, a thioacyl enzyme is formed as an intermediate. Papain has less specificity than chymotrypsin.

Papain is used in an immobilized state for the hydrolysis of chitosan in order to obtain oligomers [5]. Crystalline chymotrypsin is a proteolytic enzyme preparation obtained from the pancreas of cattle. In some cases, chymotrypsin produces deeper protein hydrolysis than trypsin. Trypsin is more resistant, and slower inactivation. Molecular mass from 21600 to 27000 Yes, pH optimum 7.0-9.0, temperature optimum 50 ° C, pI 8.1-8.2.

The closest in technical essence is the method of obtaining NMH by partial depolymerization of heparin α-glycosidic bonds using heparinase. The enzyme used in this process is produced by Flavobacterium heparinum [6, 7]. Obtaining LMWH by enzymatic means minimally affecting the structure of the molecule.

We have proposed a method for producing low molecular weight heparin by enzymatic depolymerization, characterized in that papain, chymotrypsin or complexes of hydrolases contained in the prosthetics C and celloviridine preparations are immobilized on silochrome or polyvinyl alcohol, and enzymatic depolimization is mixed with the enzyme heparin at 45-50 ° C for 1.5-3 hours, the immobilized preparation is separated by centrifugation, washed with 0.5 M sodium chloride solution and water, attached to the supernatant and desalted on a Sephadex and lyophilized. In the case of using immobilized papain, a solution of heparin in acetate buffer pH 6.0 was mixed with the immobilized enzyme at a temperature of 45 ° C for 1.5 hours. The immobilized enzyme was separated either on a glass porous filter or using a centrifuge, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column. Freeze-dried. With a yield of about 70-80%, heparins were obtained with a molecular weight of 40-65% lower than that of the starting heparin.

In the case of using immobilized chymotrypsin, a solution of heparin in acetone buffer pH 6.0 was mixed with the immobilized enzyme at a temperature of 45 ° C for 3 hours. The immobilized enzyme was separated either on a glass porous filter or using a centrifuge, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column. Freeze-dried. With a yield of about 70-80%, heparins with a molecular weight of 30-35% lower than the starting heparin were obtained.

In the case of the immobilized preparation "Protease C", a solution of heparin in phosphate buffer pH 8.0 was mixed with the immobilized enzyme at a temperature of 50 ° C for 3 hours. The immobilized enzyme was separated either on a glass porous filter or using a centrifuge, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column. Freeze-dried. With a yield of about 70-80%, heparins were obtained with a molecular weight 4 times lower than that of the original heparin.

In the case of using immobilized celloviridine, a solution of heparin in acetate buffer pH 4.5 was mixed with the immobilized enzyme at a temperature of 50 ° C for 3 hours. The immobilized enzyme was separated either on a glass porous filter or using a centrifuge, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column. Freeze-dried. With a yield of about 70-80%, heparins were obtained with a molecular weight of 40-60% lower than that of the starting heparin.

The immobilized enzymes papain, chymotrypsin, preparations of “protease C” and celloviridine for the purpose of heparin depolymerization were used for the first time. It was found that the anti-Xa activity of the target product is 160-290 units / mg, which corresponds to the specific activity of such an LMWH preparation as fraxiparin (160-280 units / mg).

The immobilization of papain, chymotrypsin, celloviridine and the preparation “protease C” on silochrome was carried out using glutaraldehyde.

To 1 g of aminosilochrom was added 8 ml of a 12.5% solution of glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2. Stirred for 30 min at room temperature. Then the sorbent was washed from excess glutaraldehyde with water and 0.1 M sodium phosphate buffer, pH 7.2, and an enzyme solution (10-15 ml) was added at a concentration of 1 mg / ml in the same buffer, stirred for 3 h at room temperature and left under cooling at 4 ° C for another 16 hours. Washed on a glass porous filter with water (200 ml), 1M NaCl (200 ml) and again with water. The amount of immobilized protein was determined by the difference in its content in the initial enzyme solution and in the wash solutions. Immobilized enzymes were stored in a refrigerator in water. In the case of papain, an enzyme stock solution was prepared in 0.1 M sodium phosphate buffer, pH 7.2, containing 5 mM L-tank and 2 mM EDTA.

The immobilization of papain, chymotrypsin, celloviridine and the “protease C” preparation on an organic sorbent polyvinyl alcohol containing aldehyde groups (150-200 μmol / g crude sorbent) was carried out according to the following procedure: to 4 g of raw (squeezed out on a glass porous filter) sorbent 55 ml of a solution of the enzyme in 0.1 M phosphate buffer with a pH of 7.1 with a protein concentration of 0 75 mg / ml was added, stirred for 3 hours at room temperature and left to cool at 4 ° C for another 16 hours. Washed on a glass porous filter water (200 ml), 1M NaCl (200 ml) and vn Bb water. The amount of immobilized protein was determined by the difference in its content in the initial enzyme solution and in the wash solutions. Blocking of unreacted aldehyde groups on the sorbent after covalent binding by the enzyme was carried out by treating with 0.1 M Tris-HCl buffer with a pH of 7.5 with stirring for 2 hours

Immobilized enzymes were stored in a refrigerator in water.

Example 1. To 6 ml of a 1% solution of heparin (porcine mucosa, molecular weight 7.5 kDa) in 0.05 M acetate buffer pH 4.5 was added 25 mg of papain immobilized on silochrome (protein content 8.5 mg / g). The weight ratio of the enzyme to the sample of heparin is 1: 1000. The suspension was stirred at 45 ° C for 1.5 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 42 mg (70% of the original) with a molecular weight of 4.7 kDa.

Example 2. To 6 ml of a 1% solution of heparin (from cattle, a molecular weight of 13.8 kDa) in 0.05 M acetate buffer pH 4.5 was added 0.055 g of papain immobilized on silochrome (protein content 11.8 mg / g ) The weight ratio of the enzyme to the sample of heparin is 1: 100. The suspension was stirred at 45 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 49.7 mg (82.8% of the original) with a molecular weight of 5.8 kDa.

Example 3. To 6 ml of a 1% solution of heparin (from cattle, molecular weight 13.8 kDa) in 0.05 M acetate buffer pH 4.5 was added 0.18 g of papain immobilized on PVA (protein content 3.3 mg / g). The weight ratio of the enzyme to the heparin sample is 1:50. The suspension was stirred at 45 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 50 mg (83% of the original) with a molecular weight of 6.1 kDa.

Example 4. To 6 ml of a 1% solution of heparin (from cattle, molecular weight 13.8 kDa) in 0.05 M phosphate buffer pH 8.0, 0.15 g of Protease C immobilized on PVA was added (protein content 8.0 mg / g). The weight ratio of the enzyme to the heparin sample is 1:50. The suspension was stirred at 50 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 247 mg (82% of the original) with a molecular weight of 3.4 kDa.

Example 5. To 6 ml of a 1% solution of heparin (from cattle, molecular weight 13.8 kDa) in 0.05 M phosphate buffer pH 6.0, 0.67 g of Protease C immobilized on silochrome was added (protein content 0.9 mg / g). The weight ratio of the enzyme to the sample of heparin is 1: 100. The suspension was stirred at 50 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 46 mg (77% of the original) with a molecular weight of 4 kDa.

Example 6. To 6 ml of a 1% solution of heparin (from cattle, a molecular weight of 13.8 kDa) in 0.05 M acetate buffer pH 4.5, 0.15 g of PVA immobilized celloviridine was added (protein content 9.4 mg / g). The weight ratio of the enzyme to the heparin sample is 1:50. The suspension was stirred at 50 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 52 mg (87% of the original) with a molecular weight of 5.8 kDa.

Example 7. To 6 ml of a 1% solution of heparin (from cattle, a molecular weight of 13.8 kDa) in 0.05 M acetate buffer, pH 4.5, 0.065 g of celloviridin immobilized on silochrome was added (protein content 9.3 mg / d). The weight ratio of the enzyme to the sample of heparin is 1: 100. The suspension was stirred at 50 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 46.8 mg (78% of the original) with a molecular weight of 6 kDa.

Example 8. To 6 ml of a 1% solution of heparin (porcine mucosa, molecular weight 75 kDa) in 0.05 M acetate buffer pH 4.5 was added 25 mg of chymotrypsin immobilized on silochrome (protein content 4.9 mg / g). The weight ratio of the enzyme to the heparin sample 200. The suspension is stirred at 45 ° C for 3 hours. The immobilized preparation was separated by centrifugation, washed with 0.5 M NaCl and water. Washings were attached to the supernatant and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 42 mg (70% of the original) with a molecular weight of 5.0 kDa.

Information sources

1. Linhardt R, Loganathan B, Al-Hakim A et al., J. Med. Chem., 33 (6), 1639-1645, 1990.

2. Linhardt R, Grant F, Cooney C et al., J. Biol. Chem., 257, 7310-7313, 1982.

3. Muzzarelli R.A.A., Chitin Handbook, Eds. Muzzatelli R.A.A., Peter M.G. European Chitin Society, Atec, Grottammare, Italy, 153-163, 1997.

4. Pantaleone D. Yalpani M., Scollas M., Carbohydrates and Carbohydrate Polymers, Analysis, Biotechnology, Modification, Antiviral, Biomedical and Other Aplications, Ed, Yalpani M. Shrewsbury (USA): ALT Press, 44-51, 1993.

5. Hong Lin, Haiying Wang, Changhu Xue, Mei Ye, Enzyme Microbial Technology, 31, 588-592, 2002.

6. Linhardt R, Grant F, Cooney C et al., J. Biol. Chem., 257, 7310-7313, 1982.

7. EP 0244235 b1, 11/04/1987.

Claims (1)

A method for producing low molecular weight heparin by enzymatic depolymerization, characterized in that papain, chymotrypsin or complexes of hydrolases contained in the preparations of “protease C” and celloviridine are immobilized on silochrome or polyvinyl alcohol and, when enzymatic depolymerization is carried out, the -50 ° C for 1.5-3 hours, the immunophilized preparation is separated by centrifugation, washed with 0.5 M sodium chloride solution and water, attached to the supernatant and desalted t on a column with Sephadex and freeze-dried.