RU2377993C2 - Method for making low-molecular heparin - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention concerns medicine, more exactly to drug technology intended for treatment of thrombotic conditions. There is offered method for making low-molecular heparin with using enzyme depolymerisation, characterised by adding dry lysozyme to 1% heparin in 0.1 M NaCl in the weight relation 1:100, mixing at 50°C within 3 hours, desalting with sephadex column and lyophilised.

EFFECT: invention ensures production of low-molecular heparin with using ovalbumin lysozyme enzyme.

3 ex, 2 tbl

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, thromboses of various localization).

In the last decade, low molecular weight heparins (NMH) with a narrower molecular weight distribution of 4-6 kDa have begun to displace unfractionated heparin (UFH) 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.

Previously, the authors have shown the possibility of obtaining LMWH under the action of hydrolytic enzymes that have never been used for this purpose, such as papani, chymotrypsin, “protease C”, or celloviridine [1].

The purpose of the invention is the production of LMWH using the enzyme lysozyme egg white. This goal is achieved by the fact that the enzyme lysozyme is used for hydrolysis. It is easily available, cheap. Previously, no one received LMWH with lysozyme.

Lysozyme - muraminidase, an enzyme of the hydrolase class is able to destroy the wall of a bacterial cell, as a result of which it dissolves (lysis). It acts on one of the main components of a bacterial cell - a complex polysaccharide consisting of two types of amino sugars [2]. The use of lysozyme for the hydrolysis of chitosan is known [3].

The closest in technical essence is the method of obtaining NMH by partial depolymerization under the action of immobilized hydrolases [1].

The essence of the proposed method is that in the case of using lysozyme, the enzyme was added to a solution of heparin in 0.1 M NaCl in a ratio of 1: 100 and the solution was thermostated at a temperature of 50 ° C for 3 hours, desalted on a column with Sephadex G-10. Freeze-dried. With a yield of about 70-80%, heparin was obtained with a molecular weight of 40-50% lower than that of the starting heparin.

Lysozyme, in the form of a solution for the purpose of depolymerization of heparin, was used by the authors for the first time. It was found that the anti-Xa activity of the target product is 139 ± 8 U / mg, which corresponds to the specific activity of an LMWH preparation such as fraxiparin 120-160 U / mg.

The authors investigated the effect of low molecular weight heparin (NMHy) with a molecular weight of 6.8 kDa obtained by hydrolysis of unfractionated heparin (UFH Belmedpreparaty OJSC, molecular weight 13.8 kDa) with lysozyme in solution on the change in the coagulation time of human blood plasma in the activated partial thromboplastin time (aPTT, effect on the internal blood coagulation pathway) and ReaClot NPO Renam (effect on factor Xa activity) [4]. Lyophilically dried human plasma was purchased at the Renam NGO. To calculate specific antithrombin (antifactor IIa, aIIa) and antifactor Xa (aXa) activities, the calibration curve of the 1st International Standard for Low Molecular Weight Heparin (NIBSC) was used.

The possibility of carrying out the claimed invention is shown by the following examples.

Example 1. To 10 ml of a 1% solution of heparin (molecular weight 13.8 kDa) in 0.1 M NaCl was added 1 mg of lysozyme. The weight ratio of enzyme to sample of heparin is 1: 100. The suspension was stirred at 50 ° C for 3 h and desalted on a Sephadex G-10 column (2.5 × 42 cm), eluent water, elution rate 80 ml / h, freeze-dried. Received 70 mg (70% of the original) with a molecular weight of 6.8 kDa (NMR).

Example 2. To a mixture of 0.06 ml of plasma and 0.06 ml of aPTT reagent (NPO Renam) was added 0.05 ml of solutions of UFH or NMHr at final concentrations of 0.14-2.20 μg / ml. After 3 minutes of incubation at 37 ° C, 0.06 ml of a 0.025 M CaCl ₂ solution was added and the time (sec) for the appearance of the fibrin clot was recorded. A significant elongation of the clotting time of plasma with increasing concentration of samples was noted (Table 1). Antithrombin activity (aIIa) of UFH and NMHr when compared with the standard was 128 ± 3 U / mg and 120 ± 5 U / mg, respectively.

Example 3. To a mixture of 0.099 ml of plasma and 0.025 ml of a solution of factor Xa (NPO Renam) was added 0.01 ml of solutions of UFH or NMHr at final concentrations of 0.07-0.47 μg / ml. After 2 min incubation at 37 ° C, 0.06 ml of a 0.035 M solution was added

CaCl ₂ and recorded the time (sec) the appearance of a fibrin clot. A significant elongation of the clotting time of plasma with an increase in the concentration of samples was noted (Table 2). The activity against factor Xa (aXa) for UFH and NMHG when compared with the standard was 117 ± 10 U / mg and 139 ± 8 U / mg.

Table 1 Plasma clot appearance time (sec) in the activated partial thromboplastin time test (aPTT) cipher Final concentration, mcg / ml 0 0.22 0.27 0.3 0.36 0.43 0.54 0.72 1,1 2.2 UFG 30.0 ± 1.8 32.3 ± 2.7 33.9 ± 2.5 36.1 ± 3.0 * 37.2 ± 3.3 * 38 ± 4.0 * 43.4 ± 3.8 ** 51.4 ± 4.4 ** 69.5 ± 6.7 *** 114.7 ± 12.3 *** NMHr 31.7 ± 1.5 32.8 ± 3.7 35.8 ± 2.8 36.7 ± 3.5 * 37.5 ± 3.5 * 42.6 ± 3.9 ** 49.9 ± 4.1 ** 68.7 ± 5.9 *** 110 ± 9.9 *** * - p <0.05, ** - p <0.01 and *** - p <0.001 significance of differences with indications at a final heparin concentration of 0 μg / ml

table 2 Plasma clot appearance time (sec) in ReaClot test cipher Final concentration, mcg / ml 0 0,07 0.14 0.18 0.23 0.28 0.35 0.47 UFG 19.4 ± 1.3 20.0 ± 2.3 20.2 ± 2.1 20.8 ± 1.7 22.1 ± 1.9 22.5 ± 2.0 28.8 ± 2.4 ** 37.9 ± 2.8 *** NMHr 20.9 ± 1.5 21.8 ± 1.9 24.5 ± 2.1 * 26.1 ± 2.3 ** 27.2 ± 1.9 ** 33.6 ± 2.5 *** 43.8 ± 3.1 ** * - p <0.05, ** - p <0.01 and *** - p <0.001 significance of differences with indications at a final heparin concentration of 0 μg / ml

Thus, upon depolymerization of unfractionated heparin (activity ratio aXa / aIIa = 0.9) with lysozyme in solution, we obtain heparin with a molecular weight lower in comparison with the initial UFH. The ratio of the activity of aXa / aIIa increases by 1.2 times.

Bibliography

1. G.E. Bannikova, V.P. Varlamov, N.N. Drozd, V.A. Makarov, V.E. Tikhonov, K. G. Skryabin, RF patent No. 2295538 dated March 20, 2007, Bull . Open., No. 5 (2007).

2. Bernhard S., Structure and function of enzymes. M., 1971.

3. Ilyina A.V., Varlamov V.P. Enzymatic depolymerization of N-succinylchitosan. Bioorg.Chem. 2007; 33 (1): 156-9.

4. Bates SM, Weitz JI // Circulation. 2005. V 112. N 4. P 53-60.

Claims (1)

A method of producing low molecular weight heparin by enzymatic depolymerization, characterized in that lysozyme is added in dry form to a 1% solution of heparin in 0.1 M NaCl in a weight ratio of 1: 100, stirred at 50 ° C for 3 hours, desalted column with Sephadex and freeze-dried.