RU2257224C1 - Method for preparing human plasmin concentrate and product for its using in medicine - Google Patents

Abstract

FIELD: medicine, biotechnology, pharmaceutical industry, veterinary science.

SUBSTANCE: method involves addition of tris-HCl buffer (pH 7.5) containing 0.1 M of lysine and 5 mM of EDTA to the Cohn's fraction followed by incubation and centrifugation. Prepared suspension is incubated with 10% PEG-3000 and after centrifugation the solution is applied onto column with lysine-Sepharose at pH 7.5 and plasminogen is eluted with buffer solution at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine and 5 mM of caprylic acid. Then plasminogen is incubated with streptokinase in tris-buffer (pH 7.5) containing 15 mM of lysine, 10 mM of ε-aminocaproic acid and 50% of glycerol. Then method involves chromatography on column with benzamidine-Sepharose at pH 8.5 and elution with buffer at pH 3.5 containing 0.1 M of glycine, 30 mM of lysine, and the end product is lyophilized that comprises about 30 IU/ml of plasmin with purity above 98%, at least 10 mM of lysine and 10 mM of glycine in an aqueous solution at pH about 3.5. The prepared product shows apyrogenic property, absence of toxicity in experiments in laboratory animals and it doesn't cause allergic or other adverse response reactions in intracutaneous or intravenous administrations, and the preparation doesn't cause defects in the vision field after its an intraorbital route of administration.

EFFECT: improved preparing method, valuable medicinal properties of plasmin.

7 cl, 1 ex

Description

Technical field

The invention relates to the field of biotechnology, pharmaceutical industry, veterinary medicine, and relates to the production of a concentrate of human plasmin and a product that can be used as a medicine for use in medicine, for example in ophthalmology or as a thrombolytic agent for coagulological disorders in various veins and arteries caliber and various localization. The product can also be used in ophthalmology as adjuvant therapy in the treatment of hemophthalmus of various origins, hyphema, reactive fibrinoid syndrome after cataract extraction and lensvitrectomy, involutional central chorioretinal dystrophy with hemorrhagic component, thrombosis of the central retinal vein and its branches of preretinosis, preretinosis with fibrinous-plastic iridocyclitis for the prevention of fistula overgrowth in antiglaucomatous operations fistulizing the second type.

State of the art

Plasmin is a serine protease that plays a key role in thrombolysis. It is the formation of plasmin that ultimately blocks all thrombolytic agents. Naturally, to dissolve blood clots, you can use the plasmin itself. Technically, the delivery of this enzyme to an occluded vessel can be accomplished by catheterizing either the proximal or distal main trunk (Novokhatny V.V. et al. US patent 6355243). Since the procedure for dissolving a blood clot is long, in addition to the high purity of the drug, it is necessary to create conditions in which plasmin is not inactivated during its duration. Plasmin can be quite competitive in use compared to a tissue plasminogen activator, one of the main “disadvantages” of which is its high cost. Other thrombolytic agents have a number of side effects and therefore are not real competitors for the tissue plasminogen or plasmin activator. However, there are a number of limitations in the use of traditional thrombolytic agents, which can be excluded in the case of direct use of plasmin (Novokhatny V.V. et al. US patent 6355243). So, in some cases, it is necessary to maintain a high local concentration of plasmin for a long time. The use of modern thrombolytics either does not allow this, or their use makes therapy very expensive. All this takes the plasmin drug to a competitive level. Naturally, in this case, the method for producing plasmin should produce a highly purified preparation, be technologically advanced and have a relatively low cost, and the final preparation should retain the necessary activity during its use.

The greatest storage stability of the drug is achieved due to its freeze drying. The stability of the drug during the thrombus dissolution procedure is achieved by lowering the pH of the solution of the used plasmin to 3.7-4.0 units (Novokhatny VV et al. US patent 6355243) and / or the addition of amino acids (Jensen VT US patent 3950513), primarily lysine or its derivatives (Aisina RB, et al. Bioorganic chemistry, 1994, 20, 182-189; Ueshima S. et al. Clin.Chim.Acta 1996, 245, 7-18).

If there is certainty in terms of stabilizing the final drug, then there is no such certainty regarding the methods for purification of plasmin, which could become the basis of the technology for its industrial production.

Any method for obtaining plasmin includes three main blocks: 1) obtaining plasminogen; 2) plasminogen activation (conversion of plasminogen to plasmin); 3) fine purification of plasmin. As additional stages, it is necessary to consider the stage of viral inactivation and the stage of stabilization of the final drug. Since, ultimately, it is a question of obtaining a highly purified preparation, only suitable teaching methods were analyzed. Blocks 1 and 3 are long and well established in world practice. The main attention is paid to the second block, which will determine the purity of the final preparation and the cost of the technology as a whole. The present invention is directed to the solution of this problem.

The plasmin preparation Fibrinolysin, produced in the USSR since the mid-70s, was a very poor quality product that caused a wide range of complications, since it was obtained from low-purity plasminogen by treatment with trypsin of animal origin. The final preparation was not only of low purification, but also unstabilized, therefore, in principle, could not solve the tasks that were set for it. The method of drug delivery was ineffective.

The closest in essence to the proposed invention is a method for producing plasmin, described Novokhatny V.V. et al. in US patent 6355243.

In the prototype method (Novokhatny V.V. et al. US patent 6355243) the preparation of plasmin consists in the fact that the plasminogen is purified starting from the Cohn fraction II + III. After solubilization of the paste, the insoluble portion is separated by centrifugation, and the resulting solution is mixed with lysine-sepharose. The plasminogen bound to the affinity carrier is eluted and then precipitated with ammonium sulfate. This precipitate is about 80% pure and serves as a source of plasmin.

Plasmin is obtained using urokinase immobilized on sepharose. This is the most expensive stage of purification, because, firstly, an expensive plasminogen activator is used, and secondly, despite immobilization, each subsequent cycle using this reagent gives less effect and therefore, as the authors of the patent themselves note, it requires constant monitoring and correction . After separating the immobilized urokinase and changing the buffer, the solution was applied to benzamidine-sepharose for selective purification of plasmin. After elution of plasmin and diafiltration of the resulting solution, the final preparation was obtained in acidified with acetic acid water with a pH of 3.7. The purity of the drug was more than 95%. The authors of the patent received about 200 g of the final preparation from 200 g of paste of the Cohn II + III fraction. Naturally, the low-tech and high cost of the second stage forces us to look for other solutions.

Disclosure of the invention.

The proposed invention includes a method for producing a plasmin concentrate, said concentrate comprising lysine and glycine, each in an amount of at least 10 mM, from human blood plasma, comprising isolating plasminogen from the Cohn II + III fraction, and activating it with any suitable agent known from prior art, viral inactivation using conventional methods (for example, using well-known viral inactivators - chemical agents or keeping at a certain temperature), removing its product chromatographic methods and fine purification using chromatography.

As a feedstock, the Cohn II-III fraction was used. For solubilization, 0.2 M Tris pH 7.5 buffer containing 0.1 M lysine and 5 mM EDTA was used. After two hours of incubation at + 4 ° C, the suspension was centrifuged at 6000 g for 1 hour.

PEG 3000 was used to precipitate impurity proteins, lipids, and lipid-associated viruses. After a two-hour incubation of the supernatant with 10% PEG at room temperature, the mixture was centrifuged at 6000 g for one hour. After replacing the buffer with 50 mM phosphate pH 7.5, containing 0.15 M sodium chloride, the solution was applied to a lysine-sepharose column. Plasminogen was eluted with a buffer solution, pH 3.5, containing 0.1 M glycine, 30 mM lysine and 5 mM caprylic acid. The proposed concentrations of glycine and lysine allow the drug to be stabilized as much as possible without losing its activity. The eluted protein was incubated for an hour at + 4 ° C, which corresponded to another stage of viral inactivation.

After changing the buffer to 20 mM Tris, pH 7.5, containing 15 mM lysine, 10 mM e-aminocaproic acid and 50% glycerol, streptokinase was added to the solution based on the molar ratio plasminogen: streptokinase = 100: 1. The mixture was incubated for 18 hours at + 4 ° C. The reaction was stopped by adding 0.1 M Tris, pH 8.5, containing 1 M sodium chloride.

For final purification of plasmin, the mixture was applied to a benzamidine-Sepharose column, equilibrated with 50 mM Tris buffer, pH 8.5, containing 0.5 M sodium chloride. Plasmin was eluted with a buffer containing 0.1 M glycine, 30 mM lysine, pH 3.5. Under these conditions, at + 4 ° C, the drug remains stable for a month. Lyophilization of the final plasmin preparation prolongs its stability.

The purity of the final preparation is at least 98%. The yield of the active drug is about 40%. Also, the present invention includes a product obtained by this method, which is a concentrate containing plasmin with a purity of more than 98%, at least 10 mM lysine and at least 10 mM glycine, with a pH of about 3.5. This pharmaceutical product can be used in medicine, veterinary medicine and / or for research purposes. The product may be a solution, lyophilized powder (for which the above concentrate is transferred into vials and dried by freezing in vacuo), it may additionally contain traditional pharmaceutical excipients, carriers, solvents and / or excipients suitable for parenteral (e.g., intravenous, intramuscular , intraarticular) introduction. The listed excipients are mixed with a dry concentrate or added to the solution according to conventional methods.

Excipients are, for example, components (inorganic and / or organic salts) necessary to maintain a pH of about 3.5, if necessary also salts or other well-known substances that regulate the osmotic pressure, optionally antioxidants, traditional for injectable pharmaceutical compositions or for ophthalmic drops or gels.

The solvents may be distilled pyrogen-free sterile water, physiological saline, buffered saline, a solution of 1,3-butanediol, glycosaminoglycans, dextrose, and the solution may also contain, if necessary, blood serum proteins, for example, albumin and / or ceruloplasmin (the latter may also to be excipients in the lyophilized finished product).

You can also lyophilize a ready-made solution containing a concentrate of plasmin, glycine and lysine and any of the listed auxiliary substances or their combinations.

If necessary, the concentrate containing plasmin, glycine and lysine according to this invention can be dissolved before administration in the blood plasma, in the blood serum of the patient or mixed with solutions and / or fractions of protein (proteins).

The product can also be made in the form of a single dosage form, for example, in the form of vials or ampoules with a solution or with a lyophilized concentrate containing plasmin with a purity of more than 98%, glycine, lysine in the indicated concentrations and, optionally, excipients or their combinations, while this plasmin activity in each single dose may be 1, 5, 10, 20, 50, 100, 200 or 500 units; the product may additionally have appropriate packaging and, optionally, instructions for use.

The best embodiment of the invention

To 50 g of the Cohn II-III fraction, 500 ml of 0.2 M Tris pH 7.5 buffer containing 0.1 M lysine and 5 mM EDTA were added (plasminogen activity was 1630 ME and was taken as 100% to estimate the yield). After two hours of incubation with constant stirring (+ 4 ° C), the suspension was centrifuged at 6000 g for 1 hour.

PEG 3000 was used to precipitate impurity proteins, lipids, and lipid-associated viruses. After a two-hour incubation of 560 ml of the suspension obtained at the previous stage (plasminogen content 1330 ME) with 10% PEG at room temperature, the mixture was centrifuged at 6000 g for an hour. The precipitate was discarded, and the resulting supernatant (about 500 ml, 1230 ME, 75% yield) was ultrafiltered. After replacing the buffer with 50 mM phosphate, pH 7.5, containing 0.15 M sodium chloride, the solution was applied to a lysine-sepharose 4B column (12 × 2.3 cm). Then the column was washed with equilibration buffer to an optical density of less than 0.05 units at 280 nm. Plasminogen was eluted with 120 ml of a buffer solution, pH 3.4, containing 0.1 M glycine, 30 mM lysine and 5 mM caprylic acid. The eluted protein (120 ml, 1000 ME, 61% yield) was incubated for one hour at + 4 ° C.

After changing the buffer to 20 mM Tris, pH 7.5, containing 15 mM lysine, 10 mM b-aminocaproic acid, 50% glycerol, the preparation was concentrated and streptokinase was added to the solution based on the molar ratio of plasminogen: streptokinase = 100: 1. The mixture was incubated for 18 hours at + 4 ° C. The reaction was stopped by adding 0.1 M Tris, pH 8.5, containing 1 M sodium chloride (activity of plasmin 630 ME, yield 40% of the initial level).

For final purification of plasmin, a mixture in a volume of 40 ml was applied to a column with benzamidine-sepharose 4B (2.6 × 5 cm), balanced with 50 mM Tris buffer, pH 8.5, containing 0.5 M sodium chloride. Plasmin was eluted with a buffer containing 0.1 M glycine and 30 mM lysine, pH 3.5. (600 ME, yield 37%). At the final stage, the diafiltration of the preparation was carried out, replacing it with a buffer solution, pH 3.4, containing 10 mM glycine and 10 mM lysine, while concentrating the preparation up to 20 ml (plasmin activity 600 ME, 37% yield). Under these conditions, at + 4 ° C, the drug remains stable for a month.

The purity of the final preparation was 99%.

The resulting product was pyrogen-free, did not show toxicity in experiments on laboratory animals (rats, rabbits), did not cause allergic or other adverse reactions with intradermal or intravenous administration, and also with intraorbital administration, the drug does not cause visual field defects.

Claims (7)

1. A method of obtaining a human plasmin concentrate, characterized in that Tris-HCL pH 7.5 buffer containing 0.1 M lysine and 5 mM EDTA is added to the Cohn II + III fraction, incubated for 2 hours, centrifuged, the resulting suspension is incubated with 10% PEG 3000 at room temperature, centrifuged at 6000 g for 1 h, the solution is applied to a lysine-sepharose column at pH 7.5, plasminogen is eluted with a buffer solution at pH 3.5 containing 0.1 M glycine, 30 mm lysine and 5 mm caprylic acid, plasminogen is incubated in the presence of streptokinase in a ratio of 100: 1 in three a pH 7.5 c-buffer containing 15 mM lysine, 10 mM e-aminocaproic acid and 50% glycerol, then chromatography on a benzamidine-sepharose column at pH 8.5 is carried out, eluting with a buffer at pH 3.5 containing 0, 1 M glycine, 30 mM lysine, and lyophilize the target product containing about 30 IU / ml plasmin with a purity of more than 98%, at least 10 mM lysine and 10 mM glycine in an aqueous solution at a pH of about 3.5. 2. The product, which is a plasmin concentrate, is characterized in that it is obtained from human blood plasma by the method according to claim 1, contains about 30 IU / ml of plasmin with a purity of more than 98%, at least 10 mM lysine and 10 mM glycine. 3. The product according to claim 2, which is an aqueous solution or lyophilized powder. 4. The product according to claim 2, which further comprises excipients, carriers, solvents and / or excipients that are pharmaceutically acceptable for parenteral administration. 5. The product according to claim 3, which has a pH of an aqueous solution of about 3.5. 6. The product according to claim 2, which is intended for use in medicine, veterinary medicine and / or for research purposes. 7. The product according to claim 2, which is intended for the treatment or prevention of conditions occurring with activation of the blood coagulation system and with thrombosis.