RU2388486C2 - Stabilised protease composition - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to medicine and concerns a composition containing serine protease, reversible inhibiter of the serine protease and a stabilising agent M characterised by formula (I).

EFFECT: invention enables creating serine protease composition suitable for direct utilisation without obligatory previous stages of preparing solutions from the material which has been subjected to deep freezing or freeze drying.

58 cl, 10 ex, 5 tbl

Description

This invention relates to an enzyme composition in which the enzyme is stabilized with certain additives in the combination of the invention. More specifically, the present invention relates to a serine protease composition comprising a reversible serine protease inhibitor and an additional stabilizing agent M, as defined below.

State of the art

Serine proteases are a group of proteolytic enzymes characterized by the fact that they contain serine and histidine residues in their active centers. Many well-known enzymes belong to this group, for example trypsin, kallikrein, thrombin and plasmin. Some of them have practical applications. Trypsin is used in the leather industry. Thrombin is used as a hemostatic agent to stop bleeding from wounds. Urokinase and tissue plasminogen activator, two other serine proteases are used clinically as thrombolytic agents in the treatment of acute myocardial infarction. Many of these enzymes are widely used as research tools, for example, to determine the structure of a protein. Moreover, enzymes are used in various diagnostic kits.

A common feature for most serine proteases is their limited stability in solution. This is mainly due to autodecomposition when left in solution, caused by their property as proteases. Such limited stability is a problem in cases where the product must be stored in solution. The currently available commercial serine proteases are almost always in the form of frozen solutions or lyophilized powders with obvious drawbacks. The extra time required to dissolve the powder or thaw the frozen solution to a suitable temperature is the most important point. However, there are other problems associated with these drugs. For frozen solutions, there is a need for temperature control (-20 ° C) at all stages, from production and transportation to storage. For lyophilized powders, there is a need to reconstitute a solution with an acceptable degree of purity and stability. It is also often necessary to prepare the product aseptically (by mixing two parts) in an environment that may be uncontrolled (for example, adverse weather conditions or lack of clean water), and it is necessary to check whether the powders were mixed properly. These are all the main disadvantages of modern products, added to the complexity of their application and their cost.

For thrombin, which should preferably be immediately available for use to stop bleeding, stability issues have forced manufacturers to use lyophilized thrombin or deep-frozen solutions. They take some time to prepare for use. Two thrombolytic agents, urokinase and a tissue plasminogen activator, are sold as lyophilized preparations, which must be dissolved before use. Since thrombolytic treatment of acute myocardial infarction should be started as soon as possible after the onset of a heart attack, any delay caused by the preparation of such drugs is a problem.

Many attempts have been made to find ways to stabilize various serine proteases. For trypsin, which self-degrades rather quickly, a simple and effective stabilizing agent is calcium ion (Sipos T and Merkel J, Biochemistry 9: 2766 (1970)). Lowering the pH to below 4 is also a method that works with certain enzymes, such as trypsin and plasmin, but is not possible for thrombin, since it is irreversibly inactivated at pH below 5. Reversible protease inhibitors may be used, but they are less popular since they cause harm by interfering with the action of the enzyme if they are used on their own (see below).

To stabilize the tissue plasminogen activator (tPA), the amino acid arginine is usually added. The currently clinically used tPA product contains arginine as a stabilizer.

Also, many attempts were devoted to finding ways to stabilize thrombin solutions. The following suggestions can be mentioned as examples of stabilizing additives: carboxylic acids in high concentrations, EDTA, various amino acids, albumin, polymers such as polyethylene glycol, polyvinylpyrrolidone and polyvinyl alcohol, glycerin, various inorganic salts, carbohydrates, gelatin, collagen.

Japanese patent application JP2004191367 describes a stabilized thrombin assay reagent for testing blood coagulation ability. The analytical reagent contains thrombin and a thrombin inhibitor, and may also contain one or more thrombin stabilizing compounds selected from calcium ion, organic acid, surfactant, and protein.

WO 02/100830, WO 02/22575, WO 00/20394, WO 99/11658, WO 02/37937 and US 5409927 describe various serine protease inhibiting compounds and their use in pharmaceutical compositions for treating various disease states such as thrombosis in which inhibition of the corresponding serine proteases is indicated.

Nakamura et al. (J. Chrom. A, 1009, (2003), 133-139) describes the use of an immobilized protease inhibitor for affinity chromatography of trypsin-like proteases.

Turner et al. (Biochemistry, 25, (1986), 4929-4935) describes three p-amidinophenyl esters that irreversibly inhibit human factor IXa.

Tsung Fu Yang et al. (Biomacromolecules, 25, (2004), 1926-1932) describes the synthesis of a cationic polymer, N, N-diethylethylenediaminopolyurethane for use in transgenosis.

In patent application US 2001/0033837 (corresponding to EP 1136084 A1) describes the preparation of thrombin containing a non-covalently bound inhibitor as a stabilizer. Moreover, the inhibitor is combined with other stabilizing additives, such as sugars or carboxylic acids, which were previously described in patents or other publications.

JP 2000300250 describes the stabilization of thrombin solutions by the addition of polyvinyl alcohol, gelatin or polyvinylpyrrolidone in various buffer solutions.

In GB 1354761, proteases and amylases are stabilized to various degrees using a variety of substances, such as aliphatic alcohols, carboxylic acids, heterocyclic compounds containing hydroxyl groups, and aliphatic or alicyclic amines.

Thus, stabilization of a serine protease using inhibitors is described (for example, in US 2001/0033837 and JP 2004191367 above). The problem with this approach is that the inhibitor greatly weakens the action of the enzyme if it is not removed before using the drug. If a potent inhibitor is used, most of the enzyme activity is lost. The best approach is to use a reversible medium strength inhibitor. However, even in this case, a significant part of the initial enzyme activity will be lost with increasing concentration of the inhibitor to obtain a good stabilizing effect.

Disclosure of invention

Therefore, the objective of this invention is to obtain a serine protease composition that is stable in solution and retains a degree of enzymatic activity that is sufficient for the practical use of the composition.

Another objective of this invention is to provide a serine protease composition that is available for direct use without preliminary preparation steps from a deeply frozen or lyophilized material.

Another objective of this invention is to make possible the practical use of reversible inhibitors of serine proteases for stabilization by adding an additional stabilizing component.

These and other objects obvious from the text presented are achieved through various claimed aspects of the present invention.

Thus, in one aspect of the present invention, there is provided a stabilized serine protease composition comprising: a) a serine protease; b) a reversible inhibitor of said serine protease; and c) a stabilizing agent M having the formula I:

Where

n is 0, 1 or 2;

X is O, N or CH ₂ ;

R ¹ -R ⁴ are the same or different and are selected from H, -CH ₂ -R ⁶ , -CH ₂ -OR ⁶ , -CH ₂ -SR ⁶ , -CH ₂ -NH-R ⁶ , -CO-OR ⁶ , -CO-NH-R ⁶ , -CH ₂ -NH-CO-R ⁶ ,

—CH ₂ —O — CO — R ⁶ , —CH ₂ —NH — CO — NHR ⁶ , —CH ₂ —NH — CO — OR ⁶ , —CH ₂ —NH — CS — NHR ^6, and —CH ₂ —O— CO-NHR ⁶ ;

R ⁵ is the same as R ¹ -R ⁴ or PQ;

P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;

Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;

each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, or a mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic ring (s) with one or more heteroatoms and non-aromatic heterocycles, where the substituents of the substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic iCal heterocycles, alkyloxy, alkylamino;

or its pharmaceutically acceptable salts.

This invention is based on the initial results of thrombin stability studies, in which it was unexpectedly discovered that the combination of the invention of a reversible enzyme inhibitor and a stabilizing agent M, such as described above, has a strong stabilizing effect on the enzyme in solution. The thrombin inhibitor and the stabilizing agent M individually have a stabilizing effect on thrombin, but their combination is several times better than any of them (see example 1). Thus, if a low concentration of the enzyme inhibitor is combined with morpholine, MOPS or related compounds, a very strong stabilizing effect on the enzyme is obtained. Some test compounds were stable, which showed a less than 30% decrease in activity, for more than 2 months at a temperature of 37 ° C. This, according to data in previous publications, confirmed by the authors of this invention, corresponds to 6 months at room temperature or 2.5 years at a refrigerator temperature. The results from the initial study were expanded to include experiments with other serine proteases, and an unexpected stabilizing effect was also noted in these experiments.

As confirmed by the examples below, the composition in accordance with this invention demonstrates significantly improved stability compared to enzyme compositions without a combination of ingredients b) and c) according to the invention. Using the approach of this invention, a low concentration of a serine protease inhibitor can be used with a satisfactory degree of stabilization. For example, the concentration of the inhibitor may be less than previously proposed, for example, in US 2001/0033837. With such a low concentration of inhibitor, the bulk of the enzyme activity remains in a stabilized enzyme solution.

It should be noted that increased stabilization due to the combination of a reversible serine protease inhibitor and stabilizing agent M should not be considered as an additional inhibitory effect provided by M. In fact, M, as described in illustrative example A, may not be able to inhibit any serine protease. Without pretending to be a theory, the authors of the present invention believe that the unexpectedly increased stabilizing effect detected is achieved due to the favorable synergy between reversible serine protease inhibitors and the stabilizing agents M of the composition in accordance with this invention. The present invention provides such a combination of a reversible serine protease inhibitor and a stabilizing agent M in a stabilized serine protease composition, and the use of such a combination to stabilize a serine protease composition.

In one embodiment of the invention, the serine protease in the composition is selected from the group of trypsin, kallikrein, thrombin, plasmin, urokinase, tissue plasminogen activator, active form of factor IX, active form of factor X and active form of factor XI. In a more specific embodiment, the serine protease is thrombin. In another specific embodiment, the serine protease is plasmin. In yet another specific embodiment, the serine protease is trypsin.

Reversible serine protease inhibitors are known to those skilled in the art, and which one is optimal for use depends on the particular serine protease used. In general, for the intended action, it is important that the inhibitor does not have significant strength. In other words, the inhibitory effect must be moderate enough so that the enzyme activity remains efficiently high. As an indication, it has been found that inhibitors having a K _{i of} from 0.01 mM to 2 mM are suitable for use in the composition of this invention, a preferred range is from 0.04 mM to 0.5 mM.

In one embodiment, wherein the serine protease is thrombin, a reversible inhibitor may be selected from N- (2'-phenoxy) -4-aminopyridine and its derivatives, benzamidine, N, N-diethylethylenediamine, aminobenzamidine, amidinopyridine and tert-butylamidine. In another embodiment, wherein the serine protease is thrombin, the reversible inhibitor is selected from N- (2'-phenoxy) -4-aminopyridine and its derivatives, N, N-diethylethylenediamine, amidinopyridine and tert-butylamidine. In a more specific embodiment, in which the serine protease is thrombin, the reversible inhibitor is N- (2'-phenoxy) -4-aminopyridine or a derivative thereof. In another embodiment, wherein the serine protease is plasmin, the reversible inhibitor is selected from N, N-diethylethylenediamine, aminobenzamidine and benzamidine. In another embodiment, in which trypsin is a serine protease, the reversible inhibitor is selected from aminobenzamidine and benzamidine. These combinations of enzymes and inhibitors are illustrative and should not be construed as limiting.

In one embodiment of the present invention, the value of n in formula I is 1 or 2. In a more specific embodiment, n in formula I is 1.

The composition in accordance with this invention contains a stabilizing agent M of the General formula I given above. In certain embodiments of the invention, the stabilizing agent M is a compound of formula II:

Where

R ¹ -R ⁴ are the same or different, and are selected from H, —CH ₂ —R ⁶ ;

R ⁵ is the same as R ¹ -R ⁴ or PQ;

P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;

Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;

each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, or a mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic ring (s) with one or more heteroatoms, and non-aromatic heterocycles, where the substituents of the substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic heterocycles; alkyloxy; alkylamino;

or its pharmaceutically acceptable salts.

Therefore, in some embodiments, the stabilizing agent M is a compound of formula III:

Where

R ⁵ is —CH ₂ —R ⁶ or PQ;

P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;

Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;

each R ^{6 is} individually selected from substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl or mono, substituents of substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds non-aromatic heterocycles, alkyloxy, alkylamino;

or its pharmaceutically acceptable salts.

In some embodiments of the invention, the stabilizing agent M is selected from the group consisting of morpholine, 3- (N-morpholino) propanesulfonic acid (MOPS), morpholinobutyl sulfonic acid, morpholinopropylcarboxylic acid, morpholinoethyl alcohol and morpholinoethyl sulfonic acid. Thus, examples of compounds M for use in the compositions of this aspect of the present invention include 3- (N-morpholino) propanesulfonic acid (MOPS). In a particular embodiment of the invention, the stabilizing agent M is morpholine.

A composition in accordance with this invention that has a stabilizing effect is one in which the serine protease is thrombin, the reversible inhibitor is N- (2'-phenoxy) -4-aminopyridine, and the stabilizing agent M is morpholine.

Another composition in accordance with this invention that has a stabilizing effect is one in which the serine protease is thrombin, the reversible inhibitor is N- (2'-phenoxy) -4-aminopyridine, and the stabilizing agent M is - (N-morpholino) propanesulfonic acid (MOPS).

Another composition in accordance with this invention that has a stabilizing effect is one in which the serine protease is thrombin, the reversible inhibitor is aminobenzamidine, and the stabilizing agent M is morpholine.

Another composition in accordance with this invention that has a stabilizing effect is one in which the serine protease is plasmin, the reversible inhibitor is N, N-diethylethylenediamine, and the stabilizing agent M is morpholine.

Another composition in accordance with this invention that has a stabilizing effect is one in which the serine protease is plasmin, the reversible inhibitor is aminobenzamidine, and the stabilizing agent M is morpholine.

In serine protease compositions for topical administration, for example, for application to a wound, it is a problem that the composition can easily leak or rinse out of the wound on which it is applied. To solve this problem, it is possible to add an adhesive polymer to the enzyme composition, which is needed in order to make the composition more viscous and stick to the skin or wound. As an embodiment of the invention, such an addition of an adhesive polymer to the composition of the invention may have an additional unexpected and beneficial effect on its stability. Adding a polymer has two goals: increasing the viscosity and adhesiveness of the composition, and, at the same time, helping to further stabilize the enzyme.

In some embodiments of the invention, the composition further comprises a viscous and adhesive polymer selected from polysaccharides and gelatin. Thus, the polymer can be, for example, a polysaccharide selected from starch, its derivatives, cellulose, its derivatives, and mixtures thereof. Specific non-limiting examples of starches used as additives to the compositions of this invention include corn starch and potato starch, and mixtures thereof, non-limiting examples of useful cellulose derivatives include carboxymethyl cellulose and ethyl hydroxyethyl cellulose and mixtures thereof. In a specific embodiment, the polysaccharide is carboxymethylchitosan. In other embodiments of the invention, said polysaccharide is present in a concentration of 0.1-5%. However, it is also contemplated that the polymer is gelatin, such as gelatin from cold-water fish. In some embodiments of the invention, said gelatin is present in a concentration of 0.5-20%.

In one embodiment of the invention, said serine protease is present at a concentration of 0.001-2 mg / ml. In a more specific embodiment, said serine protease is present at a concentration of 0.01-1 mg / ml.

In one embodiment of the invention in which the serine protease is thrombin, the thrombin concentration is 5-3500 activity units / ml.

In one embodiment of the invention in which the serine protease is thrombin, the thrombin concentration is from 200 to 1000 activity units / ml. In one embodiment of the invention in which the serine protease is thrombin, the thrombin concentration is from 5 to 20 activity units / ml.

In one embodiment of the invention, said reversible inhibitor of said serine protease is present at a concentration of 0.1-10 mM. In a more specific embodiment, said reversible inhibitor of said serine protease is present at a concentration of 0.5-2 mM.

In one embodiment of the invention, said stabilizing agent M is present at a concentration of 0.02-0.5 M. In a more specific embodiment, said stabilizing agent M is present at a concentration of 0.1-0.3 M.

According to another aspect, the invention provides the use of the composition described above as a medicament.

Another aspect of the present invention relates to the use of said composition, in which the serine protease is thrombin, for the preparation of a medicament for hemostasis in a patient suffering from bleeding. In a related aspect of the invention, there is provided a method for hemostasis in a patient suffering from bleeding, comprising applying a composition in accordance with this invention, where the serine protease is thrombin in the composition, to the site of bleeding in an amount sufficient to reduce or stop said bleeding.

In connection with the indicated use or the method in which the thrombin composition of the invention is used as a medicine, the stability of the composition of the invention has advantages under the conditions in which it is used. Often, thrombin compositions are used in the context of critical situations where it is critical to stop bleeding in a patient. In the same situations, the use of conventional hemostatic thrombin preparations is difficult, as they often require laborious and time-consuming thawing operations (if they are frozen) and / or dissolution (if they are lyophilized). This invention makes it possible to obtain, for example, such hemostatic agents in the form of solutions whose stability is such that they can be easily stored for extended periods of time, for example, in an ambulance or emergency helicopter, until the need arises. its use at the scene of an accident or the like. At this time, they can be used as such, without delay in cooking.

Conventional drugs used to stop bleeding contain rather high concentrations of thrombin, from 200 to 1000 activity / ml units. In plastic surgery, this can cause a risk of increased scarring. Solutions with a low concentration of thrombin are currently being prepared at the clinic by diluting concentrated thrombin solutions. There are no ready-to-use preparations. Therefore, in another aspect, the invention provides a stabilized thrombin composition with significantly lower concentrations of thrombin, from 5 to 20 activity units / ml, and their use in plastic surgery.

In another aspect of the invention, the known properties of plasmin urokinase or tPA as thrombolytic agents are used. Thus, the present invention provides the use of a composition, such as described above, in which the serine protease is selected from plasmin, urokinase and a tissue plasminogen activator, for the preparation of a medicament for thrombolytic treatment. A related aspect is a method of thrombolytic treatment in a patient in need thereof, comprising administering a composition, such as described above, wherein the serine protease is selected from plasmin, urokinase and tissue plasminogen activator in the composition in an amount sufficient for such treatment. In these two related aspects, the considered thrombolytic treatment, as unlimited examples, can be carried out for the treatment of myocardial infarction or for the treatment of stroke.

As stated in the context of the compositional aspect of the present invention, increased stabilization due to the combination of a reversible serine protease inhibitor and stabilizing agent M does not occur due to the additional inhibitory effect provided by M. In fact, M, as described in illustrative example A, may not have any or the ability to inhibit serine protease. Without claiming to be a theory, the authors of the present invention believe that the unexpectedly increased stabilizing effect obtained is achieved due to favorable synergies between reversible serine protease inhibitors and M stabilizing agents of the composition in accordance with this invention.

Therefore, in another aspect, the invention provides the use of a combination

a) a reversible serine protease inhibitor, and

b) a stabilizing agent M of the formula I:

Where

n is 0, 1 or 2;

X is O, N or CH ₂ ;

R ¹ -R ⁴ are the same or different, and are selected from H. —CH ₂ —R ⁶ , —CH ₂ —OR ⁶ , —CH ₂ —SR ⁶ , —CH ₂ —NH — R ⁶ , —CO-OR ⁶ , -CO-NH-R ⁶ , -CH ₂ -NH-CO-R ⁶ ,

—CH ₂ —O — CO — R ⁶ , —CH ₂ —NH — CO — NHR ⁶ , —CH ₂ —NH — CO — OR ⁶ , —CH ₂ —NH — CS — NHR ^6, and —CH ₂ —O— CO-NHR ⁶ ;

R ⁵ is the same as R ¹ -R ⁴ or PQ;

P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;

Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;

each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, or a mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic ring (s) with one or more heteroatoms and non-aromatic heterocycles, where the substituents of the substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic heterocycles; alkyloxy; alkylamino; or its pharmaceutically acceptable salts;

to stabilize the serine protease composition, wherein the reversible serine protease inhibitor and the stabilizing agent M act in synergy to provide a stabilizing effect on the serine protease.

In the use of a combination of a reversible serine protease inhibitor and a stabilizing agent in accordance with this invention to stabilize a serine protease composition, the selection of specific components that can be used and substituents for compounds M are as described above in the composition aspect of the present invention.

In yet another aspect, the present invention provides a method for stabilizing a serine protease, which comprises mixing a serine protease with a) a reversible inhibitor of said serine protease; and b) a stabilizing agent M of the formula I:

Where

n is 0, 1 or 2;

X is O, N or CH ₂ ;

R ¹ -R ⁴ are the same or different, and are selected from H. —CH ₂ —R ⁶ , —CH ₂ —OR ⁶ , —CH ₂ —SR ⁶ , —CH ₂ —NH — R ⁶ , —CO-OR ⁶ , -CO-NH-R ⁶ , -CH ₂ -NH-CO-R ⁶ ,

—CH ₂ —O — CO — R ⁶ , —CH ₂ —NH — CO — NHR ⁶ , —CH ₂ —NH — CO — OR ⁶ , —CH ₂ —NH — CS — NHR ^6, and —CH ₂ —O— CO-NHR ⁶ ;

R ⁵ is the same as R ¹ -R ⁴ or PQ;

P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;

Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;

each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, or a mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic ring (s) with one or more heteroatoms and non-aromatic heterocycles, substituents of substituted groups selected from lower alkyl, halogens, where substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic iCal heterocycles, alkyloxy, alkylamino; or its pharmaceutically acceptable salts.

In a method for stabilizing a serine protease composition in accordance with this invention, the selection of specific components that can be used and substituents for compounds M are as described above in the composition aspect of the present invention.

Another aspect of the present invention relates to the use of a composition, such as described above, for adsorption onto a solid object so that said solid object can provide the enzymatic activity discussed. In particular, in many surgical areas it is of interest to enter and, in particular, exit the arteries, causing as little damage as a result of bleeding. To stop bleeding from an artery, it was previously proposed to use the form of “arterial tampons” (such objects are also known as vascular seals, locking devices for entering the thigh (if the femoral artery is used for entering, for example, angiography), devices for hemostasis of vessels and locking devices for puncture ) obtained, for example, from collagen or other biodegradable material. According to this aspect of the invention, such a tampon can advantageously be coated with a composition according to the invention in which the serine protease is thrombin. This swab allows you to more quickly close the hole in the artery due to the fact that the thrombin from the composition helps coagulate the blood around the swab. Thus, the present invention provides, in this aspect, a device for vascular hemostasis containing a certain amount of the composition in accordance with this invention, in which the serine protease is thrombin absorbed on it. A device for vascular hemostasis is preferably made from a biodegradable solid or semi-solid material, such as collagen, chitosan or another biological polymer.

Another aspect of the present invention relates to a new identification of N, N-diethylethylenediamine as a serine protease inhibitor. Thus, in this aspect, the invention provides the use of N, N-diethylethylenediamine as a serine protease inhibitor, as well as a method of inhibiting serine protease, comprising mixing together an inhibitory amount of N, N-diethylethylenediamine with it. In some embodiments of this aspect of the invention, the serine protease is plasmin. In other embodiments of this aspect of the invention, the serine protease is thrombin.

It is generally preferable to realize all the advantages of various aspects of the invention so that the composition of the invention is in a form selected from solution and gel. In this regard, aqueous solutions and aqueous gels are more preferred.

Definitions

As used herein, the term “lower alkyl” means an unbranched or branched, cyclic, saturated or unsaturated (alkenyl or alkynyl) hydrocarbon radical which may or may not be substituted. If it is cyclic, the alkyl group preferably contains C3-C12, more preferably C5-C10, most preferably C5-C7. If it is alicyclic, the alkyl group preferably contains C1-C10, more preferably C1-C6, more preferably methyl, ethyl, propyl (n-propyl, isopropyl), butyl (branched or unbranched) or pentyl, most preferably methyl .

As used herein, the term “aryl” means an aromatic group such as phenyl or naphthyl, or a mono-, bi- or tricyclic heteroaromatic group containing one or more heteroatoms (s), preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl, triazolyl, tetrazolyl, isoxazolyl, isothiazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyldinyl, pyrindiolinyl benzthiazolyl , purinyl, cinnolinyl, pterdinyl.

In this description, the term “functional group” means if it is not protected: hydroxy-, thiolo-, amino-functional group, carboxylic acid, and if it is protected: lower alkoxy, N-, O-, S-, acetyl, carboxylic acid ester .

As used herein, the term “heteroaryl” means an aromatic group containing one or more heteroatoms (s), preferably selected from N, O and S, such as pyridyl, pyrrolyl, quinolinyl, furanyl, thienyl, oxadiazolyl, thiadiazolyl, thiazolyl, oxazolyl, pyrazolyl , triazolyl, imidazolyl, pyrimidinyl, indolyl, pyrazinyl or indazolyl.

As used herein, the term “non-aromatic heterocycle” means a non-aromatic cyclic group containing one or more heteroatoms (s), preferably selected from N, O and S, such as a cyclic amino group, such as pyrrolidinyl, piperidyl, piperazinyl, morpholinyl or cyclic ether, such as tetrahydrofuranyl, monosaccharide.

As used herein, the term “halogen” means fluoro, chloro, bromo or iodo.

As used herein, the term “substituted” means that the group is substituted with a functional group such as hydroxyl, amine, sulfide, silyl, carboxylic acid, halogen, aryl, etc.

Examples of pharmaceutically acceptable addition salts for use in the compositions of this invention include those derived from mineral acids such as hydrochloric, hydrobromic, phosphoric, metaphosphoric, nitric and sulfuric acids, and organic acids such as tartaric, acetic, citric, malic, lactic, fumaric, benzoic, glycolic, gluconic, succinic and arylsulfonic acids. Pharmaceutically acceptable excipients described herein, for example excipients, adjuvants, carriers or diluents, are well known to those skilled in the art and are readily available. A pharmaceutically acceptable excipient may be one that is chemically inert to the active compounds and which does not cause harmful side effects or toxicity under the conditions of use. The pharmaceutical compositions may be found, for example, in The Science and Practice of Pharmacy, 19 ^{th edition, Mack Printing Company, Easton} , Pennsylvania ( 1995).

As detailed in the description of the invention, it is possible to select an inhibitor for use in the compositions and methods of this invention, “N- (2'-phenoxy) -4-aminopyridine and its derivatives”. By this is meant a compound having the formula IV:

Where

R ^{1 is} selected from H, C1-C6 alkyl, C3-C7 cycloalkyl, phenyl, benzylacetyl and benzoyl;

X is selected from oxygen, nitrogen and sulfur;

R ² and R ^{3 are} each individually selected from H, halogen, hydroxyl, C1-C6 alkyl, C3-C7 cycloalkyl, C1-C6 alkyloxy; and

R ^{4 is} selected from H, C1-C6 alkyl, arylalkyl and acyl.

Preferred inhibitors that have the formula V:

Where

R ^{1 is} selected from C1-C6 alkyl, C3-C7 cycloalkyl, phenyl and benzyl;

R ² and R ^{3 are} each individually selected from H, halogen, hydroxyl, C1-C6 alkyl, C3-C7 cycloalkyl, and C1-C6 alkyloxy.

Examples

the examples below illustrate the invention and should not be construed as limiting.

In the following description of experiments carried out in accordance with this invention, the time required to achieve 70% of the initial activity is used as a digital indicator of the stability of the enzyme solution. This indicator, designated “T 70%”, was chosen because it corresponds to what can be taken as the maximum permitted loss of activity during the period of operation of a commercial product.

In experimental studies apply high temperature (37 ° C), as well as high concentrations of the enzyme. They are used in order to obtain stability data in a reasonably short period of time, and not wait months or years. The temperature dependence of stability was studied, and the results are given in Example 1. This study showed that the inactivation process is about 3 times slower at room temperature, and about 20 times slower at refrigerator temperature than the process actually measured (at 37 ° FROM).

Moreover, the inactivation process is concentration dependent and is faster at higher enzyme concentrations. The thrombin concentration used in example 1 is 1 mg / ml (or 3300 units / ml), i.e. higher than 0.1-0.3 mg / ml used in modern commercially available preparations and / or devices. Studies of the dependence on the concentration showed that the inactivation process is 3-4 times slower at such concentrations, compared with the concentration used in example 1.

The combination of these indicators gives a factor of 10 to 12 by which the T value of 70% is multiplied to obtain data that correspond to storage conditions at room temperature for a commercial product containing serine protease, such as a hemostatic preparation containing thrombin. In table 1, the composition with N- (2'-phenoxy) -4-aminopyridine and MOPS has a T% of 70% equal to 120 days. This corresponds to the value for 0.1-0.3 mg / ml of the product, more than 1200 days at room temperature, i.e. more than three years. This undoubtedly exceeds any indicators obtained previously regarding the stabilization of thrombin in solution.

Example 1 - Stabilization of Human Thrombin

To determine the clotting activity of thrombin solutions, the clotting time of a fibrinogen solution (1.3 mg / ml) was measured after various dilutions of a solution of a specific human thrombin (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden) were added. Coagulation time was measured using an Amelungen Kc 1 coagulometer (Amelungen, Germany). To study the stability of thrombin solutions with various additives, the samples were incubated in a thermostat chamber at a temperature of 37 ° C. Aliquots were taken at different time intervals and the remaining thrombin activity in these aliquots was measured. From the obtained values, activity decline curves were constructed.

The activity decline curves of 1 mg / ml thrombin solutions in 10 mM HEPES, 0.13 M NaCl buffer, pH 7.4, showed T values of 70% for about 1.6 days at a temperature of 37 ° C. Corresponding experiments at room temperature (about 21 ° C) showed a T value of 70% for 5.4 days, and after storage in a refrigerator (temperature about 5 ° C), a T value of 70% was 36 days. Thus, as expected, there is a strong temperature dependence.

Solutions containing 1 mg / ml thrombin in 10 mM HEPES and 0.13 M NaCl at pH 7.4 with the indicated stabilizing additives were placed in a thermostat chamber and their activity decay curves were determined. The results are shown in table 1. Data for the corresponding 1 mg / ml thrombin solution without additives are included for comparison.

Obviously, all tested compounds have a stabilizing effect. However, there is a synergistic effect of the combinations of the inhibitor and the morpholine-containing compound in accordance with this invention, which confirms the excellent results obtained for such combinations. As the table above illustrates, the addition of 0.20 M pure MOPS gives a 4.7-fold increase in stabilization, and the addition of 0.5 mM reversible thrombin inhibitor aminobenzamidine gives a 12.5-fold increase in stabilization. The combinations in accordance with this invention, however, stabilize the thrombin composition much better, 42.5 times. The invented combination of MOPS and N, N-diethylethylenediamine also stabilizes the enzyme better than the individual components. Also, the combination of 0.20 M MOPS and 1.9 mM N- (2'-phenoxy) -4-aminopyridine provides a very significant increase in stabilization by 75 times, while individual components increase stability by 4.7 times and 22 times, respectively.

The thrombin used in the study is human thrombin obtained from plasma. Recombinant human thrombin was also investigated, and it showed almost the same behavior.

Example 2 - Stabilization of bovine thrombin

Investigated stabilization of bovine thrombin. The experimental parameters were the same as in example 1, but the applied concentration of bovine thrombin (Baxter) is 0.4 mg / ml When stored at 37 ° C, a thrombin solution in HEPES buffer showed a T value of 70% 1.3 days. A solution of thrombin in HEPES buffer plus 3.0 mM N- (2'-phenoxy) -4-aminopyridine and 0.20 M MOPS showed a T value of 70% for 54 days.

The results showed that bovine thrombin is somewhat more unstable than the studied preparations of human thrombin, but that, nevertheless, the use of the compositions in accordance with this invention showed a very good stabilizing effect.

Example 3 - Low Thrombin Concentrations

The stabilization of thrombin was investigated in compositions containing low concentrations of thrombin. It is shown that the stabilizing effect of the compositions in accordance with this invention also work for relatively low concentrations of thrombin.

A solution of human thrombin of 15.0 activity units / ml (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden) in HEPES buffer, pH 7.4, showed a T value of 70% for 23 days. An appropriate solution in HEPES buffer, pH 7.4 plus 2.0 mM N- (2'-phenoxy) -4-aminopyridine and 0.20 M MOPS showed a T value of 70% for 92 days.

Example 4 - stabilization of plasmin

Tested the stabilization of plasmin solutions in accordance with this invention. Plasmin activity was determined using a Chromozym TH chromogenic peptide substrate (Pentapharm, Switzreland) and a change in absorbance at 405 nm in a spectrophotometer. Solutions containing 100 μg / ml plasmin (specific activity 3.2 units / mg, Sigma-Aldrich) in 10 mM HEPES and 0.13 M NaCl, pH 7.4, as well as the stabilizers shown in table 2 below, were incubated at temperature of 37 ° C, and samples were taken at various points in time to determine activity. The results obtained are shown in table 2.

From the presented results it is obvious that a very strong stabilization was obtained when applying the combination in accordance with this invention. 0.20 M morpholine increases the stability of the plasmin composition 4 times, 0.13 M N, N-diethylethylenediamine 2.7 times and 1.3 mm aminobenzamidine 17 times. However, the combination of morpholine and N, N-diethylethylenediamine increases the stability of the plasmin composition by 7.3 times, and the combination of morpholine and aminobenzamidine increases stability by 72 times.

Example 5 - Stabilization of trypsin

tested the stabilization of trypsin solutions in accordance with this invention. Trypsin activity was determined using tosylarginine methyl ester (TAME) as a substrate, and the change in absorbance at 247 nm was measured in a spectrophotometer. Solutions containing 100 μg / ml trypsin (treated with TRSC, Sigma-Aldrich) in 10 mM HEPES and 0.13 M NaCl, pH 7.4, and also the stabilizers indicated in Table 3 were incubated at 37 ° C, and samples were taken in various time intervals for determining activity. The results obtained are shown in table 3.

Again, the stabilizing effect is greatest for the composition in accordance with this invention. Thus, 0.5 M of pure morpholine gives a stabilization increase of 13 times, and 1 mM of pure benzamidine gives a stabilization increase of 72 times. The combination of the invention, on the other hand, provides an increase in stabilization of 147 times.

Example 6 - stabilization of thrombin with CMC

Tests of thrombin solutions containing from 1.0 to 2.0% carboxymethyl cellulose (CMC) for adherence (adhesion) to human skin showed that the addition of CMC greatly increases both viscosity and adherence. However, it was unexpectedly found that the stability of such thrombin solutions increases even more. 1 mg / ml solution of human thrombin (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden) in 0.5 mm aminobenzamidine, 0.20 M MOPS, 10 mm HEPES, 0.13 M NaCl, 2.0% relative to CMC, they were incubated at a temperature of 37 ° C, and activity decline curves were determined. The obtained T value of 70% was 175 days.

Example 7 - Stabilization with other adhesive polymers

Four other polymers were also investigated: ethyl hydroxyethyl cellulose (EHEC), potato starch, corn starch and gelatin of cold-water fish. All four polymers increased the adhesion and viscosity of thrombin solutions. The compatibility and stability of thrombin solutions with polymers was further studied by incubating at a temperature of 37 ° C 1 mg / ml solutions of human thrombin (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden) in 0.20 M MOPS, 0.5 mm aminobenzamidine , 10 mm HEPES, 0.13 M NaCl, pH 7.4, containing various polymers. The concentrations of the polymers used were: EHEC, 0.6%; two different starches, 4.0%; and gelatin, 12.8%. EHEC was fully compatible with thrombin, and the T value of 70%, i.e. about 65 days, was the same as for the solution without EHEC. Starch-containing solutions had T values of 70% for 22 and 26 days. The thrombin stability was very good in gelatin, at a T value of 70% for more than 90 days, which demonstrates the additional stabilizing effect of gelatin in cold-water fish.

Example 8 - Experiments with bleeding

The ability of the compositions in accordance with this invention to stop bleeding was tested in a series of experiments on rabbits. The selected model was incisions in the liver, which are a commonly used model. The abdominal cavity of the rabbits was opened and the liver exposed. Standard incisions of 3 mm were made on the surface of the liver, and 0.10 ml of the test solution was applied to the wound using a syringe. Blood coagulation time was measured. For each solution, 10-12 experiments were performed. Mean bleeding times were calculated after discarding the fastest and slowest values in each series of experiments. In comparison, the widely used hemostatic agent Tisseel (Baxter), a fibrin glue, was also included in the study. Tisseel was used in accordance with the manufacturer's recommendations. 0.2 ml of the solution was applied to each wound using a double syringe with a mixing chamber. The results obtained are given in table 4 below.

From these results, it is obvious that a thrombin solution stabilized in accordance with this invention is most effective for rapid blood coagulation in a patient with bleeding that is comparable to or better than widely used agents.

Example 9 - Compatibility with porous materials

A solution containing 0.4 mg / ml of human thrombin (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden) in 10 mm HEPES, 0.14 M NaCl, 0.5 mm aminobenzamidine, 0.20 M MOPS with pH 7.4 was absorbed into a piece of polyurethane gypsum (sold under the name Ligasone from Hartmann Scamdicare AB, Anderstorp, Sweden). A sufficient amount of solution was used to saturate a piece of polyurethane. The piece was transferred to a test tube, which was then closed to prevent evaporation. The tube was stored at 37 ° C, and samples of the solution were taken at intervals by gentle pressure on a piece of polyurethane. The activity decline curves showed a T value of 70% for 74 days, which corresponds to a 46-fold increase in stability.

Example 10 - Adsorption of the enzyme in the solid phase

tested the adsorption of thrombin in stabilized solutions in the surface. Solid chitosan flakes (at least 85% deacetylated, Sigma-Aldrich) about 3 × 3 mm, were incubated for 10 minutes in solutions of 400 units / ml of human thrombin (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden ) in 10 mM HEPES, 0.13 M NaCl, pH 7.4. The solutions have the following additives: 1) no, 2) 0.10 M morpholine, 2 mM N- (2'-phenoxy) -4-aminopyridine, 3) 0.10 M morpholine, 2 mM N- (2'-phenoxy) -4-aminopyridine, 0.5% carboxymethyl cellulose. Then the flakes were removed and dried on filter paper. To measure the clotting effect of thrombin, the flakes were placed in a test tube and 0.4 ml of a 1.3 mg / ml fibrinogen solution was added. To improve clot detection, the tube also contains a small steel ball. The clotting time obtained initially for flakes from various incubation mixtures ranged from 1 to 4 minutes. After incubation in Eppendorf tubes at 37 ° C for 7 days, the clotting time for flakes incubated in solution 1) was significantly longer. Values ranged from 24 to 27 minutes. On the contrary, the clotting time for flakes incubated in solutions 2) and 3) was in the range from 1 to 2.5 minutes, i.e. same as the original values. Obviously, a strong stabilization of thrombin activity can be obtained by using solutions 2) and 3). To test the hemostatic effect in vivo, chitosan flakes incubated in solution 3) were applied to wounds on the rabbit liver according to the animal model described in Example 8. The average blood coagulation time was 27 seconds (based on six experiments).

Illustrative example A - Morpholine is not a thrombin inhibitor

The possibility that morpholine is a thrombin inhibitor was evaluated. The thrombin clotting effect of fibrinogen was usually measured in clotting tests in which the clotting time of a fibrinogen solution was determined using mechanical or optical devices. Coagulation tests in these experimental structures were carried out in 0.01 M HEPES, 0.13 M NaCl buffer with a pH of 7.4, which corresponds to the standard procedure (United States Pharmacopeia). A solution of human thrombin (obtained from plasma, 3300 units / mg, Biovitrum AB, Sweden) containing 89 units / ml was used, and dilutions 1/5, 1/10 and 1/16 were tested. Solutions of various concentrations of morpholine were prepared in HEPES buffer by adding a concentrated solution of morpholine adjusted to pH 7.4. When morpholine was dissolved in water, the pH reached 9-10, so HCl was added to obtain a pH of 7.4. It also increased the ionic strength of the solution. Table I shows the results. The resulting clotting time was converted to thrombin concentration using a standard curve.

As is evident from these results, there is a prolongation of coagulation time with an increase in the concentration of morpholine up to a certain level. However, the same thing is observed when the ionic strength increases due to NaCl and the same profile is obtained. Thus, the prolongation effect was most likely caused by an increase in ionic strength. Further, it is known that an increase in ionic strength from 0.15 M to 0.22 M causes changes in the polymerization of fibrin (B. Blombäck, Thrombosis Research, vol. 83, (1996), p.1-75).

This actually corresponds to the interval studied in this series of experiments, in which the initial concentration of NaCl was 0.13 M and then increased to 0.18 M and further to 0.33 M. This also corresponds to the obtained plateau level. Conclusion: morpholine itself is not a thrombin inhibitor.

Claims (58)

1. The composition of the stabilized serine protease containing a) serine protease; b) a reversible inhibitor of said serine protease; and c) a stabilizing agent M having the formula I

where n is 0, 1 or 2;
X is O, N or CH ₂ ;
R ¹ -R ⁴ are the same or different and are selected from H, -CH ₂ -R ⁶ , -CH ₂ -OR ⁶ , -CH ₂ -SR ⁶ , -CH ₂ -NH-R ⁶ , -CO-OR ⁶ , -CO-NH-R ⁶ , -CH ₂ -NH-CO-R ⁶ ,
—CH ₂ —O — CO — R ⁶ , —CH ₂ —NH — CO — NHR ⁶ , —CH ₂ —NH — CO — OR ⁶ , —CH ₂ —NH — CS — NHR ^6, and —CH ₂ —O— CO-NHR ⁶ ;
R ⁵ is the same as R ¹ -R ⁴ or PQ;
P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;
Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;
each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or
unsubstituted aryl or mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic rings (rings) with one or more heteroatoms and non-aromatic heterocycles, where the substituents of the substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic heterocycles, alkyloxy, alkylamino;
or its pharmaceutically acceptable salts. 2. The composition according to claim 1, in which the serine protease is selected from the group comprising trypsin, kallikrein, thrombin, plasmin, urokinase, tissue plasminogen activator, the active form of factor IX, the active form of factor X and the active form of factor XI. 3. The composition according to claim 2, where the reversible inhibitor has a value of K; from 0.01 to 2 mm, for example, from 0.04 mm to 0.5 mm. 4. The composition according to claim 3, where the serine protease is thrombin. 5. The composition according to claim 4, where the reversible inhibitor is selected from N- (2'-phenoxy) -4-aminopyridine and its derivatives, benzamidine, N, N-diethylethylenediamine, aminobenzamidine, amidinopyridine and tert-butylamidine. 6. The composition according to claim 4, where the reversible inhibitor is selected from N- (2'-phenoxy) -4-aminopyridine and its derivatives, N, N-diethylethylenediamine, amidinopyridine and tert-butylamidine. 7. The composition according to claim 4, where the reversible inhibitor is N- (2'-phenoxy) -4-aminopyridine or its derivative. 8. The composition according to claim 3, where the serine protease is plasmin. 9. The composition of claim 8, where the reversible inhibitor is selected from N, N-diethylethylenediamine, aminobenzamidine and benzamidine. 10. The composition according to claim 3, where the serine protease is trypsin. 11. The composition of claim 10, wherein the reversible inhibitor is selected from aminobenzamidine and benzamidine. 12. The composition according to claim 1, where n in the formula I is 1 or 2. 13. The composition according to p. 12, where n in the formula I is 1. 14. The composition according to item 13, where the stabilizing agent M is a compound of formula II

where R ¹ -R ⁴ are the same or different, and are selected from H, —CH ₂ —R ⁶ ;
R ⁵ is the same as R ¹ -R ⁴ or PQ;
P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m , where m is 1-6 and Y is O, NH or S;
Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;
each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, or a mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic ring (s) with one or more heteroatoms, and non-aromatic heterocycles, substituents of substituted groups selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic FIR heterocycles, alkyloxy, alkylamino;
or its pharmaceutically acceptable salts. 15. The composition of claim 14, wherein the stabilizing agent M is a compound of formula III

where R ⁵ is —CH ₂ —R ⁶ or PQ;
P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;
Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;
each R ^{6 is} individually selected from substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, where the substituents of the substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic heterocycles, alkyloxy, alkylamino;
or its pharmaceutically acceptable salts. 16. The composition of claim 15, wherein the stabilizing agent M is selected from the group consisting of morpholine, 3- (N-morpholino) propanesulfonic acid (MOPS), morpholinobutyl sulfonic acid, morpholinopropylcarboxylic acid, morpholinoethyl alcohol and morpholinoethyl sulfonic acid. 17. The composition according to clause 16, where the stabilizing agent M is selected from morpholine and 3- (N-morpholino) propanesulfonic acid. 18. The composition of claim 17, wherein the stabilizing agent M is morpholine. 19. The composition according to claim 1, where the serine protease is thrombin, the reversible inhibitor is N- (2'-phenoxy) -4-aminopyridine, and the stabilizing agent M is morpholine. 20. The composition according to claim 1, where the serine protease is thrombin, the reversible inhibitor is N- (2'-phenoxy) -4-aminopyridine, and the stabilizing agent M is 3- (N-morpholino) propanesulfonic acid. 21. The composition according to claim 1, which further comprises a viscous and adhesive polymer selected from polysaccharides and gelatin. 22. The composition according to item 21, where the viscous and adhesive polymer is a polysaccharide. 23. The composition according to item 22, where the polysaccharide is selected from starch, its derivatives, cellulose, its derivatives, and mixtures thereof. 24. The composition of claim 23, wherein the polysaccharide is selected from carboxymethyl cellulose, ethyl hydroxyethyl cellulose, and mixtures thereof. 25. The composition of claim 22, wherein the polysaccharide is carboxymethylchitosan. 26. The composition according to any one of paragraphs.22-25, where the specified polysaccharide is present in a concentration of 0.1-5%. 27. The composition according to item 21, where the viscous and adhesive polymer is gelatin. 28. The composition according to item 27, where the gelatin is gelatin from cold-water fish. 29. The composition according to any one of paragraphs.27-28, where the specified gelatin is present in a concentration of 0.5-20%. 30. The composition according to claim 1, where the specified serine protease is present in a concentration of 0.001-2 mg / ml 31. The composition according to claim 1, where the serine protease is thrombin, and the concentration of thrombin is 5-3500 units of activity / ml 32. The composition according to p, where the serine protease is thrombin, and the concentration of thrombin is from 200 to 1000 units of activity / ml 33. The composition according to p, where the serine protease is thrombin, and the concentration of thrombin is from 5 to 20 units of activity / ml 34. The composition according to claim 1, where the specified reversible inhibitor of the specified serine protease is present in a concentration of 0.1-10 mm. 35. The composition according to claim 1, where the specified stabilizing agent M is present in a concentration of 0.02-0.5 M. 36. The composition according to claim 1, which has a form selected from a solution and a gel, for example, an aqueous solution and an aqueous gel. 37. The use of the composition according to any one of the preceding paragraphs as a medicine. 38. The use of a composition according to any one of claims 1 to 36, wherein the serine protease is thrombin, for the preparation of a medicament for hemostasis in a patient suffering from bleeding. 39. A method of hemostasis in a patient suffering from bleeding, comprising applying the composition according to any one of claims 1 to 36, wherein the serine protease is thrombin in the composition at the site of bleeding in an amount sufficient to reduce or stop said bleeding. 40. The use of the composition according to p. 33 in plastic surgery. 41. The use of a composition according to any one of claims 1 to 36, wherein the serine protease is selected from plasmin, urokinase and tissue plasminogen activator, for the preparation of a medicament for thrombolytic treatment. 42. A method of thrombolytic treatment in a patient in need thereof, comprising administering a composition according to any one of claims 1 to 36, wherein the serine protease is selected from plasmin, urokinase and tissue plasminogen activator in a composition in an amount sufficient for such treatment. 43. The use of a combination of a reversible serine protease inhibitor and a stabilizing agent M, as defined in claim 1, for stabilizing a serine protease composition, wherein said reversible serine protease inhibitor and said stabilizing agent M act in synergy to provide a stabilizing effect on the serine protease. 44. The use of claim 43, wherein the serine protease is as defined in any one of claims 2, 4, 8, 10, 30, 31, 32, and 33. 45. The use of claim 43, wherein the reversible inhibitor is as defined in any one of claims 3, 5, 6, 7, 9, 11, and 34. 46. The application of claim 43, wherein the stabilizing agent M is as defined in any one of claims 12-18 and 35. 47. The use of claim 43, wherein said combination further comprises a viscous and adhesive polymer selected from polysaccharides and gelatin. 48. The application of clause 47, where the viscous and adhesive polymer is as defined in any of paragraphs.22-29. 49. The use of claim 43, wherein said reversible serine protease inhibitor is N- (2'-phenoxy) -4-aminopyridine and said stabilizing agent M is 3- (N-morpholino) propanesulfonic acid. 50. A method for stabilizing a serine protease, which comprises mixing a serine protease with a) a reversibly active inhibitor of said serine protease; and b) a stabilizing agent M of the formula I

where n is 0, 1 or 2;
X is O, N or CH ₂ ;
R ¹ -R ⁴ are the same or different, and they are selected from H. —CH ₂ —R ⁶ , —CH ₂ —OR ⁶ , —CH ₂ —SR ⁶ , —CH ₂ —NH — R ⁶ , —CO-OR ⁶ , -CO-NH-R ⁶ , -CH ₂ -NH-CO-R ⁶ ,
—CH ₂ —O — CO — R ⁶ , —CH ₂ —NH — CO — NHR ⁶ , —CH ₂ —NH — CO — OR ⁶ , —CH ₂ —NH — CS — NHR ^6, and —CH ₂ —O— CO-NHR ⁶ ;
R ⁵ is the same as R ¹ -R ⁴ or PQ;
P is selected from - (CH ₂ ) _m - and - (CH ₂ ) _m —Y— (CH ₂ ) _m -, where m is 1-6 and Y is O, NH or S;
Q is selected from H, —SO ₃ , —COOH, —NH ₂ , —OH, and —CONH ₂ ;
each R ^{6 is} individually selected from H, substituted or unsubstituted lower alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted benzyl, substituted or unsubstituted aryl, or a mono-, bi- or tricyclic unsubstituted or substituted heteroaromatic ring (s) with one or more heteroatoms and non-aromatic heterocycles;
substituents of substituted groups are selected from lower alkyl, halogens, substituted or unsubstituted aryl, substituted or unsubstituted heteroaromatic compounds, non-aromatic heterocycles, alkyloxy, alkylamino;
or its pharmaceutically acceptable salts. 51. The method according to claim 50, wherein the serine protease is as defined in any one of claims 2, 4, 8, 10, 30, 31, 32, and 33. 52. The method of claim 50, wherein the reversible inhibitor is as defined in any one of claims 3, 5, 6, 7, 9, 11, and 34. 53. The method according to item 50, where the stabilizing agent M is as defined in any one of paragraphs 12-18 and 35. 54. The method according to p. 50, which further includes mixing a serine protease with a viscous and adhesive polymer selected from polysaccharides and gelatin. 55. The method according to item 54, where the viscous and adhesive polymer is as defined in any one of paragraphs.22-29. 56. The method according to any one of claims 50 to 55, wherein said reversible serine protease inhibitor is N- (2'-phenoxy) -4-aminopyridine and said stabilizing agent M is 3- (N-morpholino) propanesulfonic acid. 57. Device for vascular hemostasis, on which the composition according to any one of claims 1 to 36, where the serine protease in the composition is thrombin is adsorbed. 58. The device for hemostasis of blood vessels according to § 57, which contains a biodegradable material selected, in particular, from chitosan and collagen.