JP2002533473A - Pharmaceutical formulations containing hyaluronan degrading enzymes of microbial origin - Google Patents

Abstract

  (57) [Summary] The present invention relates to pharmaceutical formulations which are particularly suitable as injectable preparations which are applied therapeutically in vascular diseases resulting from atherogenic deposits on the inner wall of arteries. The injectable preparation contains hyaluronic acid lyase of bacterial origin, or a hyaluronan degradable fragment prepared from this enzyme. Hyaluronate degrading fragments of bacterial hyaluronate lyase are also an object of the present invention. Such fragments can be prepared by treating the holoenzyme with a specifically degrading protease.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] With age or in pathological conditions, hydrophobic plaques are deposited on the inner wall of the artery (intimal lining) in humans and mammals. This hydrophobic plaque results in arterial stiffness, lesions, and reduced lumen due to neointimal formation in blood vessels. Pathological conditions such as arrhythmias, clot formation and thrombosis, cerebral infarction, cerebral thrombosis, hypertension and reduced blood supply are closely associated with such vascular changes.

[0002] The present invention relates to a pharmaceutical composition having the property of reducing the area of the intima covered by plaque or reducing its effect on blood flow when used in an artery or vein. Composition. Such effects are closely related to the presence of intact enzymes (holoenzymes) and / or novel small enzyme fragments that can be made from such enzymes in the pharmaceutical formulations of the present invention.

[0003] The proposed holoenzyme is hyaluronate lyase, a fragment of which also has hyaluronan degrading activity. This activity leads in particular to a favorable degradation of hyaluronic acid. Furthermore, the present invention relates to new enzyme fragments obtained by a special enzymatic degradation process.

[0004] Hyaluronic acid belongs to the glycosaminoglycans along with chondroitin sulfate, dermatan sulfate, heparin and heparan sulfate. With the exception of hyaluronic acid, the glycosaminoglycans contain sulfate groups (which are sulfated glycosaminoglycans). Glycosaminoglycans are present in all vertebrate tissues. Hyaluronic acid is present, inter alia, in intracellular matrix, skin and cartilage, and body fluids such as joint fluid and aqueous humor of the eye. Hyaluronic acid, along with other glycosaminoglycans, is contained in the vascular wall and is probably also present in atherosclerotic deposits or atherosclerotic plaques on arterial lining. Hyaluronic acid has the property of combining with water, forming a viscous hydrocolloid liquid, and forming a poorly soluble complex with strongly basic proteins. Hyaluronic acid is composed of glucuronic acid and acetylated glucosamine linked by β- (1-3) -glycosidic bonds. The disaccharide units are then linked by β- (1-4) -glycosidic bonds. Hyaluronic acid is degraded by enzymes that are grouped under the generic name hyaluronidase.

[0005] The generic name hyaluronidase describes three hyaluronic acid-degrading enzymes that are not correct in a systematic enzymatic sense, but exhibit different actions (J. Ludowieg, "The Mechanism of Hyaluronidase"). , JBC, 236, 33
3-339 (1961)). These are endohydrolases that hydrolytically degrade β- (1-3) -bonds by adding water. These include most hyaluronidases from higher organisms, such as medically used hyaluronidases from bovine testes. These enzymes are also called hyaluronic acid-glycan hydrolases (EC 3.2.1.35/36),
It hydrolyzes hyaluronic acid and, to a limited extent, other glycosaminoglycan linkages. An endo-β-hyaluronidase from leech that degrades β- (1-4) -linkage very specifically is a further type.
All of these enzymes mentioned are hyaluronidases with hydrolytic activity, ie hyaluronidases in a more strict sense.

A third type of enzyme, hyaluronate lyase (EC 4.2.2.1)
Degrades hyaluronic acid at the β (1-4) -bond by an elimination mechanism involving the formation of a double bond at the (4-5) position of glucuronic acid. Thus, hyaluronate lyase is not a hyaluronidase. Hyaluronate lyase is present in microorganisms. Hyaluronic acid lyase has been found in, for example, Streptomyces actinomycetes and other bacteria. Its common characteristic property is its narrow specificity for hyaluronic acid. Degradation of other glycosaminoglycans is usually negligible (J.-H. Ozegowski et al., "Purification and characterization of hyaluronidase from Streptococcus agalactiae", Zbl. Bakt. 280, 497). ~ 50
6 (1994); Linker et al., "Production of unsaturated uronides by bacterial hyaluronidase", JBC, 219, 13-25 (1956); Ohya, Y
. Kaneko, "A new hyaluronidase of the genus Streptomyces", BBA
198 (1970), 607-609; Gase, J.M. -H. Ozego
wski and H.W. Malke, "HypB gene of Streptococcus agalactiae encoding hyaluronate lyase: complete sequence and expression analysis",
BBA, 1998, 86-98).

[0007] Most bacterial hyaluronate lyases have a molecular weight of 116,000 daltons to 1
It is a high molecular weight protein that is between 20,000 daltons. These high molecular weight hyaluronate lyases are generally relatively susceptible to denaturants and extreme physiological conditions. In particular, when preparing pharmaceutical and veterinary formulations, a significant degree of activity may be lost due to the denaturing process. Furthermore, the diffusivity of relatively large enzymes is significantly less than that of smaller molecules. As a result, the diffusion and simultaneous transport (eg, similarly into plaques) of high molecular weight hyaluronate lyase (holoenzyme) into tissues by permeation and / or diffusion is limited in theory. The immunogenic effect is believed to be lower.

[0008] Degrading proteins with the help of proteases in an enzymatic process to obtain protein fragments for sequencing that generally do not have enzymatic activity is state of the art in the art. Therefore, it should be theoretically possible to reduce an enzyme (holoenzyme) obtained from a microorganism by partial digestion with a protease having a specific activity. However, to date, the origin of enzyme fragments, particularly hyaluronate lyase, can be identified by the same portion of the amino acid sequence, or enzymatically active fragments of microbial hyaluronate lyase prepared from hyaluronate lyase were unknown. . Therefore, there is no known method by which enzymatically active fragments can be prepared from hyaluronate lyase.

Hyaluronidase from bovine testis has been used to increase the permeability of animal or human tissues. This hyaluronidase acts as a penetration enhancer or absorption enhancer for drugs administered subcutaneously or intramuscularly, or to facilitate perfusion of larger volumes of fluid and to regress edema more quickly. are doing. The good effect is already in the 1960's, in the case of acute tendonitis and paratenitis crepitans,
For venous treatment of plantar heel spines, and in dermatology, it has been obtained using a high dose of bovine testicular ylase "Dessau" administered intravenously. For example, in the case of progressive scleroderma, focal scleroderma and keloids, the symptoms are significantly reduced after hyaluronidase treatment.

In the 1970's, attempts were made to use hyaluronidase to affect the late sequelae of myocardial infarction in animal models. The purpose of this study was to improve peripheral blood circulation by reducing the necrotic area of the heart due to the accelerated formation of new blood vessels after infarction.

[0011] Since the 1960's, there are several references related to the use of bovine testicular hyaluronidase to improve the therapeutic effects of arterial vascular disease in humans, in particular, to improve blood circulation in peripheral blood vessels. (For example, Thrunherr and Koch, quoted by Wolff, "Results of Treatment with Mixed Intravenous Injection of Magnesium Composition / Hylas in Cases of Pelvic Type II Arterial Disease"
, Z. arztl. Fortbild. , 1972, 66, 446-447).
For example, Wolff investigated pelvic type II arterial disease by injecting intravenously (300 IU, three times a week, a combined injection over six weeks) hyaluronidase from bovine testes combined with magnesium ions. 90% of patients reported improved vascular blood circulation. Grosshennig describes ("Results of the treatment of degenerative arterial vascular disorders of the lower limbs by intravenous administration of magnesium composition" Schaffenberg "and hyaluronidase" (Dtsch. Ges.-w
Esen, 1965, 21, 869-872), performed a similar mixed procedure in patients with degenerative arterial vascular disorders of the lower limbs and large seated degenerative arterial occlusion of the pelvis type, with "brilliant results". Grosschennig is 300 IU
Was administered three times a week for a period of about 5 weeks. In a few severe cases, a total of 30 injections were given. The achieved exacerbation of the symptoms is 6 weeks to 8 weeks after the treatment.
It was not detected during the weekly follow-up test.

The hyaluronidase available from bovine testis was only available with relatively low activity, and it was therefore obviously very disadvantageous that it could only be administered with low enzymatic activity. For example, one month intravenous, intraperitoneal or subcutaneous administration of very low 300 IU of hyaluronidase activity in hypercholesterolemic rabbits had no effect on atherosclerotic vascular changes . Repeated administration of this amount over a period of up to three months reduces blood cholesterol content and plasma fibrin content, increases plasma free heparin content,
Increased vascular permeability in the tissue direction and also resulted in a marked reduction in atherosclerotic plaque (AI Persovskii, SI Koval'c).
huk, V .; A. Baronenko, R.A. N. Gold'man, S.M. Ya.
Guz and T.W. L. Fonbarova (1973), "Effect of hyaluronidase on the course of experimental atherosclerosis", Kardiologij
a, 13, 37-40).

[0013] An enzyme called hyaluronate lyase, which is described in British Patent No. 1,060,51.
G. 3, "Enzymes considered to be substantially homogeneous, are particularly effective hyaluronidases due to their broad specificity" obtained from animal tissues.
otlieb et al. This enzyme is described, for example, in British Patent 1,098.
, 957 and in mixture with hyaluronidase of animal origin in UK Patent 1,179,787 for the treatment of allergic conditions. Gottlieb also proposed that this hyaluronidase or hyaluronic acid lyase treat vascular diseases in humans, such as arrhythmias, thrombosis, cerebral infarction, cerebral thrombosis and myocardial infarction (particularly atherosclerosis) (US Patent No. 3,708,575). For example, 10,000 IU / mL of sterile isotonic solution is injected intravenously, intraarterially or intrathecally. This enzyme was obtained from animal material according to the information given in the patent (US Pat. No. 3,708,575). It is evident from the description of the present invention that the enzyme used was testicular hyaluronidase. The esterase activity observed for the enzyme, the broad specificity claimed, as well as the method used to measure the activity, which is not specific for the lyase reaction, are related to animal hyaluronidase. Therefore,
Enzymes with the specificity of degrading hyaluronate lyase obtained from animal testes have not been described in very recent literature (review: GL Frost, T. Csoka).
And R. Stern, Trends Glycosci. Glycotech
nol. 8, 419-434).

According to the information given in the literature, hyaluronidase obtained from bovine testes is capable of dissolving, degrading or permeable atherosclerotic deposits, and possibly also the components of blood clots and clots. It is suitable for increasing the permeability of blood vessels to blood flow due to the fact that A component of a blood clot that can be lysed can be a clot formed plaque particle that is separated from the intima. Furthermore, it has been shown that the use of hyaluronidase makes the blood vessel wall more permeable to the tissue as well as to its possible adjacent tissues.

[0015] However, a disadvantage of using hyaluronidases of animal origin, especially hyaluronidases obtained from bovine testes, is that, due to their origin, the enzymes, viruses, BSE (bovine spongiform encephalopathy) ) And other infectious agents. As a further disadvantage, for the production of hyaluronidase, a great deal of purification effort is required to remove impurities in the form of components derived from animal material, especially foreign proteins. Moreover, in order to obtain a sufficient amount of hyaluronidase, a large amount of starting material must therefore be collected under non-perishable conditions.

[0016] Hyaluronidase from mammalian testis also hydrolyzes sulfated glycosaminoglycans present on the vessel wall due to its non-specificity. However, dermatan sulphate or chondroitin sulphate, heparin and heparan sulphate are thought to prevent the clot from adhering to the inner wall of blood vessels due to its anticoagulant properties. Thus, the hydrolysis of these compounds may increase the secondary formation of coagulated blood on the vascular lining, and thus testicular hyaluronidase may also have undesirable side effects. is there.

Sawyer et al. (EU 193330 B1) describe the preparation and use of a special endo-β-glucuronidase with a molecular weight of 28,500 D obtained from leech to stimulate the flow of physiological fluids in the case of eye diseases. did. This enzyme is very specific for hyaluronic acid, and due to its greater stability at high temperatures and extreme pH values, it has been found that Hirudo medicinal
is different from the known endo-β-glucuronidase obtained from is).

DISCLOSURE OF THE INVENTION It is an object of the present invention to provide an enzyme that degrades plaque by using an enzyme obtained from a microorganism in a pharmaceutical formulation suitable for the treatment and prevention of atherosclerotic vascular changes. To avoid the shortcomings of previously proposed uses of hyaluronidases of animal origin. In principle, microbial enzymes are readily available because they can be produced by biotechnological means.

It is also an object of the present invention to produce in a simple manner an enzymatically active enzyme fragment having plaque-degrading activity from a suitable microbial holoenzyme, and to provide such a fragment as a pharmaceutical formulation Is to use. Fragments are more stable in pharmaceutical formulations, are less immunogenic when used, and can be expected to penetrate better.

Furthermore, the enzymes available according to the invention should not have a negative effect on living mammalian organisms when greater enzymatic activity is administered over a prolonged period. The enzyme should also degrade sulfated glycosaminoglycans to a limited extent, while lysing plaque and coagulated blood components comparable to that obtained by hyaluronidase obtained from animal testes. Should bring. On the other hand, the degradation of sulfated glycosaminoglycans in the intima is so limited that the risk of secondary formation of coagulated blood is due to the absence of sulfated glycosaminoglycans. Should not be too large.

Thus, according to the present invention, of microbial origin, it degrades plaques and reduces the plaque or clot components or the lining of blood vessels covered with coagulated blood as a component of pharmaceutical formulations used in the vasculature Sometimes it can be comparable to or better than the effect achieved with a formulation of hyaluronidase from bovine testis, and has a favorable effect on the development of vascular disease However, enzymes should be found that are non-toxic and have no other negative effects when used in humans or animals as injectable preparations.

A still further object of the present invention is to make available smaller proteins or fragments with hyaluronate lyase activity and to show how to make them. Such a fragment should have less enzymatic activity properties than the holoenzyme or have the same enzymatic activity properties as the holoenzyme.

Accordingly, the present invention includes pharmaceutical formulations suitable for the treatment of vascular diseases that occur directly or as a result of the formation of atherosclerotic plaque.

According to the present invention, hyaluronic acid lyase (EC 4.2.2.1), especially a microorganism of the genus Streptococcus, preferably Streptococcus agalactiae species b or Streptococcus sp. Aximilis (Streptococcus a
Pharmaceutical formulations containing one or more bacterial enzymes having the activity of a hyaluronate lyase secreted by fermentation into a steeping medium by fermentation, or an enzyme fragment made from such a hyaluronate lyase, It has been found that under in vitro conditions, material is released from isolated plaque deposits obtained from atherosclerotic vessels of mammals or humans, or to dissolve thin plaque sections.

In addition, an intravenous injection of a pharmaceutical formulation of the invention containing relatively large enzymatic activity and hyaluronate lyase and / or enzymatic fragments can be administered to live Watanabe rabbits, which have been treated in the aorta due to genetic defects. Repeated intravenous injections (with large deposits of plaque), it was found that the extent of plaque deposits in the blood vessels was significantly reduced after treatment. The interior of the blood vessels of the untreated animals had their surfaces covered with a thin whitish layer, as well as thicker plaques. On the other hand, the inside of the blood vessels of the treated animals was free of such thinner plaque deposits and was a deep red color on the healthy inner surface of the blood vessels. The area coverage of the thicker plaque deposits was also reduced.

This effect is demonstrated in pharmaceutical formulations where the molecular weight is between 114,000 D and 124.0
It is not limited to the presence of the holoenzyme in the range of 00D, and the molecular weight is, for example,
It is surprising that 84,000-86,000 can also be obtained with highly active and stable fragments of the holoenzyme.

Furthermore, it has been found that the fragments of the present invention can be prepared by proteolytic digestion of bacterial hyaluronate lyase with a protease, preferably a protease that cleaves the C-terminal peptide bond of aromatic amino acids. Was done. In a preferred embodiment, a 116,000 dalton hyaluronate lyase obtained from Streptococcus agalactiae is degraded into an enzymatically active fragment of hyaluronate lyase. This fragment has a molecular weight of 84,000 to 86,000 daltons. In its degradation specificity, the fragments are not different from undigested hyaluronate lyase (holoenzyme). However, in comparison with the holoenzyme, the fragments are distinguished by a distinctly greater stability in aqueous solution and in the presence of denaturing agents. This enzyme has a specific activity between 400,000 IU / mg and 800,000 IU / mg.
This is the same as, or even greater than, the specific activity of the holoenzyme of 400,000 IU / mg.

Accordingly, the present invention relates to the use of a microbial hyaluronate lyase as a holoenzyme
At least one stabilizing agent and / or a pharmaceutically safe carrier or an inert substance, each in a highly purified form, as a fragment thereof which degrades hyaluronic acid, or as a mixture of both forms, And a pharmaceutical formulation containing a pharmaceutically safe diluent, as needed.

The pharmaceutical formulations of the present invention are primarily used in mammals (humans and animals)
Suitable for the treatment of vascular diseases attributable to the presence of atherosclerotic plaque in blood vessels such as arrhythmias, atherosclerosis, cerebral infarction, cerebral thrombosis, coronary thrombosis and myocardial infarction.

Pharmaceutical formulations prepared according to the invention are injectable preparations, especially for intravenous or arterial injection, wherein the highly purified enzyme protein of the holoenzyme and / or fragments or both thereof Is an injectable preparation consisting of a sterile isotonic aqueous solution of a defined mixture of active enzyme species:
It is between 00 IU / mL and 4,000,000 IU / mL. The enzyme protein is conveniently used in the form of a lyophilized solid, usually containing stabilizing additives.
Examples of stabilizing additives include sodium chloride, glucose, magnesium salt, polyvinylpyrrolidone, amino acids, albumin (particularly, ovalbumin) and its hydrolyzate, or cereal protein and its hydrolyzate. The amount of hyaluronate lyase and / or fragment thereof used in the injection can range from 2,000 IU / kg body weight of the animal or human being treated.
It is between 12,000 IU.

The enzymes or fragments thereof used according to the invention do not show a negative effect on the health of the experimental animals. The proposed enzymes and / or fragments thereof are not limited to the actual removal of deposits, but circulatory diseases such as myocardial ischemia, coronary infarction, myocardial infarction, arrhythmias, atherosclerosis and similar manifestations It is evident that it also has advantageous effects in the sense of prevention and treatment of and is part of the protection scope of the present invention. In the application of the present invention, fragments have the advantage over holoenzymes because they can penetrate more rapidly into the solid layer of plaque as a result of their smaller size, and thus can result in their degradation more quickly. I was

The present invention is not limited to hyaluronate lyase or a fragment thereof derived from a particular microorganism. However, the present invention is illustrated using, as an example, the commonly available Streptococcus agalactiae enzyme. The resulting hyaluronate lyase has an isoelectric point of about 8.6, a molecular weight of about 116,000 D, and acts as an endoglycanase. This enzyme also has the advantageous property of being inhibited by sulfated glycosaminoglycans such as hyaluronic acid. According to the purification methods described below, hyaluronate lyase is obtained as a highly purified enzyme for injection purposes, or the purified hyaluronate lyase is converted to a fragment by a protease of specific activity. While not limiting the invention, the microorganism Streptomyces hygroscopicus (Strept
Acidic metalloprotease MO / 2 (DD270924) obtainable from Omyces hygroscopicus AP40 can be used, for example, as a specifically acting protease. This protease contains about 14,
It is distinguished by a molecular weight of 000 kD and an isoelectric point between 3.85 and 4.0.

The following detailed description of the production of holoenzymes as well as the production of fragments, in particular the sequence of purification steps, is also given by way of example and is not intended to limit the scope of the invention. Production of highly pure hyaluronate lyase and highly purified fragments in a pharmaceutical formulation suitable for injection can be performed, for example, as follows. With respect to the order and the selection of the purification steps, the scope of the invention is not limited to the described method.

The fermentation of microorganisms (eg, any of the above microorganisms) is carried out in a stirred fermentor at pH
It is carried out at a constant pH with an acidity of 6.5 to 7.5. Lactic acid produced during fermentation is neutralized by adding dilute sodium hydroxide. The medium consists of mineral salts, yeast hydrolysate, casein or soy peptone, as required, and glucose. The cells are separated after about 20 hours of fermentation. The cell mass is discarded. The culture filtrate is concentrated and purified by an ultrafiltration device. The exclusion limit (cutoff) of the ultrafiltration filter module is between 30 kD and 50 kD. A pre-purified enzyme solution is obtained, which must be subjected to a further purification step and can then be used as a preparation for injection. The absence of pyrogen in the injectable preparation is assured by the purification step and by using an inert substance which does not contain pyrogen.

After increasing the ionic strength by adding ammonium sulfate until the saturation reaches 40%, the enzyme is adsorbed on phenyl sepharose. Subsequently, the enzyme is eliminated with a buffered neutral aqueous solution containing 25% of the amount of ammonium sulfate required for 100% saturation. After the dialysis step to remove impurities, the solution was washed with Q-Sepharose (Phara
macia). Thereafter, the dye is specifically adsorbed to a dye such as aminophenyl oxamic acid. The final purification, together with the molecular weight measurement, was performed with Superdex (Pha
rmacia).

[0036] Streptococcus equi
When S. similis is used to obtain the enzyme, the purification step for adsorption to the dye can be omitted. This enzyme acts as an exoglycanase. Its isoelectric point is between pH 4.5 and pH 4.8. This enzyme is only inhibited to a very small extent by sulfated glycosaminoglycans.

The partial digestion of the holoenzyme into fragments is performed using a specific protease. Such a protease is immobilized or not.
The reaction with the immobilized protease is convenient because the protease can be selectively digested without contamination by the protease itself. In the case of a direct conversion performed with the addition of a protease, the digestion with the protease must be followed by an inactivation step, the separation of the protease proteins and a high degree of purification. The enzyme fraction can be used to generate fragments in the following manner.

The specifically degrading protease MO / 2 is bound in a known manner, for example to Sepharose. The immobilized protease is mixed with an aqueous solution of hyaluronate lyase, for example, at a pH of 7.5 and a temperature of 37 ° C. After digestion is complete, the fragment is precipitated with ammonium sulfate and obtained in high purity form by molecular weight chromatography. After immunization with rabbits, only highly purified enzymes or enzyme fractions show specific precipitation. All bands detectable on the blot are stained with the monoclonal antibody. Holoenzyme is about 400,0
It has a specific activity of 00 IU / mg and the specific activity of the fragment is 400,000 IU / mg.
mg to 800,000 IU / mg. For stabilization, at least one stabilizing agent such as albumin (preferably, ovalbumin) and an inorganic salt is added to the enzyme or fragment.

One method for obtaining the fragments is described, but the invention is not limited thereby. According to the present invention, a protease that cleaves a peptide bond at the C-terminal side of an aromatic amino acid is used for digestion. In a preferred embodiment, metalloprotease MO / 2 obtained from a culture filtrate of Streptomyces hygroscopicus (strain AP40) is used. MO / 2 is a metalloprotease with Co ⁺⁺ at the active center (DD270924). The protease is preferably used in purified form. Protease MO / 2 is, for example, phenyl sepharose, DEAE sepharose, Q-sepharose and sephacryl (Sephacryl).
acryl) Purified by chromatography on S100 at a 20-fold concentration factor. Specific activity was 0.25 Kunitz units / mg (U / mg
), With the maximum reaction occurring at an acidity of pH 4.2 and a maximum reaction temperature of 65
° C. SDS gel electrophoresis and Sephadex G5
0 Molecular weight of enzyme determined by molecular weight chromatography with Superfine (
Mm) is from 14,000D to 15,000D. Protease MO / 2 is 3
. Endopeptidase with an isoelectric point of 85 and an isoelectric point of 3.92. This enzyme hydrolyzes natural polypeptides such as casein, hemoglobin, bovine serum albumin and ovalbumin.

It is advantageous to use protease MO / 2, which degrades the enzyme protein very specifically and has little digestive activity on the protein. This enzyme
Almost exclusively cleaves peptide bonds at the C-terminal group of aromatic amino acids. This enzyme has transesterification activity on N-benzoyl-L-proline nitroanilide and outstanding milk precipitation properties. Milk sedimentation properties, which can be detected by the formation of casein gels with long-term stability comparable to that of gels precipitated by laboratory enzymes with very specific actions, are comparable to those of fragments. The limited activity of protease MO / 2, which specifically degrades the binding of holoenzymes without resulting in, can be evaluated as a further indicator of very advantageous activity in the context of the present invention.

In a further embodiment of the invention, the protease MO / 2 is optionally bound to a suitable insoluble immobilized support and used in this form to degrade holoenzymes. This approach has the advantage that transfer of the dissolved protease to the fragment solution is prevented in this way.

According to the present invention, the fragment is obtained by partial digestion or proteolytic partial degradation of the holoenzyme by a protease having a specific cleavage activity, which preferably cleaves the peptide bond at the C-terminal side of the aromatic amino acid. It is made.

The high-purity enzyme solution or fraction containing the fragments can be concentrated by partially removing water and, if necessary, stabilizing agents, inert substances or pharmaceutically active substances. After the substance has been added and sterile filtered, it can be used as an injectable preparation. High purity solutions of enzymes or fragments can be lyophilized after sterile filtration and addition of, for example, 5 mM magnesium chloride and 1% ovalbumin. The lyophilized enzyme or fragment can be dissolved in a salt solution to make an isotonic injectable preparation. The invention also relates to the use in mammals and humans of hyaluronic acid lyase of microbial origin, fragments thereof that degrade hyaluronic acid or a mixture of both forms, comprising arrhythmia, atherosclerosis, cerebral infarction, cerebral thrombosis , Coronary artery thrombosis and myocardial infarction, for use in treating vascular diseases attributable to the presence of atherosclerotic plaque in blood vessels.

A further object of the invention relates to a fragment of a bacterial hyaluronate lyase having hyaluronate lyase activity.

The following examples serve to illustrate the invention but do not limit it in any way.

Examples Example 1 Fermentation is carried out in a stirred fermenter with a total volume of 30 L. The working capacity is 20L. For inoculation, Braunschweig's Deutschen Sammlun
g von Mikroorganismen und Zellkultur
No. 2134 ^γ (= ATCC13813 = NCT)
A commonly available strain of Streptococcus agalactiae sp. Stored in C8181) is used. The storage for storage is performed using Kryokonserve (Mas
(t-diagnostica). The spheres covered with the stock are added to the microbe counting agar slant agar tube and brought into contact with the agar surface by moving (
Culture A). Subsequently, the rolled spheres are added to 3 mL of heart-brain broth to check for sterility (Culture B). Both cultures were grown upright at 37 ° C. for 2 hours.
The cells were cultured for 4 hours.

Initial Pre-Culture A medium (2 × 50 mL) containing 5 g / L casein peptone and 10 g / L yeast extract (Difco) was added to two 100 mL Steilbruss
t "flask and sterilize at a pH of 7.0. In addition to 50 mL of culture medium,
5 ml of Eagle's medium are added in each case immediately before inoculation. Inoculation is performed by rinsing the slant agar tube (Culture A) with the culture solution of Culture B. Incubate the “Steilburst” flask with shaking (at 150 rpm) at 37 ° C. for 24 hours.

Second Preculture 20 g / L yeast extract and 10 g / L pancreatic peptone, 1.75 g / L sodium acetate, 7 g / L sodium bicarbonate (dissolve separately), 7 g / L
(2 x 1 L) of disodium hydrogen phosphate dihydrate and 3.5 g / L sodium dihydrogen phosphate is adjusted to pH 6.2 with 20% sulfuric acid and then sterilized. After autoclaving, the pH is about 7.0. A 6.0 g / L glucose solution is autoclaved separately and 100 mL of this is added to the second pre-culture prior to inoculation. Inoculation is performed by adding 50 mL of the first preculture (
5% v / v). Medium at 32 ° C. with gentle shaking at about 45 rpm
For 24 hours.

Main Culture The medium of the main culture consists of 20 g / L yeast extract, 10 g / L pancreatic peptone and 25 g / L glucose. Glucose is sterilized separately. Inoculation is 1L
Using the second preculture. The operating conditions for the fermentation were 34 ° C., pH 7.0,
Headspace ventilation (23 l / min air) and 150 rpm stirring speed.
The pH is kept constant by dropwise addition of a 40% sodium hydroxide solution.
Glucose is added manually so that the glucose concentration does not fall below 5 g / L. The culture is stopped after 20 hours.

Example 2 A culture solution (50 L) having a hyaluronate lyase activity of 400 IU / mL obtained by fermenting a Streptococcus agalactiae species is subjected to sterile filtration with a 0.2 μm filter to remove viable cells. Subsequently, the clear culture filtrate was cut off at 60
Concentrate to 2 L by ultrafiltration at 2,000 D. Solid ammonium sulfate (480 g)
Is added to this 2 L culture concentrate. Proteins that precipitate at this concentration are removed by clarification filtration using filter aids. The clear concentrate is then applied to a 2.5 × 20 cm phenyl sepharose column previously equilibrated with a phosphate buffered 40% ammonium sulfate solution at pH 6.5. After washing with a phosphate buffered pH 6.5 35% ammonium sulfate solution, the hyaluronate lyase is eluted with a phosphate buffered pH 6.5 25% ammonium sulfate solution. The eluted fractions are pooled and added by adding solid ammonium sulfate.
Adjust to 0% saturation. The precipitate is collected and 100 mL of 0.03 M Tris buffer (pH
8. Dissolve in 2) and dialyze against the same buffer. Subsequently, the dialyzed crude hyaluronate lyase is added to an equilibrated 2.2 x 15 cm Q-Sepharose column. Hyaluronate lyase is not adsorbed and is found in the eluate. The eluate is brought to 80% saturation by adding solid ammonium sulfate. The precipitated protein is collected, dissolved in 0.05 M Tris buffer (pH 8.7), and ammonium sulfate is removed using a Sephadex G10 column. The hyaluronate lyase solution so equilibrated is transferred to a properly equilibrated affinity column (0.8 cm × 10 cm) of N- (p-aminophenyl) oxamate-agarose and
. Elute with 1M sodium carbonate solution (pH 9.7).

The eluted fractions containing hyaluronate lyase are combined and precipitated by bringing the saturation to 80% with ammonium sulfate. The precipitate was collected, dissolved in 1 mL of 0.1 M Tris buffer (pH 7.5), and dissolved in a Superdex 200 column (16 mL).
x60). The protein band of 116,000 D hyaluronate lyase was collected and 0.02 M Tris buffer containing 0.005 MgCl ₂ (pH 7.0).
Dialyze against 5). Hyaluronate lyase was obtained in a yield of 10 mg and 400 mg.
Obtained at a specific activity of 2,000 IU / mg. Ovalbumin is added to the dialyzed hyaluronate lyase solution at a concentration of 1% and the solution is lyophilized.

Example 3 Corresponding to Examples 1 and 2, pre-culture and cultivation of a microorganism of the species Streptococcus eximiris is carried out to obtain the enzyme in pure form. Purification using N- (p-aminophenyl) oxamic acid is omitted.

Example 4 Purified protease MO / 2 (10 mg) was dissolved in 2 mL of 0.1 M sodium bicarbonate solution and 1 mL of bromocyan sepharose (Pharmaci)
Add a) and shake at 4 ° C for 12 hours. Subsequently, the solution containing unbound MO / 2 is separated from the resulting MO / 2 protease Sepharose using a sintered glass filter. Subsequently, the unsaturated bromocyan sepharose binding sites are blocked by reaction with 2 mL of a 0.1 M glycine solution. After blocking, the MO / 2 Sepharose is washed with 0.1 M acetic acid (pH 4.5) and 0.1 M sodium bicarbonate solution.

The hyaluronate lyase obtained from Streptococcus agalactiae (1
0 mg) is dissolved in 1 mL of 0.05 M phosphate buffer (pH 7.0), and the temperature is adjusted to 37 ° C. in a thermostat. Immobilized MO / 2 protease Sepharose (0.25 g) conjugated with 0.1 Kunitz units of proteolytic activity is added to this solution.
After 15 minutes, the protease Sepharose is separated and the resulting fragment is precipitated from solution by 80% saturation with ammonium sulfate. After 18 hours, collect the precipitate and dissolve in 0.05 Tris buffer (pH 7.5). F fragment
Purify by molecular weight chromatography on upperdex 200. The active fragment fractions are pooled, dialyzed against 0.05M Tris buffer, and lyophilized with albumin. This fragment has a specific activity of 600,00
0 IU / mg.

Example 5 Hyaluronate lyase obtained from Streptococcus agalactiae (4
mg) containing 1 mL of Tris hydrochloride buffer (pH 7.8) in a thermostat at 37 ° C.
Temperature. To this solution was added a protease MO having a specific activity of 0.1 Kunitz units / mg.
Add 80 μg of / 2. This mixture is digested at 37 ° C. for 30 minutes. To stop the reaction, add 10 μL of 0.1 M EDTA (pH 7.0). The mixture is then transferred to a 3 mL affinity column of N- (p-aminophenyl) oxamic acid-agarose and eluted with 0.05 M sodium carbonate (pH 9.7). The fragments were collected by precipitation by 80% saturation with ammonium sulfate and purified by molecular weight chromatography. The active fragment fractions were pooled, dialyzed against 0.05 M Tris buffer, and lyophilized with albumin. This fragment has a specific activity of 460,000 IU / mg.

Example 6 A solution (0.5 mL) of a hyaluronate lyase fragment (Example 4) was added to 0.1 mL of a solution.
Add to 5 mL of 0.1 M acetate buffer (pH 6.0) and dilute in an isometric series. Subsequently, 0.5 mL of a hyaluronic acid solution containing 0.2 mg of hyaluronic acid per mL of 0.1 M acetate buffer (pH 6.0) is added to each dilution. Subsequently, the mixture is incubated at 37 ° C. for 30 minutes. The enzymatic reaction is stopped by treating each dilution with 2 mL of 2.5% cetyltrimethylammonium bromide solution / 2% sodium hydroxide solution. After standing at room temperature for 20 minutes, the turbidity of each dilution is measured at 600 nm in a 1 cm cuvette, and the amount of degraded hyaluronic acid is determined using a calibration curve. Five International Units (IU) of a hyaluronic acid lyase fragment degrades 16 μg of hyaluronic acid per minute.

Example 7 The holoenzyme (20,000 IU) and the 20,000 IU fragment (Example 4) are dissolved in 3 mL of distilled water. Both solutions are kept at room temperature for 24 hours. Subsequently, the enzyme activity is measured. The activity of the holoenzyme solution drops to 60% during this time, while the activity of the fragment solution is 95% of the activity of the starting solution.

Example 8 Activity measurement of hyaluronic acid lyase or a fragment thereof 1. Optical test at 232 nm Storage solution of hyaluronic acid (HA) and sulfated hyaluronic acid (mg / mL)
And store at 4 ° C.

The standard test stylization was performed as follows, using a 3 mL quartz cuvette (d = 1c
m) at a wavelength of 232 nm and a temperature of 26 ° C. 1.8 mL of 0.1 mL
Add 200 μL of a stock solution of hyaluronic acid to 1 M acetate buffer (pH 6.0) and start the reaction with 2-4 U / mL (50 mL) of hyaluronic acid lyase. Final volume is 2050 μL.

For the inhibition experiments, the above standard test substances were modified as follows. 1.5m
The reaction is started by adding 300 μL-500 μL of sulfated hyaluronic acid and 200 μL of hyaluronic acid to L-1.8 mL of buffer and adding hyaluronate lyase. Hyaluronate lyase is partially inhibited non-antagonistically. The Ki value is 5.5 × 10 ⁻⁴ mg / mL.

The hyaluronate lyase linearly increases the absorbance at 232 nm, and the activity can be determined from the slope. The calculation is based on the absorption coefficient (ε = 6.0 × 10 ⁻³ L / mole / cm) of the generated Δ ^−1.5 unsaturated uronide (L. Ludowi).
g, "Mechanism of hyaluronidase", JBC, 236, 333-339 (1991)
)).

[0062] 2. B. Optical test at 600 nm in microplate Gerlach, W.C. Kohler, "Streptococcus pyogenes (Streptoco
hyaluronic acid lyase of C. pyogenes II. Characterization of hyaluronate lyase (EC 4.2.2.1) ", Zbl. Bakt. Hyg.
, I. Abt. Orig. , A221, 296-302 (1972) Measurement of lyase activity The hyaluronate lyase solution was mixed with a stock solution (50,000 U / mL) at a ratio of 1: 5.
Prepared by dilution at 0. Hyaluronate lyase solution (0.5 mL)
Is added to 0.5 mL of 0.1 M acetate buffer (pH 6.0) and diluted in an isometric series. Subsequently, 0.5 mL of a hyaluronic acid solution containing 0.2 mg / mL of 0.1 M acetate (pH 6.0) is added to each dilution. Subsequently, the mixture is incubated at 37 ° C. for 30 minutes. The enzymatic reaction is stopped by treating each dilution with 2 mL of 2.5% cetyltrimethylammonium bromide solution / 2% sodium hydroxide solution. After 20 minutes at room temperature, the turbidity of each dilution
The amount of degraded hyaluronic acid is determined using a calibration curve by measuring with a 1 cm cuvette at m. The five international units (IU) of the hyaluronate lyase fragment
Decomposes 16 μg of hyaluronic acid per minute.

Example 9 Hyaluronate lyase (holoenzyme, 10 mg) is dissolved in 1 mL of 0.05 M phosphate buffer (pH 7.0) and brought to a temperature of 37 ° C. in a thermostat. To this solution is added 0.25 g of a specifically degrading protease Sepharose having a proteolytic activity of 0.1 Kunitz units / mg. After 15 minutes, the resulting fragment, except for the insoluble protease Sepharose, was removed from the solution with ammonium sulfate.
Precipitate by% saturation. After 18 hours, the precipitate is removed, and the cells are dissolved in 0.05 M Tris buffer (pH 7.5). The fragment is purified by molecular weight chromatography on Superdex 200. The active fraction is purified, dialyzed against 0.05M Tris buffer and lyophilized with albumin.

Example 10 To detect the plaque lytic activity of hyaluronate lyase, an approximately 0.3 mm whitish yellow tissue piece (plaque fragment) obtained from the neointima of human carotid intima ) With a hyaluronate lyase solution (1 mL of Tris buffer (10 mM, pH 7.0).
0) at about 20,000 IU) and incubated at room temperature for 12 hours. The previously sharp phase boundaries of the plaque pieces are partially degraded, releasing a suspended fine dispersion. In a concurrent test, plaque pieces were treated with Tris buffer only. In the latter case, there was no sign of degradation.

Example 11 Attempt to Degrade Atherosclerotic Vascular Deposits of Human Origin by Hyaluronate Lyase Fragment Approximately 0.3 mm of whitish yellow tissue obtained from the neointima of human carotid intima A piece (plaque piece) was added to the fragment solution (1 mL of Tris buffer (10 mM,
(pH 17.0) at about 15,000 IU) and incubated for 12 hours at room temperature. The previously sharp phase boundaries of the plaque pieces are extensively degraded, releasing a suspended fine dispersion. In a concurrent test, plaque pieces were treated with Tris buffer only. In the latter case, there was no sign of degradation.

Example 12 Watanabe rabbit Ibm: Effect of hyaluronate lyase on atherogenic vascular deposits in WHHL (Watanabe hereditary hyperlipidemia). This rabbit is 1981
Year after, Y. Watanabe (Kobe University, Japan) and was bred to Charles River Savo in 1992 as a breeding line, and since 1994 the Broekman Institute (Som
eren, The Netherlands).

[0067] Watanabe rabbits have hereditary hyperlipidemia caused by a genetic defect in the LDL receptor. Hypercholesterolemia (> 400mg / 100
As a result, all animals develop aortic atherosclerosis early.

Fourteen days prior to the start of the experiment, 10 watanabe rabbits (I
bm: WHHL; 4 females and 6 males) from Broekman Institute
ute (Someren, The Netherlands). Animals are caged one by one (0
. 5 × 1 m) and bred at room temperature of 20 ° C. ± 2 ° C. Standard feed for animals (
ssniff Kanichen-Haltung) and drinking water were available ad libitum. Before starting the experiment, ie before the first clinical chemistry study, 10 rabbits were gendered, randomly removed and numbered 1-10.

The animals received infusion three times a week (Monday, Wednesday and Friday). Four control group rabbits (1-4) were given 20 ml of pyrogen-free 0.9% sodium chloride solution within one hour in each case of intravenous (iv) administration. Six experimental animals (5-10) were in each case 1 hour
30,000 IU of highly purified hyaluronate lyase was injected intravenously with a 20 mL injection volume of pyrogen-free 0.9% sodium chloride solution. This corresponds to a dose of 10,000 IU / kg body weight.

During the experiment, overall behavior, body weight, body temperature and clinical chemistry parameters (cholesterol, triglycerides and amylase) were examined.

The overall behavior of the animals was not significantly affected by treatment with either 0.9% sodium chloride solution or hyaluronate lyase. Fully grown Watanabe rabbits (control group or hyaluronate lyase treated group) weighed 9
There was only a slight change during the treatment period from November 18, 2006 to February 3, 1997. Body temperature measured before and after each infusion is -0.3 ° C to + 1.2 ° C
Range. There were no significant differences between control group rabbits and hyaluronate lyase treated rabbits. The serum cholesterol level of the control group animals did not change significantly by the injection of 0.9% sodium chloride. In one of the four animals, serum cholesterol levels were halved. Hyaluronate lyase treatment also did not significantly alter serum cholesterol levels. Under the infusion procedure, occasionally very large serum glyceride levels were reduced in all animals. After the 20th injection, the value of the control group was 570 mg / dL.
７740 mg / dL, the value of hyaluronate lyase treated rabbits was 200
mg / dL to 290 mg / dL.

In two of the four control group rabbits, the aortic arch was severely atheromatous and in two of the four birds moderate to severe atheromatous. In two of the six hyaluronate lyase-treated rabbits, the aortic arch was moderately atheromatous and in four of the six birds mild to moderately atheromatous. In two of the four control rabbits, the thoracic aorta was severely atheromatous and two of the four were moderately atheromatous. In four of the six hyaluronate lyase-treated rabbits, the thoracic aorta was mildly atheromatous and two out of six
Almost no coating could be detected on the wings. In two of the four control rabbits, the abdominal aorta was severely atheromatous and two out of four
Feathers were moderately atheromatous. Moderate in two of six hyaluronate lyase-treated rabbits, and weak to mild film could be detected in four of the six rabbits. In two of the four control group rabbits, the pulmonary aorta was heavily covered with plaque and two of the four were mild to moderate. In one of the six hyaluronate lyase rabbits, the pulmonary aorta was heavily covered with plaque, and two of the six birds were lightly to moderately covered with plaque, but three out of six. No plaque was present in the feather.

The overall behavior, weight development, body temperature before and after injection, and clinical chemistry parameters of Watanabe rabbits treated with 0.9% sodium chloride solution and hyaluronate lyase were two injections, each of ten injections Showed no significant difference during the treatment cycle. Thus, it can be concluded that the infusion is well tolerated.

According to a subjective assessment of the gross aortic material, the extent of the atherosclerotic capsule was greater than that of the six Watanabe rabbits treated with the microbial hyaluronate lyase.
It is revealed that four Watanabe rabbits treated with 9% sodium chloride solution are considered more severe.

Example 13 A concentrated enzyme solution of high purity was diluted with a sufficient salt solution to obtain a solution having a concentration of 0.1 mM.
Contains 9% salt and 10,000 IU / mL hyaluronate lyase. This solution is sterilized and filtered through a sterilizing filter having a pore size of 0.2 μm. Add 1 mL of this solution to an ampoule under sterile conditions. The ampoule is lyophilized under aseptic conditions and sealed under aseptic conditions. Add 1 mL of sterile distilled water before injection.

Example 14 A concentrated enzyme solution of high purity was diluted with a solution of magnesium nicotinate and magnesium adipate (magnesium composition “Schaffenberg”) to give 25 mg of magnesium nicotinate and 88 mg Of magnesium adipate and 10,000 IU / mL of hyaluronate lyase. This solution is placed in an ampoule as in Example 13.

Example 15 A highly pure solution of hyaluronate lyase is diluted with a solution of salt and ovalbumin so that the solution contains 40,000 IU / mL, 0.9% salt and 1% ovalbumin . This solution is further processed as described in Example 13.

Example 16 A solution of high purity hyaluronate lyase was diluted with a solution of salt and soy protein hydrolysate to give a solution of 40,000 IU / mL, 0.9% salt and 1%
(Prepared from soybean protein hydrolyzate).
This solution is further processed as described in Example 13.

Example 17 A solution of a highly pure fragment obtained from the holoenzyme of Streptococcus agalactiae was diluted with a solution of salt and ovalbumin to give a solution of 40,000 I
U / mL, containing 0.9% salt and 1% ovalbumin. This solution is further processed as described in Example 13.

Example 18 A highly pure and highly concentrated solution of a fragment obtained from Streptococcus agalactiae was diluted with sufficient salt solution and magnesium chloride to give a solution of 1%.
Contains lyase activity of 000 IU / mL. This solution is sterilized and filtered through a sterilizing filter having a pore size of 0.2 μm. 1 mL of this solution
Add to each ampoule. The ampoule is lyophilized under aseptic conditions and sealed under aseptic conditions. Add 1 mL of sterile distilled water before injection.

SUMMARY OF THE DISCLOSURE The present invention relates to pharmaceutical formulations which are particularly suitable as injectable preparations for therapeutic application in vascular diseases resulting from atherogenic deposits on the inner wall of arteries. The injectable preparation contains hyaluronic acid lyase of bacterial origin, or a hyaluronan degradable fragment prepared from this enzyme. Hyaluronan degradable fragments of bacterial hyaluronate lyase are also an object of the present invention. Such fragments can be prepared by treating the holoenzyme with a specifically degrading protease.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61P 7/02 A61P 9/10 9/06 C12N 9/88 9/10 A61K 37/56 C12N 9/88 37 / 18 (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF , BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, TZ) , UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZA, ZW (72) Inventor Albert Hertre Germany-07749 Jena Oscar-Sachau-Strasse 10 (72) Inventor Gundera Peschel, Germany Day-07743 Jena Cross Witzer Strasse 21 game F-term (reference) 4B050 CC02 DD02 LL01 4C076 AA12 BB13 CC11 CC14 DD22 DD67 FF11 FF36 4C084 AA01 AA02 AA03 BA22 BA43 Z 45 ZA54 ZC20

Claims (22)

[Claims] 1. A hyaluronic acid lyase of bacterial origin which is contained as a holoenzyme, as a hyaluronic acid degradable fragment thereof, or as a mixture of both forms, and at least one stabilizing agent and / or a pharmaceutically safe carrier. Or a pharmaceutical formulation containing an inert substance. 2. The pharmaceutical formulation according to claim 1, wherein the hyaluronic acid lyase is derived from a microorganism of the genus Streptococcus. 3. The pharmaceutical formulation according to claim 1, wherein said hyaluronic acid lyase is derived from a microorganism of Streptococcus agalactiae. 4. The bacterial hyaluronate lyase in the form of a holoenzyme has an isoelectric point in the range of 8.6 and a molecular weight in the range of 116 kD.
A pharmaceutical formulation according to claim 1. 5. The pharmaceutical formulation of claim 1, wherein said fragment of hyaluronate lyase has a molecular weight in the range of 84 kD to 86 kD. 6. The pharmaceutical combination according to claim 1, for treating a vascular disease selected from the group consisting of arrhythmia, atherosclerosis, cerebral infarction, cerebral thrombosis, coronary thrombosis and myocardial infarction. object. 7. The method according to claim 1, which is obtained from bacterial hyaluronate lyase by partial enzymatic hydrolysis by a protease and contains a hyaluronan degradable fragment of hyaluronate lyase that cleaves the C-terminal group of aromatic amino acids. A pharmaceutical formulation as described. 8. The pharmaceutical formulation according to claim 1, which is an injection preparation for intravenous or arterial injection. 9. The activity of said hyaluronate lyase and / or fragment thereof of microbial origin is from 20,000 IU / mL to 4,000,000 IU / mL.
The pharmaceutical formulation according to claim 1, wherein the pharmaceutical formulation is between: 10. The pharmaceutical of claim 1, comprising a purified aqueous injection containing hyaluronate lyase and / or a fragment thereof and about 1% (w / w) albumin and / or a hydrolyzate thereof. Compound. 11. The pharmaceutical formulation according to claim 1, comprising a purified aqueous injection containing hyaluronate lyase and / or a fragment thereof and a magnesium salt. 12. The pharmaceutical formulation according to claim 1, comprising a purified aqueous injection containing hyaluronate lyase and / or a fragment thereof and glucose. 13. An enzymatic fragment of bacterial hyaluronate lyase having hyaluronate lyase activity. 14. The enzyme fragment according to claim 13, wherein the hyaluronate lyase is derived from a microorganism of the genus Streptococcus. 15. The enzyme fragment according to claim 13, wherein said hyaluronic acid lyase is derived from a microorganism of Streptococcus agalactiae. 16. The enzyme fragment according to claim 13, which is obtained from bacterial hyaluronate lyase by partial enzymatic hydrolysis by a protease and cleaves the C-terminal group of aromatic amino acids. 17. The method according to claim 1, wherein the bacterial hyaluronate lyase is obtained by subjecting it to partial enzymatic hydrolysis by a protease in a form immobilized on a carrier.
4. The enzyme fragment according to 3. 18. The enzyme fragment according to claim 13, which is obtained by subjecting bacterial hyaluronate lyase to a partial enzymatic hydrolysis with a metalloprotease MO / 2 in a form immobilized on a carrier. 19. The method according to claim 19, wherein the hyaluronic acid lyase obtained from the streptococcus is 6.5 to 6.5.
14. The enzyme fragment according to claim 13, obtained by subjecting it to a partial enzymatic hydrolysis with a metalloprotease MO / 2 at an acidity in the pH range of 8.0 and a temperature in the range of 25C to 45C. 20. Streptococcus agalactiae having a molecular weight of about 115,000 D to 120,000 D (Streptococcus agala).
14. The enzyme fragment according to claim 13, obtained by subjecting the hyaluronate lyase obtained from ctiae) to a partial enzymatic hydrolysis. 21. The enzyme fragment of claim 13, which has a molecular weight of about 84,000D to 86,000D. 22. A specific activity of 400,000 IU / mg to 800,000 IU.
14. The enzyme fragment according to claim 13, which is / mg.