EP1056839A1

EP1056839A1 - Pharmaceutical formulation containing a microbial enzyme which digests hyaluronic acid

Info

Publication number: EP1056839A1
Application number: EP99964464A
Authority: EP
Inventors: Norbert Presselt; Peter-Jürgen Müller; Jörg-Hermann Ozegowski; Albert Härtl; Gundela Peschel
Original assignee: Friedrich Schiller Universtaet Jena FSU; id pharma GmbH
Current assignee: Friedrich Schiller Universtaet Jena FSU; id pharma GmbH
Priority date: 1998-12-23
Filing date: 1999-12-22
Publication date: 2000-12-06
Also published as: WO2000039290A2; WO2000039290A8; JP2002533473A; CA2318356A1; HUP0103660A2; WO2000039290A3; PL344745A1; IL138051A0; AU3032400A; DE19982882D2; KR20010041235A

Abstract

The invention relates to pharmaceutical compositions which are especially suitable as injection preparations to be used for the therapy of vascular diseases which are caused by atheromatous deposits on the inner walls of arteria. Said injection preparations contain bacterial hyaluronate lyase or a hyaluronate-digesting enzyme fragment produced from said enzyme. The invention also relates to the hyaluronate-digesting enzyme fragment of the bacterial hyaluronate lyase. It may be produced by treating the holoenzyme with a specifically digesting protease.

Description

Pharmaceutical formulation containing a hyaluronic acid-cleaving enzyme of microbial origin

On the inner wall of the artery, the intima, accumulate in humans as well

Mammals remove hydrophobic plaques with increasing age or with pathological conditions, which lead to hardening of the arterial walls, to lesions and to a reduction in the inner lumen by the formation of a neointima in the vessels. With the vascular changes there are pathological phenomena, such as cardiac arrhythmias, thrombus formation and thromboses, cerebal infarcts, cerebral thromboses,

Hypertension and a decrease in blood supply are closely related.

The invention relates to pharmaceutical formulations which have the property of reducing the extent of the areas of the intima covered with plaques or their effect on the intra-arterial or intravenous application

Decrease blood flow. This effect is closely related to the presence of a complete enzyme (holoenzyme) and / or a novel, smaller enzyme fragment that can be produced from this enzyme in the pharmaceutical formulations according to the invention. The proposed holoenzyme is a hyaluronate lyase and the fragment also has hyaluronic acid-splitting activity. This activity leads above all to a preferred breakdown of hyaluronic acid.

In addition, the invention relates to new enzyme fragments produced by a special enzymatic degradation process.

In addition to chondroitin sulfates, dermatan sulfate, heparin and heparan sulfate, hyaluronic acid belongs to the glycosaminoglycans. The glycosaminoglycans mentioned, in addition to hyaluronic acid, contain sulfate groups (sulfated glycosaminoglycans). Glycosaminoglycans are found in the tissues of all vertebrates. Hyaluronic acid is particularly present in the intercellular matrix, the skin and the cartilage, as well as in body fluids such as the synovial fluid and the aqueous humor of the eyes. Together with the other glycosaminoglycans, it is contained in the walls of the blood vessels and probably also in the atheromatous deposits or plaques on the arterial walls. Hyaluronic acid has the property of binding water with the formation of viscous, hydrocoid solutions and of forming complexes that are difficult to dissolve with strongly basic proteins. It consists of glucuronic acid and acetylated glucosamine, which are connected by ß- (l-3) - glycosidic bonds. These disaccharide units are in turn connected to one another by glycosidic β- (1-4) bonds. The hyaluronic acid is split by enzymes, which are summarized with the umbrella term hyaluronidases.

The generic term hyaluronidases describes - incorrectly in an enzymologically systematic sense - three different hyaluronic acid-cleaving enzyme types (J. Ludowieg: The mechanism of hyaluronidases, JBC 236, 333-339, (1961)). On the one hand, there are the endohydrolases that hydrolytically cleave the β- (1-3) bonds with the addition of water. They include the majority of hyaluronidases from higher organisms, for example those used medically Bovine testicular hyaluronidase. These enzymes, also called hyaluronate glycan hydrolases (EC 3.2.1.35/36), hydrolyze to a limited extent other glycosaminoglycan bonds in addition to hyaluronic acid. Another type is the endo-ß-hyaluronidase from the leech, which cleaves the ß- (l-4) -binding highly specifically. All of the enzymes mentioned are hydrolytic hyaluronidases or hyaluronidases in the narrower sense.

The third type of enzyme, the hyaluronate lyases (EC 4.2.2.1), cleaves the hyaluronic acid in the ß (1-4) bonds according to an elimination mechanism with the formation of a double bond in the (4-5) position on the glucuronic acid. Hyaluronate lyases are therefore not hyaluronidases. Hyaluronate lyases occur in microorganisms. For example, they are found in Streptomycetes and other bacteria. Their common characteristic is their narrow specificity towards hyaluronic acid. Other glycosaminoglycans are usually split to a negligible extent (J.-H. Ozegowski et. Al: Purification and characterization of Hyaluronidase from Streptocoecus agalactiae, Zbl. Bakt. 280, 497-506 (1994), A. Linker et al: The production of unsaturated uronides by bacterial hyaluronidases, JBC 219 13-25, (1956), T. Ohya, Y. Kaneko: Novel hyaluronidase from Streptomyces, BBA 198 (1970), 607-609 and K. Gase, J.-H Ozegowski, and H. Malke: The Streptocoecus agalactiae hylB gene encoding hyaluronat lyase completion of the sequence and expression analysis, BBA, 1998, 86-98).

Most microbial hyaluronate lyases are high molecular weight proteins with molecular weights between 1 16,000 to 120,000 daltons. These high molecular hyaluronate lyases are mostly relatively sensitive to denaturing agents and extreme physiological conditions. Particularly in the preparation of pharmaceutical and veterinary formulations, there can be considerable loss of activity due to denaturing processes. In addition, the diffusion ability of the relatively large enzymes is significantly less than the diffusion ability of smaller molecules. Theoretically, this limits the diffusion and thus the promotion of penetration or diffusion of the high-molecular hyaluronate lyases (holoenzymes) into the tissues, for example also into the plaques. A lower immunogenic effect is likely.

It is state of the art to cleave proteins with the help of proteases within an enzymatic process in order to obtain protein fragments - generally not enzymatically active - for sequencing. Theoretically, it should be possible to reduce the enzymes (holoenzyme) formed by microorganisms by partial digestion with proteases with a specific effect.

So far, however, no enzyme fragments, in particular no enzymatically active fragments of microbial hyaluronate lyases have become known, in which an origin to a hyaluronate lyase can be recognized from identical sections in the amino acid sequence or which are produced from it. Accordingly, no processes are known with which enzymatically active fragments are produced from hyaluronate lyases. Bovine testicular hyaluronidase is used to make animal and human tissues more permeable. It serves as a penetration or absorption accelerator for subcutaneously or intramuscularly administered drugs or to facilitate the perfusion of larger quantities of fluids and faster ones Regression of edema. Good effects were already achieved in the 1960s with high-dose, intravenously administered cattle testicular hylase "Dessau" for acute tendovaginitis and paratenonitis crepitans, for the intravenous treatment of plantar heel spurs and in dermatology. In progressive scleroderma, circum-scripted scleroderma and keloids after hyaluronidase treatment, the symptoms were significantly reduced. In the 1970s, attempts were made to influence the late consequences of myocardial infarction in an animal model with hyaluronidases. The aim of the studies was to reduce the size of the cardiac necrosis due to the accelerated formation of new vessels after the infarction and to improve the peripheral blood flow.

Since the 1960s there has been literature evidence that bovine testicular hyaluronidase can be used to therapeutically influence arterial vascular diseases in humans, in particular to improve the blood flow to peripheral vessels (e.g. Thurnherr and Koch, cited at Wolff: Treatment results with intravenous magnesium comp./Hylase -Mixed injections in arteriopathies of the pelvic type in stage II. Medical training, 1972, 66, 446 - 447). Wolff reported that hyaluronidase from bovine testicles with the simultaneous use of magnesium ions, administered intravenously (mixed injection, three times 300 IU per week for 6 weeks), improved the blood flow to the vessels in 90% of the examined patients with stage II arteriopathy. Grosshennig (in: Results of the treatment of degenerative arterial vascular diseases of the lower extremities with intravenous administration of magnesium compositum "Scharffenberg" and hyaluronidase. Dtsch. Ges.-wesen, 1965, 21, 869 - 872) performed a similar mixed treatment for degenerative vascular diseases of the lower extremities as well as patients with high-seated pelvic-type degenerative vascular obliterations with "impressive success." He gave 300 IU three times a week for about 5 weeks. In some severe cases, 30 injections were made. Follow-up examinations were 6-8 weeks after the end of treatment no deterioration of the achieved state can be determined.

It was certainly very disadvantageous that the hyaluronidases from bovine testicles were only available in relatively low activities and thus only low enzyme activities could be applied. Thus, a one-month i.V., i.p. or s.c. Administration of a very low hyaluronidase activity of 300 IU in hypercholesterolemic rabbits had no effect on the atherosclerotic vascular changes. Repeated administration of this amount over a period of up to three months lowered the blood cholesterol and fibrin content of the plasma, increased the free heparin content of the plasma, increased vascular permeability to the tissue and also led to a significant decrease in the atheromatous vascular plaque (AI Pertsovskii, SI KovaFchuk, VA Baronenko, RN Gold'man, S. Ya. Guz, and TL Fonbarova (1973): Effect of hyaluronidase on the course of experimental atherosclerosis. Kardiologija, 13, 37 - 40).

An enzyme which has been declared as hyaluronate lyase and "which appears to be a substantially homogeneous material, is particulary effective hyaluronidase because of its wide specifity" has been described by Gottlieb et al. In GB 1,060,513 from animal tissues. The enzyme was described, for example, in the patents GB 1,098,957 and in a mixture with hyaluronidase of animal origin in GB 1,179,787 for the treatment of allergic symptoms suggested. Gottlieb also proposed (US 3,708,575) to treat vascular diseases in humans such as cardiac arrhythmias, thromboses, cerebral infarcts, cerebral thromboses and heart attacks, in particular atherosclerosis, with this hyaluronidase or hyaluronate lyase. An isotonic sterile solution, for example 10,000 IU / ml, is injected intravenously, intraarterially or intrathecally. The enzyme was obtained from animal material in accordance with the information in the property right (US 3,708,575). From the description of the invention it is clear that the enzyme (s) used is the use of testicular hyaluronidases. An esterase activity ascribed to the enzyme, the broad specificity stated and the activity determination method used, which is not specific for the lyase reaction, relates to an animal hyaluronidase. Accordingly, even enzymes with the cleavage specificity of a hyaluronate lyase from animal testes are not listed in the latest literature (Review: GL Frost, T. Csoka and R. Stern: Trends Glycosci. Glycotechnol. 8, 419 - 434).

According to the cited literature, hyaluronidase from bovine testicles is suitable for increasing the permeability of blood vessels to the blood stream by dissolving, breaking down or rendering atheromatous deposits and possibly constituents of blood clots and blood thrombi. The constituents of the blood thrombus that may be detached could be plaque particles that have detached from the intima and on which a blood thrombus has formed.

There are also indications that hyaluronidase applications make the vessel walls towards the tissue and possibly the adjacent tissue more permeable.

A disadvantage of using hyaluronidase of animal origin, in particular hyaluronidase from bovine testicles, is that the enzyme carries with it the risk of transmitting viruses, BSE (bovine spongioform encephalopathy) and other infectious material due to its origin. Another disadvantage is that in the production of hyaluronidase to remove impurities in the form of ingredients, in particular foreign proteins, a very large amount of cleaning is required. In order to obtain sufficient amounts of hyaluronidase, correspondingly large amounts of starting material have to be collected under conditions in which spoilage is prevented.

Due to its non-specificity, the hyaluronidase from mammalian testicles also hydrolyzes the sulfated glycosaminoglycans present in the vessel wall. Dermatan sulfate or chondroitin sulfate, heparin and heparan sulfate, however, are believed to prevent blood clots from attaching to the inner wall of the vessels due to their anticoagulant properties. There is therefore the possibility that the hydrolysis of these compounds increases the secondary formation of blood clots at the intima and thus the testicular hyaluronidases could also have an unfavorable side effect. Sawyer et. al. (EU 193330 B1) describe the production and use of a special endo-ß-glucuronidase with a molecular weight of 28,500 D from a leech to stimulate the flow of physiological fluids in eye diseases. This enzyme is highly specific for hyaluronic acid and differs from one Known endo-ß-glucuronidase from the leech (Hirudo medicinalis) due to its higher stability at higher temperatures and extreme pH values.

The aim of the present invention is to avoid the disadvantages of the previously proposed use of hyaluronidases of animal origin as a plaque-degrading enzyme by using enzymes obtained from microorganisms in suitable galenical formulations for therapy and for preventing atherosclerotic vascular changes. Microbial enzymes can in principle be obtained in a simple way through the possibilities of their biotechnological production.

It is also the object of the invention to produce enzymatically active enzyme fragments with a plaque-degrading activity in a simple way from suitable microbial holoenzymes and to use them as galenical formulations. The use of the fragments is expected to be more stable in the pharmaceutical formulations, to be less immunogenic when used and to penetrate better.

Furthermore, the enzymes provided according to the invention should not negatively influence the living mammal organism when longer enzyme activities are applied over longer periods of time. The enzymes are also said to cause only a limited cleavage of the sulfated glycosaminoglycans, in order on the one hand to bring about a dissolution of plaques and of constituents of blood clots that is comparable to hyaluronidase from animal testes. On the other hand, the severely limited breakdown of the sulfated glycosaminoglycans in the intima should not lead to the increased risk of secondary blood clots forming due to the lack of sulfated glycosaminoglycans.

The invention is therefore based on the object of finding plaque-degrading enzymes of microbial origin which, as constituents in pharmaceutical formulations, have a comparable or better effect with hyaluronidase from bovine testicles in dissolving plaques or in reducing the inner vessel walls occupied by plaques and of components of the thrombus and blood clots when used in the blood vessel system, have a favorable effect on the course of the disease in vascular diseases, are not toxic and have no other negative effects when used in humans or animals as an injection.

It is also part of the task that led to the invention to provide a smaller protein or fragment with hyaluronate lyase activity and to provide a process for its preparation. The fragment is said to have similar or identical enzymatic activity properties as its holoenzymes.

The present invention accordingly includes pharmaceutical formulations suitable for the treatment of vascular diseases which occur directly or as a result of atheromatous plaque formation. The invention also includes a new enzyme fragment, its production and use.

According to the invention, it was found that a pharmaceutical formulation which preferred one or more bacterial enzymes with the activity of a hyaluronate lyase (EC 4.2.2.1.), In particular one of the microorganisms of the genus Streptocoecus of the generally available species Streptocoecus agalactiae or Streptocoecus equisimilis, when fermented into the submerged medium, contains hyaluronate lyases excreted or contains an enzyme fragment produced from the hyaluronate lyases, in vitro from the isolated plaque pads obtained from the mammalian atheromatous vessels, and from or dissolves thin plaque sections.

Furthermore, it has been found that with often repeated intravenous injections of comparatively high enzyme activities containing pharmaceutical formulations according to the invention, which contain the hyaluronate lyase and / or the enzyme fragment, into living Watanabe rabbits, which have large plaque deposits due to a genetic defect on the aorta had significantly reduced the extent of plaque buildup in the vessels after treatment. While the inside of the vessels of the untreated animals had a surface covered with a thin whitish plaque layer and also thicker plaques, the inside of the treated animals was free from the thinner plaque deposits and had the deep red color of healthy inner surfaces of the vessels. The surface area of the thicker plaque deposits was also reduced.

It is surprising that this effect is not only limited to the presence of the holoenzymes with a molecular weight in the range from 1 14,000 D to 124,000 D in the pharmaceutical formulation, but especially also due to a highly active, stable fragments of the holoenzymes with a molecular weight of, for example, 84,000 up to 86,000 D. It has also been found that the fragments according to the invention can be produced by proteolytic digestion of the bacterial hyaluronate lyases with proteases, preferably a protease, which cleaves the C-terminal peptide bond from aromatic amino acids. In a preferred example, the hyaluronate lyase with a molecular weight of 1 16,000 daltons from Streptocoecus agalactiae is cleaved into an enzymatically active hyaluronate lyase fragment. The fragment has a molecular weight of 84,000 - 86,000 daltons. There is no difference in its cleavage specificity to the undigested hyaluronate lyase (holoenzyme). Compared to the holoenzyme, however, the fragment is distinguished by a significantly higher stability in aqueous solutions and against denaturing agents. The fragment has a specific activity between 400,000 IU / mg to 800,000 IU / mg and thus has the same or even a higher specific activity than the holoenzyme with a specific activity of 400,000 IU / mg.

The invention accordingly relates to a pharmaceutical formulation containing a hyaluronate lyase of microbial origin as a holoenzyme, a hyaluronate-cleaving fragment thereof or a mixture of both forms, in each case in highly purified form, and at least one stabilizer and / or pharmaceutically acceptable carrier or auxiliary and optionally additionally pharmaceutically acceptable Dilution * gos ^u medium.

The pharmaceutical formulations according to the invention are primarily for the treatment of vascular diseases, such as cardiac arrhythmias, atherosclerosis, cerebral infarcts, cerebral thromboses, coronary thromboses and cardiac infarcts, suitable for mammals (humans and animals) due to the presence of atheromatous plaques in the blood vessels.

Pharmaceutical formulations produced according to the invention are, in particular, injection preparations for intravenous or intraarterial injections, which consist of an isotonic, sterile aqueous solution of the highly purified enzyme protein of the holoenzyme and / or the fragment or a defined mixture of both active enzyme species, the enzyme activities being between 20,000 and 4,000. 000 IU / ml. The enzyme proteins are advantageously used in the form of a freeze-dried solid which generally contains stabilizing additives. Stabilizing additives can be, for example, sodium chloride, glucose, magnesium salts, polyvinylpyrrolidone, amino acids, albumin, in particular ovalbumin, and its hydrolysates or cereal proteins and their hydrolysates. The amount of hyaluronate lyase and / or its fragment used in an injection is between 2,000 and 12,000 IU / kg body mass of the treated mammal or human.

The enzyme or fragment used according to the invention shows no negative

Effects on animal health.

It is obvious and within the scope of protection that the effect of the proposed enzyme and / or its fragment does not only affect the removal of the current one

Deposits is limited, but a beneficial effect on circulatory diseases such as myocardial ischemia, myocardial infarction, coronary infarction, cardiac arrhythmia,

Atherosclerosis and similar phenomena in terms of prevention as well as one

Therapy. Advantageous in the use of the fragment according to the invention compared to the

Holoenzyme is that due to its smaller size, it penetrates the solid layers of the plaques faster than the holoenzyme and thereby causes them to be destroyed more quickly. Without specifying the invention on the hyaluronate lyase or its fragment on a specific microorganism, the invention is illustrated using the example of the enzyme from the generally accessible Streptocoecus agalactiae. The hyaluronate lyase formed has an isoelectric point of approximately IP = 8.6 and a molar mass of approximately 116,000 D and acts as an endoglycanase. The enzyme has the advantageous property that it is inhibited by sulfated glycosaminoglycans, such as sulfated hyaluronic acid. The purification processes listed below are used to obtain the hyaluronate lyase as a highly purified enzyme for injection purposes, or to purify purified hyaluronate lyase with the specific-acting protease to form the fragment. The specific-acting protease can be used, without restricting the invention, for example the acidic metalloprotease MO / 2 (DD 270924), which can be obtained from the microorganism Streptomyces hygroscopicus AP40. This protease is characterized by a molecular weight of approximately 14,000 kD and an isoelectric point between 3.85 to 4.0. The following detailed description of the production of the holoenzyme as well as that of the fragment, in particular the order of the purification steps, has an exemplary character and is not intended to limit the scope of protection. The production of the high-purity hyaluronate lyase and the highly purified fragment in one galenical formulation suitable for injection can be carried out, for example in relation to the sequence and also the selection of the cleaning steps, in a manner which does not restrict the scope of protection of the invention, as follows. The fermentation of the microorganism, for example one of the abovementioned, takes place in a stirred fermenter with constant pH at an acidity of pH 6.5 to 7.5. The lactic acid formed during the fermentation is neutralized by adding dilute sodium hydroxide solution. The medium consists of inorganic salts, yeast hydrolyzate, optionally casein peptone or soy peptone and glucose. After about 20 hours of fermentation, the cells are separated. The cell mass is discarded.

The culture filtrate is concentrated and purified in an ultrafiltration device. The cut-off limit of the ultrafilter module is between 30 and 50 kD. The result is a pre-cleaned enzyme solution which has to be subjected to further cleaning steps before it can be used as an injection. The cleaning steps and the use of pyrogen-free additives ensure that the injection product is free of pyrogens.

After increasing the ionic strength by adding ammonium sulfate to a saturation of 40%, the enzyme is adsorbed on phenylsepharose. The desorption then takes place with a neutral, buffered aqueous solution which contains 25% ammonium sulfate, based on 100% saturation. After a dialysis step, the solution is mixed with Q-Sepharose (Pharmacia) to remove impurities. This is followed by specific adsorption on a dye, for example aminophenyloxamic acid. The final cleaning together with the determination of the molecular weight can consist of molecular weight chromatography on Superdex (Pharmacia).

When using Streptocoecus equisimilis as an enzyme generator, the cleaning step by adsorption on the dye is not necessary. This enzyme acts as an exoglycanase, its isoelectric point is between pH 4.5 and 4.8 and it is only inhibited to a very small extent by sulfated glycosaminoglycans.

The partial digestion of the holoenzyme into the fragment takes place with immobilized or also with non-immobilized specific protease. An advantage of the implementation with immobilized protease is that the digestion can be carried out specifically and without contamination by the protease itself. In the case of direct reaction with added protease, an inactivation step of the protease, the separation of the protease protein and a high purification must take place after digestion. The enzyme fraction can be used to produce the fragment in the following way: The specifically cleaving protease MO / 2 is bound in a known manner, for example to Sepharose. The immobilized protease is mixed for example at pH 7.5 and a temperature of 37 ° C. with an aqueous hyaluronate lyase solution. After digestion is complete, the fragment is precipitated with ammonium sulfate and obtained in highly pure form by molecular weight chromatography. The highly purified enzyme or the enzyme fragment show only a specific precipitation after immunization in the rabbit. All detectable bands are stained in the blot with monoclonal antibodies. The holoenzyme has a specific activity of approximately 400,000 IU / mg and the specific activity of the fragment is approximately between 400,000 to 800,000 IU / mg. To stabilize the enzyme or fragment at least one of the stabilizers, for example albumin, preferably ovalbumin and inorganic salts.

Without thereby restricting the invention, a way of obtaining the fragment will be described by way of example. According to the invention, digestion proteases are used which cleave the C-terminal peptide bond of aromatic amino acids. In a preferred embodiment, the metalloprotease MO / 2 is used, which is produced from the culture filtrate from Streptomyces hygroscopicus (strain AP 40). MO / 2 is a metalloenzyme with Co ++ in the active center (DD 270 924). The proteases are preferably used in purified form. The protease MO / 2 is purified, for example, by chromatography on phenylsepharose, DEAE-Sepharose, Q-Sepharose and Sephacryl S100 with an enrichment factor of 20. The specific activity is 0.25 Kunitz Units / mg (U / mg ), the maximum reaction at an acidity of pH = 4.2 and the maximum reaction temperature at 65 ° C. The molecular weight (mm) of the enzyme, determined by SDS gel electrophoresis and molecular weight chromatography on Sephadex G50 superfine, is Mm = 14,000 to 15,000 D. The protease MO / 2 is an endopeptidase with an isoelectric point at IP = 3.85 and one at I = 3.92. The enzyme hydrolyzes natural polypeptides such as casein, hemoglobin, bovine serum, albumin and ovalbumin. An advantage of using protease MO / 2 is that the enzyme cleaves proteins very specifically or has a very low general digestive activity towards proteins. The enzyme almost exclusively cleaves only the peptide bonds of the C-terminal residue from the aromatic amino acids. It has an esterolytic activity against N-benzoyl-L-proline nitroanilide and pronounced milk-digestion properties. The properties of milk precipitation, which can be demonstrated by the formation of a long-term stable cheese gel, comparable to the gel which occurs in milk precipitation with the very specific-acting labenzyme, are further indications of the limited activity of the protease which is very advantageous for the invention To evaluate MO / 2, specifically cleave the bonds of the holoenzyme without leading to a breakdown of the fragments.

In a further embodiment of the invention, the protease MO / 2 is optionally bound to suitable insoluble immobilization supports and used in this form to cleave the holoenzyme. The advantage of this procedure is that the transition of dissolved protease into the solutions of the fragments is prevented in this way.

According to the invention, the fragment is produced by partial digestion or partial proteolytic degradation of the holoenzyme with a protease which acts in a cleavage-specific manner and which cleaves the peptide bond preferably on the C-terminal side of the aromatic amino acids.

The highly pure enzyme solutions or the fragment-containing fractions can be used as injection preparations after concentration with partial removal of water and optionally with the addition of stabilizers, auxiliaries or pharmacologically active substances and sterile filtration. The highly pure enzyme or fragment solution can also be freeze-dried after sterile filtration, for example with the addition of 5 mM magnesium chloride and 1% egg albumin. The freeze-dried enzyme or fragment can be dissolved with saline as an isotonic injection. The invention also relates to the use of a hyaluronate lyase of microbial origin, a hyaluronate-cleaving fragment thereof or a mixture of both forms for the treatment of vascular diseases, such as cardiac arrhythmias, atherosclerosis, cerebral infarcts, cerebral thromboses, coronary thromboses and cardiac infarcts, which indicate the presence of atheromatous plaques in blood vessels are attributable to mammals and humans.

Another object of the present invention relates to fragments of bacterial hyaluronate lyases with hyaluronate lyase activity.

The following exemplary embodiments serve to explain the invention, but they are not intended to limit it in any way.

Experimental part

Example 1 The fermentation is carried out in a stirred fermentor with a gross filling volume of 30 l. The working volume is 20 1. A generally available strain of the species Streptocoecus agalactiae is used for inoculation, which is kept in the German Collection of Microorganisms and Cell Cultures GmbH (DSM) in Braunschweig under number 2134 ^{τ (} = ATCC 13813 = NCT C8181). The trunk preservation is carried out with a cryopreserve (mast diagnostics). A ball covered with stock material is placed in an agar slant tube with bacterial agar and brought into contact with the agar surface by movement (culture A). The rolled ball is then placed in 3 ml of a heart-brain broth (culture B) for sterile control. Both cultures are cultivated as stand cultures for 24 h at 37 ° C.

1. Pre-culture

2 x 50 ml of a medium containing 5 g / 1 casein peptone and 10 g / 1 yeast extract (Difco) are filled into two 100 ml steep-breast bottles and sterilized at pH pH 7.0. In addition to the 50 ml culture medium, 5 ml Eagle medium is added shortly before inoculation. The inoculation is carried out by washing away the slant agar tube Culture A with the culture solution from Culture B. The steep breast bottles are cultivated with shaking (150 rpm) at 37 ° C for 24 h.

2Nork culture

2x1 1 of a medium consisting of 20 g / 1 yeast extract and 10 g / 1 pancreatic peptone, 1, 75 g / 1 Νa acetate, 7 g / 1 Νa hydrogen carbonate (extra dissolved), 7 g / 1 di-Νa- Hydrogen phosphate x 2H ₂ O and 3.5 g / 1 Νa-di-hydrogen phosphate is adjusted to pH 6.2 before sterilization with 20% H ₂ SO ₄ . After autoclaving, the pH is around 7.0. A solution of a glucose solution containing 6.0 g / l is autoclaved separately. 100 ml of this is added before seeding the 2nd pre-culture. The inoculation takes place by adding 50 ml of the 1st preculture (inoculation with 5% v / v). The cultivation is carried out with slow shaking at approx. 45 rpm at 32 ° C for 24 hours.

Main culture

The medium of the main culture consists of 20 g / 1 yeast extract, 10 g / 1 pancreatic peptone and 25 g / 1 glucose. The glucose is specially autoclaved. The inoculation is carried out with one liter of the second pre-culture. The fermentation parameters are 34 ° C., pH 7.0, aeration via head space (25 1 / min air) and stirring speed 150 rpm. The pH is kept constant by titration with a 40% sodium hydroxide solution. Glucose is added manually so that the glucose concentration does not drop below 5 g / 1. The cultivation is ended after 20 hours.

Example 2

50 l culture solution of a Streptocoecus agalactiae species with a hyaluronate lyase activity of 400 IU / ml are freed from the living cells by sterile filtration in a 0.2 μm filter. The clear culture filtrate solution is then concentrated by ultrafiltration at a cut off of 60,000 D to a culture concentrate of 2 l. 480 g of solid ammonium sulfate are added to this 2 1 culture concentrate. The proteins precipitating at this concentration are separated off by clear filtration using a filter aid and the clear concentrate is applied to a 2.5 x 20 cm phenylsepharose column previously equilibrated with 40% phosphate-buffered ammonium sulfate solution pH 6.5. After washing with a 35% phosphate-buffered ammonium sulfate solution pH 6.5, the hyaluronate lyase is eluted with a 25% phosphate-buffered ammonium sulfate solution pH 6.5. The eluted fractions are combined and adjusted to a saturation of 80% by adding solid ammonium sulfate. The precipitate is collected and dissolved in 100 ml of 0.03 M Tris buffer pH 8.2 and dialyzed against the same buffer.

The dialyzed crude hyaluronate lyase solution is then passed through an equilibrated Q-Sepharose column of 2.2 x 15 cm. The hyaluronate lyase is in the run. The run is saturated to 80% by adding solid ammonium sulfate. The precipitated protein is collected, dissolved in 0.05 M Tris buffer pH 8.7 and freed of ammonium sulfate on a Sephadex G 10 column. The solution containing hyaluronate lyase, thus equilibrated, is applied to an appropriately equilibrated affinity column (0.8 cm × 10 cm) of N- (p-aminophenyl) oxamic acid agarose and eluted with 0.1 M NaCO ₃ solution pH 9.7. The eluted fractions containing hyaluronate lyase are combined and precipitated by saturation to 80% with ammonium sulfate. The precipitate is collected, dissolved in 1 ml 0.1 M Tris buffer pH 7.5 and applied to a Superdex 200 column (16 x 60). The hyaluronate lyase protein band at 116,000 D is collected, dialyzed against 0.02 M Tris buffer pH 7.5 containing 0.005 M MgCl ₂ . 10 mg hyaluronate lyase with a specific activity of 400,000 IU / mg are obtained. The dialyzed hyaluronate lyase solution is added to ovalbumin in a concentration of 1% and the solution is dried lyophilically. Example 3

According to Examples 1 and 2, a microorganism of the species Streptocoecus ecμtisimilis is precultivated, cultivated and the enzyme is obtained in pure form. The work-up step of purification over the N- (p-aminophenyl) oxamic acid agarose is omitted.

Example 4

10 mg of purified protease MO / 2 is dissolved in 2 ml of 0.1 M sodium hydrogen carbonate solution. For this, 1 ml of bromine cyan Sepharose (Pharmacia) is added and shaken at 4 ° C for 12 hours. The uncoupled MO / 2-containing solution is then separated from the resulting MO / 2 protease sepharose with a frit. Then unsaturated binding sites on the bromine cyan Sepharose are blocked by reaction with 2 ml of 0.1 M glycine solution. After blocking, the MO / 2-Sepharose is washed with 0.1 M acetic acid of pH 4.5 and 0.1 M sodium hydrogen carbonate solution.

10 mg hyaluronate lyase from Streptocoecus agalactiae are dissolved in 1 ml 0.05 M phosphate buffer pH 7.0 and the temperature in the thermostat is 37 ° C. 0.25 g of fixed MO / 2 protease sepharose are added to this solution, to which a proteolytic activity of 0.1 Kunitz units / g was bound. After 15 minutes, the protease sepharose is separated off and the resulting fragment is precipitated from the solution to 80% by saturation with ammonium sulfate. After 18 hours the sediment is collected and dissolved in 0.05 M Tris buffer pH 7.5. The fragment is purified by molecular weight chromatography on Superdex 200. The active fragment fractions are pooled, dialyzed against 0.05 M Tris buffer and lyophilized with the addition of albumin. The fragment has a specific activity of 600,000 IU / mg.

Example 5

4 mg hyaluronate lyase from Streptocoecus agalactiae are dissolved in 1 ml 0.05 M Tris-HCl buffer pH 7.8 and the temperature in the thermostat is 37 ° C. 80 μg of the protease MO / 2 with a specific activity of 0.1 Kunitz units / mg are added to this solution and the mixture is digested at 37 ° C. for 30 minutes. To stop the reaction, 10 μl of 0.1 M EDTA pH 7.0 are added to the mixture. The mixture is then applied to a 3 ml affinity column of N- (p-aminophenyl) oxamic acid agarose and eluted with 0.05 M Na _j CO _j pH 9.7. The

Fragment was precipitated by saturation on 80% ammonium sulfate, collected and purified by molecular weight chromatography. The active fragment fractions are pooled, dialyzed against 0.05 M Tris buffer and lyophilized with the addition of albumin. The fragment has a specific activity of 460,000 IU / mg. Example 6

0.5 ml solution of the hyaluronate lyase fragment (Example 4) are added to 0.5 ml 0.1 M acetate buffer pH 6.0 and diluted in the form of a geometric series. Then 0.5 ml of a hyaluronic acid solution containing 0.2 mg hyaluronic acid / ml 0.1 M acetate buffer pH 6.0 is added to each dilution. This mixture is then incubated at 37 ° C for 30 minutes. The enzyme reaction is stopped by diluting each with 2ml of a 2.5%

Cetyltrimethylammoniumbromidlösung in 2% sodium hydroxide solution is added. After standing at room temperature for 20 minutes, the turbidity is measured in each dilution at 600 nm in 1 cm cuvettes and the amount of split hyaluronic acid is determined via a calibration curve. Five international units (IU) of the hyaluronate lyase fragment split 16μg hyaluronic acid / min.

Example 7

20,000 IU each of the holoenzyme and the fragment (Example 4) are dissolved in 3 ml of distilled water. Both solutions were kept at room temperature for 24 hours and then the enzyme activity was measured. The activity of the

Holoenzyme solution drops to 60% during this period, while the activity of the fragment solution is 95% of the activity of the starting solutions.

Example 8

Activity determination for hyaluronate lyase or the fragment

1. Optical test at 232 nm. Stock solutions for hyaluronic acid (HA) and sulfated hyaluronic acid (mg / ml) are prepared and stored at 4 ° C.

The standard test batch is carried out as follows in 3 ml quartz cuvettes (d = 1 cm) at a wavelength of 232 nm and a temperature of 26 ° C. 200 μl of stock hyaluronic acid are added to 1.8 ml of 0.1 M acetate buffer pH 6.0 and the reaction is started with 2 to 4 U / ml (50 ml) of hyaluronate lyase. The final volume is 2050 μl.

For the inhibition tests, the standard test approach described above is varied as follows. 300 to 500 μl of sulfated hyaluronic acid and 200 μl of hyaluronic acid are added to 1.5 to 1.8 ml of buffer and the reaction is started by adding hyaluronate lyase. The hyaluronate lyase is partially non-competitively inhibited. The Ki value is 5.5 x 10 ^"4 mg / ml.

Hyaluronate lyase shows a linear increase in absorbance at 232 nm, from whose increase the activity can be calculated. An extinction coefficient of e = 6.0 x 10 ³ lxmor'cm ^{"1 is used as a basis} for the Δ ^4.5 -unsaturated uronide formed (J. Ludowig: The mechanism of hyaluronidases. JBC 236, 333-339 (1991)). 2. Optical test at 600 nm on microplates

Determination of lyase activity according to D. Gerlach, W. Köhler "Hyaluronate lyase from Streptocoecus pyogenes. II. Characterization of hyaluronate lyase" (E.C. 4.2.2.1.) Zbl. Bakt.Hyg., I. Dept. Orig. A 221, 296-302 (1972)

A hyaluronate lyase solution is prepared from a stock solution (50,000 U / ml) by diluting 1:50. 0.5 ml of hyaluronate lyase solution are added to 0.5 ml of 0.1 M acetate buffer pH 6.0 and diluted in the form of a geometric series. Then 0.5 ml of a hyaluronic acid solution containing 0.2 mg / ml 0.1 M acetate buffer pH 6.0 is added to each dilution. This mixture is then incubated at 37 ° C for 30 minutes. The enzyme reaction is stopped by adding 2 ml of a 2.5% cetyltrimethylammonium bromide solution in 2% sodium hydroxide solution to each dilution. After 20 minutes at room temperature, the turbidity is measured in 1 cm cuvettes in each dilution at 600 nm and the amount of split hyaluronic acid is determined via a calibration curve. 5 international units (IU) hyaluronate lyase split 16μg hyaluronic acid / min.

Example 9

10 mg hyaluronate lyase (holoenzyme) are dissolved in 1 ml 0.05 M phosphate buffer pH 7.0 and the temperature in the thermostat is 37 ° C. 0.25 g of a specifically cleaving protease sepharose with a proteolytic activity of 0.1 Kunitz units / mg are added to this solution. After 15 minutes, the insoluble protease sepharose is separated off and the resulting fragment is precipitated from the solution to 80% by saturation with ammonium sulfate. After 18 hours, the sediment is separated off and dissolved in 0.05 M Tris buffer pH 7.5. The fragment is purified by molecular weight chromatography on Superdex 200. The active fractions are combined, dialyzed against 0.05 M Tris buffer and lyophilized with the addition of albumin.

Example 10

To demonstrate the plaque-dissolving activity of hyaluronate lyase, approximately 0.3 mm large pieces of white-yellow tissue (plaque pieces) taken from the neointima of the internal carotid artery in humans are dissolved in solutions of hyaluronate lyase (approximately 20,000 IU in 1 ml Tris buffer , 10 mM, pH 7.0) and incubated for 12 hours at room temperature. There is a partial dissolution of the previously sharp phase boundaries of the plaque pieces and a release of suspended, finely divided material. In parallel, plaque pieces were only treated with Tris buffer. There are no signs of disintegration.

Example 11

Experiment with the fragment of hyaluronate lyase to dissolve atheromatous vascular Deposits of human origin:

Approximately 0.3 mm large pieces of whitish-yellow tissue (plaque pieces) taken from the neointima of the internal carotid artery in humans were dissolved in the fragment (about 15,000 IU in 1 ml Tris buffer, 10 mM, pH 7.0). suspended and incubated for 12 hours at room temperature. There is an extensive dissolution of the previously sharp phase boundaries of the plaque pieces and a release of suspended, finely divided material. In parallel, plaque pieces were only treated with Tris buffer. There are no signs of disintegration.

Example 12

Influence of hyaluronate lyase on atheromatous vascular deposits in Watanabe

Rabbit Ibm: WHHL (Watanabe heritable hyperlipidemic). The rabbit has been bred by Y. Watanabe, Kobe University, Japan since 1981 and came in 1992 as

Breeding line to Charles River Savo and has been used by the Broekman Institute in

Someren, Netherlands, bred.

The Watanabe rabbit has an inherited hyperlipidemia caused by an inherited deficit in LDL receptors. As a result of hypercholesterolemia (> 400 mg / 100 ml plasma), all animals develop prematurely

Aortic arteriosclerosis.

14 days before the start of the experiment, 10 Watanabe rabbits (Ibm: WHHL; 4? And 6σ ") aged 9 to 11 months were purchased from the Broekman Institute in Someren, Netherlands. The animals were individually caged (0.5 x 1 m) kept at room temperatures of 20 ± 2 ° C and supplied with standard food (ssniff rabbit keeping) and drinking water ad libitum.

Before the start of the experiment, i.e. before the first clinical-chemical examination, the 10 rabbits were randomized according to gender and then identified with the numbers 1 to 10.

The animals received infusions 3 times a week (Mondays, Wednesdays and Fridays). 4 control rabbits (1 - 4) were each given 20 ml of pyrogen-free 0.9% NaCl solution intravenously (IV) within 1 hour. The 6 experimental animals (5-10) were each given 30,000 IU of highly purified hyaluronate lyase per hour with the infusion volume of 20 ml of pyrogen-free 0.9% NaCl solution. injected. This corresponds to a dosage of 10,000 IU / kg body mass

General behavior, body mass (BM), body temperature and clinical-chemical parameters (cholesterol, triglycerides, and amylase) were checked during the test period.

The general behavior of the animals was not visibly impaired by the treatments with 0.9% NaCl solution or with hyaluronate lyase. The body masses of the adult Watanabe rabbits, controls or

Hyaluronate lyase-treated changes only slightly during the treatment period from November 18, 1996 to February 3, 1997

The body temperature changes, measured before and after each infusion, were in the Fluctuation range from - 0.3 to + 1.2 ° C. No noticeable differences between control and hyaluronate lyase-treated rabbits were measured. The serum cholesterol values of the control animals did not change significantly as a result of the 0.9% NaCl infusions. In one before four (1/4) animals, the cholesterol concentration in the serum was halved.

The hyaluronate lyase treatment also did not lead to any noticeable shifts in the serum cholesterol concentrations

Under the infusion treatment, the z. T. very high Se mtriglycerid values. After the 20th infusion, the controls were between 570 and 740 mg / dl and the hyaluronate lyase-treated rabbits were between 200 and 290 mg / dl.

In (2/4) control rabbits, the aortic arches were highly atheromatous and in 2/4 moderate to high grade. In 2/6 rabbits treated with hyaluronate lyase, the aortic arches were moderate and in 4/6 mild to moderate atheromatous.

In 2/4 control rabbits, the thoracic artery was highly atheromatous and in 2/4 moderately atheromatous. In 4/6 hyaluronate lyase rabbits, the thoracic artery was slightly atheromatous and in 2/6 plaque was barely noticeable. The abdominal aorta was severe in 2/4 control rabbits and atheromatous in 2/4 mild to moderate. In 2/6 hyaluronate lyase rabbits, moderate to moderate and in 4/6 weak to slight atheromatous deposits were recognizable.

The Aa were in 2/4 control rabbits. pulmonalis heavily covered with plaques and 2/4 mild to moderate in 1/6 hyaluronate lyase rabbits, the pulmonary aorta was high, in 2/6 mild to moderate plaques and 3/6 no plaques.

General behavior, body mass development, body temperature before and after

Infusions and clinical chemical parameters of 0.9% NaCl solution and

Hyaluronate lyase-treated Watanabe rabbits showed no noticeable differences during two treatment cycles with 10 infusions each, so that it can be concluded that the infusion fluids are well tolerated.

A subjective assessment of the macroscopic aortic material shows that the extent of the atheromatous deposits in the 4 Watanabe rabbits treated with 0.9% NaCl solution appears to be more severe than in the 6 Watanabe rabbits treated with microbial hyaluronate lyase.

Example 13

Highly pure concentrated enzyme solution is diluted with so much saline that the resulting solution contains 0.9% saline and 10,000 IU / ml hyaluronate lyase. The solution is sterile filtered through a sterile filter with a pore size of 0.2 μm. 1 ml of this solution is filled into ampoules under sterile conditions. The ampoules are freeze-dried under sterile conditions and under sterile

Conditions closed. 1 ml of sterile distilled water is added before the injection. Example 14

Highly pure concentrated enzyme solution is diluted with a solution of magnesium nicotinate and magnesium adipinate (magnesium compositum "Scharffenberg") so that the resulting solution contains 25 mg magnesium nicotinate and 88 mg magnesium adipinate and 10,000 IU / ml hyaluronate lyase. The solution is ampouled according to example 13.

Example 15

A solution of a high purity hyaluronate lyase is diluted with a solution of table salt and egg albumin so that the solution is 40,000 IU / ml, 0.9% table salt and 1% egg albumin. The solution is processed as described in Example 13.

Example 16

A solution of a high-purity hyaluronate lyase is diluted with a solution of sodium chloride and soy protein hydrolyzate so that the solution is 40,000 IU / ml, 0.9% sodium chloride and 1% of a hydrolyzate made from soy protein. The solution is processed as described in Example 13.

Example 17

A solution of a highly pure fragment from the holoenzyme of Streptocoecus agalactiae is diluted with a solution of table salt and egg albumin so that the solution is 40,000 IU / ml, 0.9% table salt and 1% egg albumin. The solution is processed as described in Example 13.

EXAMPLE 18 A highly pure concentrated solution of a fragment from Streptocoecus agalactiae is diluted with so much saline and magnesium chloride that the resulting solution contains 0.9% saline and 100,000 IU / ml lyase activity. The solution is sterile filtered through a sterile filter with a pore size of 0.2 μm. 1 ml of this solution is filled into ampoules under sterile conditions. The ampoules are freeze-dried under sterile conditions and closed under sterile conditions. 1 ml of sterile distilled water is added before the injection.

Claims

Claims:

1. Pharmaceutical formulation containing a hyaluronate lyase of bacterial origin as a holoenzyme, a hyaluronate-cleaving fragment thereof or a mixture of both forms, and at least one stabilizer and / or pharmaceutically acceptable carrier or auxiliary.

2. Pharmaceutical formulation according to claim 1, characterized in that the hyaluronate lyase originates from a microorganism of the genus Streptocoecus.

3. Pharmaceutical formulation according to claim 2, characterized in that the hyaluronate lyase comes from the microorganism Streptocoecus agalactiae.

4. Pharmaceutical formulation according to claim 1, characterized in that the bacterial hyaluronate lyase in the form of the holoenzyme has an isoelectric point in the range of IP = 8.6 and a molecular weight in the range of 116 kD.

5. Pharmaceutical formulation according to claim 1, characterized in that the fragment of hyaluronate lyase has a molecular weight in the range from 84 to 86 kD.

6. Pharmaceutical formulation according to claim 1 for the treatment of vascular diseases selected from the group cardiac arrhythmias, atherosclerosis, cerebral infarcts, cerebral thromboses, coronary thromboses and cardiac infarcts.

7. Pharmaceutical formulation according to claim 1 containing a hyaluronate-cleaving fragment of a hyaluronate lyase obtainable from bacterial hyaluronate lyase by means of an enzymatic partial hydrolysis by a protease which cleaves the peptide bond of the C-terminal residue of aromatic amino acids.

8. Pharmaceutical formulation according to claim 1, characterized in that it is in the form of an injection for intravenous or intraarterial injections.

9. Pharmaceutical formulation according to claim 1, characterized in that the activity of the hyaluronate lyase of microbial origin and / or its fragment is between 20,000 to 4,000,000 IU / ml.

10. A pharmaceutical formulation according to claim 1 comprising a purified aqueous solution for injection containing hyaluronate lyase and / or its fragment and about 1% (w / w) albumin and / or its hydrolysates.

11. A pharmaceutical formulation according to claim 1 comprising a purified aqueous solution for injection containing hyaluronate lyase and / or its fragment and magnesium salts.

12. Pharmaceutical formulation according to claim 1, comprising a purified aqueous solution for injection containing hyaluronate lyase and / or its fragment and glucose.

13. Enzyme fragment of bacterial hyaluronate lyase, characterized in that it has hyaluronate lyase activity.

14. Enzyme fragment according to claim 13, characterized in that the hyaluronate lyase comes from a microorganism of the genus Streptocoecus.

15. Enzyme fragment according to claim 13, characterized in that the hyaluronate lyase comes from the microorganism Streptocoecus agalactiae.

16. Enzyme fragment according to claim 13, characterized in that it is produced from a bacterial hyaluronate lyase by enzymatic partial hydrolysis using a protease that cleaves the peptide bond of the C-terminal residue of aromatic amino acids.

17. Enzyme fragment according to claim 13, characterized in that it is produced by subjecting a bacterial hyaluronate lyase to the enzymatic partial hydrolysis by a protease in carrier-immobilized form.

18. Enzyme fragment according to claim 13, characterized in that it is produced by subjecting a bacterial hyaluronate lyase to the enzymatic partial hydrolysis by the metalloprotease MO / 2 in carrier-immobilized form.

19. Enzyme fragment according to claim 13, characterized in that it is produced by a hyaluronate lyase from streptococci of an enzymatic partial hydrolysis by the metalloprotease MO / 2 at an acidity in the pH range from pH 6.5 to pH 8.0 and temperatures in Range from 25 ° C to 45 ° C is subjected.

20. Enzyme fragment according to claim 13, characterized in that it is produced by a hyaluronate lyase from Streptocoecus agalactiae, with a molecular weight of about 1 15,000-120,000 D, is subjected to an enzymatic partial hydrolysis.

21. Enzyme fragment according to claim 13, characterized in that it has a molecular weight of about 84,000 - 86,000 daltons.

22. Enzyme fragment according to claim 13, characterized in that it has a specific activity of 400,000 to 800,000 IU / mg.