HUP0103660A2 - Pharmaceutical formulation containing a microbial enzyme which digests hyaluronic acid - Google Patents

Abstract

The subject of the invention is a medicinal preparation, which is particularly suitable as an injection preparation for the treatment of vascular diseases attributable to atheromatous deposits. The injection preparation contains hyaluronate-lyan of bacterial origin and a hyaluronate-decomposing enzyme fragment produced from hyaluronate-lyan. The latter fragment is also the subject of the invention. The fragment is produced by treating the aholoenzyme with a protease that specifically degrades it. HE

Description

the holoenzyme is treated with a protease that specifically degrades it.

main

A pharmaceutical preparation containing a hyaluronic acid-degrading enzyme of microbial origin

On the inner wall of the arteries of humans and mammals, the so-called intima, hydrophobic plaques are deposited with increasing age or in pathological conditions, which lead to hardening of the artery wall, lesions and a decrease in the internal cross-section due to the formation of neointima in the vessels. Changes in blood vessels are closely related to pathological phenomena, such as cardiac arrhythmia, thrombus formation and thrombosis, cerebral infarction, hypertension and poor blood supply.

The invention relates to pharmaceutical compositions which have the property of reducing the surface area of the intima covered with plaques when administered intraarterially or intravenously, and of reducing the effect of plaque surfaces on blood flow. This effect is closely related to the fact that the pharmaceutical compositions according to the invention contain a complete enzyme (holoenzyme) and/or a novel, smaller enzyme fragment which can be produced from this enzyme.

The proposed enzyme is a hyaluronate lyase, and the fragment also has hyaluronic acid degrading activity. This activity mainly leads to the preferential degradation of hyaluronic acid. In addition, the invention also relates to enzyme fragments produced by a novel, specific enzymatic degradation process.

Hyaluronic acid belongs to the glycosaminoglycans, along with chondroitin sulfates, dermatan sulfate, heparin and heparan sulfate. With the exception of hyaluronic acid, the aforementioned glycosaminoglycans contain sulfate groups (sulfated glycosaminoglycans). Glycosaminoglycans occur in all vertebrate tissues. Hyaluronic acid occurs mainly in the intercellular matrix of skin and cartilage tissue, as well as in body fluids, such as synovial fluid and aqueous humor. Atheromatous deposits and plaques on the walls of blood vessels and probably on the inner walls of arteries also contain other glycosaminoglycans in addition to hyaluronic acid. Hyaluronic acid is able to bind water during the formation of viscous, hydrocolloid solutions, and forms insoluble complexes with strongly basic proteins. Hyaluronic acid consists of glucuronic acid and acetylated glucosamine, which are linked to each other by P-(1-3)-glycosidic bonds. The resulting disaccharide units are linked to each other by glycosidic-(1-4) bonds. The collective term for the enzymes that break down hyaluronic acid is hyaluronidases.

Hyaluronidases is a collective term - not correctly in an enzymological systematization sense - describing three different types of hyaluronic acid-degrading enzymes with different mechanisms of action (J. Ludowieg: The mechanism of hyaluronidases, JBC 236, 333339, 1961). One type is the endohydrolases, which hydrolytically cleave the P-(l-3) bond while absorbing water. This group includes most hyaluronidases from higher organisms, such as bovine testicular hyaluronidase used in medicine. These enzymes, also called hyaluronate glycan hydrolases (E.C. 3.2.1.35/36), hydrolyze other glycosaminoglycan bonds in addition to hyaluronic acid to a limited extent. Another type is represented by the endo-p-hyaluronidase from leech, which cleaves the P~(l-4) bond very specifically. These enzymes listed here are hydrolytically acting hyaluronidases, or hyaluronidases in a narrower sense.

The third type of enzymes, hyaluronate lyases (EC 4.2.2.1), cleave the p_(l-4)-bond of hyaluronic acid by an elimination mechanism, during which a double bond is formed at the (4-5)-position of the glucuronic acid. Accordingly, hyaluronate lyases are not hyaluronidases. Hyaluronate lyases occur in microorganisms, for example, they have been found in Streptomycetes and other bacteria. A common feature is their close specificity for hyaluronic acid; other glycosaminoglycans are usually only degraded to a negligible extent (J.-H. Ozegowski et al.: Purification and characterization of Hyaluronidase from Streptococcus 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, K. Gase, J.-H. Ozegowski and H. Malke: The Streptococcus agalactiae hylB gene encoding hyaluronat lyase completion of the sequence and expression analysis, BBA, 1998, 8698). Most microbial hyaluronate lyases are high molecular weight proteins, with molecular weights between 116,000 and 120,000 Daltons. These high molecular weight hyaluronate lyases are generally relatively sensitive to denaturing agents and extreme physiological conditions. Thus, denaturing processes during the production of pharmaceutical and veterinary preparations can lead to a significant loss of activity. In addition, the ability of the relatively large enzyme molecule to diffuse is significantly lower than that of smaller molecules. This theoretically limits the diffusion of high molecular weight hyaluronate lyases (holoenzymes) into tissues (including plaques) and thus their penetration and diffusion-promoting effect. A weak immunogenic effect is likely.

It is known in the art that proteins can be broken down by proteases in an enzymatic process, so that protein fragments - usually without enzymatic activity - are available for sequencing. Theoretically, it should therefore be possible that enzymes (holoenzymes) produced by microorganisms can be reduced in size by partial digestion with a specifically acting protease. However, no enzyme fragment of microbial hyaluronate lyase, in particular an enzyme fragment with enzymatic activity, has been known so far, the origin of which from hyaluronate lyase could be recognized on the basis of identical sections of the amino acid sequence, or which would have been produced from hyaluronate lyase. Accordingly, no process is known by which fragments with enzymatic activity can be produced from hyaluronate lyases.

Hyaluronidase from bovine testicles is used to make human or animal tissue more permeable, i.e. as a substance that accelerates penetration and absorption in subcutaneously or intramuscularly administered pharmaceutical preparations, or to facilitate the perfusion of larger amounts of fluid, for faster regression of edema. Already in the 60s, good results were achieved with high doses of bovine testicular hyalase called “Dessau” administered intravenously in cases of acute tendovaginitis and Paratenonitis crepitans, in the intravenous treatment of plantar heel spurs and in dermatology. Thus, in cases of progressive scleroderma, circular scleroderma and keloids, treatment with hyaluronidase significantly reduced symptoms. In the 70s, experiments were carried out to influence the late consequences of myocardial infarction with hyaluronidases in animal models. The aim of the studies was to reduce the area of dead heart tissue by accelerating the regeneration of blood vessels after a heart attack and improving peripheral blood supply.

Since the 1960s, there have been indications in the literature that hyaluronidase from bovine testicles can be used to therapeutically influence arterial blood vessel diseases in humans, especially to improve the blood supply to peripheral tissues (e.g. Thurnherr and Koch, cited in Wolff's book: Behandlungsergebnisse mit intravenösen Mägnesüim comp./Hylase-Mischinjektionen bei Arteriopathien vöm Beckentyp im Stage II. Z. árztl. Fortbild., 1972, 66, 446448). Wolff reported there that hyaluronidase from bovine testicles, administered intravenously together with magnesium ions (mixed injection, 3x 300 IU per week, i.e. generic units for 6 weeks), improved blood flow in the vessels in 90% of patients with stage II pelvic-type arteropathy. Grosshennig (Ergebnisse der Behandlung degeneratierer arterieller GefäPerkrankungen der unteren Extremitáten mit intravenouse Gaben von magnesium compositum „Scharffenberg” und Hyaluronidase. Dtsch. Ges.-wesen, 1965, 21, 869-872) carried out a similar combination therapy in degenerative artery diseases of the lower extremities and in patients with pelvic type, high-lying vascular obliteration “with impressive success”. He administered 300 IU 3x per week for about 5 weeks. In some severe cases he gave a total of 30 injections. During a follow-up examination 6-8 weeks after the end of the treatment, no deterioration in the achieved condition was observed.

It was certainly a great disadvantage that the hyaluronidase from bovine testis, which was available, had only relatively low activity, so that only a small amount of enzyme activity could be used. For example, low hyaluronidase activity (300 IU) administered i.v., i.p. or s.c. for one month did not affect atherosclerotic vascular changes in hypercholesterolemic rabbits. Repeated administration of the aforementioned dose for three months reduced blood cholesterol levels and plasma fibrin content, increased the amount of free heparin in plasma, increased the permeability of blood vessels -> tissue and significantly reduced atheromatous plaques (A.I. Pertsovskii, S.I. Koval’chuk, V.A. Baronenko, R.N. Gold'man, S. Ya. Guz and T.L. Fonbarova (1973): Effect of hyaluronidase on the course og experimental atherosclerosis. Kardiologija, 13, 37-40).

Gottlieb et al., in GB 1 060 513, isolated an enzyme from animal tissue and declared it to be hyaluronidase, which appeared to be an essentially homogeneous substance and had hyaluronidase activity. This enzyme, for example, was recommended by the authors of GB 1 098 957, together with animal-derived hyaluronidase, by the authors of GB 1 179 787, for use against allergic symptoms. Gottlieb also suggested (US 3 708 575) that human vascular diseases, such as cardiac arrhythmia, thrombosis, cerebral infarction, cerebral thrombosis and myocardial infarction, and atherosclerosis, should be treated with this hyaluronidase or hyaluronidase. An isotonic, sterile solution containing, for example, 10,000 IU/ml of enzyme is injected intravenously, intraarterially or intrathecally. The enzyme was obtained from animal materials according to US 3 708 575. It is clear from the patent specification that the enzymes used were testicular hyaluronidases. The esterase activity attributed to the enzyme, the additional specificity given, and the method of determining the activity, which is not specific for lyase reactions, indicated an animal hyaluronidase. This is consistent with the fact that the latest literature no longer reports enzymes isolated from animal testes with the degradation specificity of hyaluronate lyases (Review: G. Frost, T. Csóka and R. Stern: Trends Glycisci. Glycorechnol. 8, 419-434).

According to the cited literature data, hyaluronidase obtained from bovine testicles is suitable for increasing the amount of blood flowing through the blood vessel by dissolving, breaking down or making permeable atheromatous deposits, possibly blood clots and incipient blood thrombi. The blood thrombus nucleus that is dissolved in a given case is perhaps a plaque particle that has detached from the intima and from which a blood thrombus has formed. There are also indications that the use of hyaluronidase makes the vessel wall more permeable to the tissues, and possibly also makes the adjacent tissue more permeable.

However, the disadvantage of animal-derived hyaluronidase, especially that obtained from bovine testicles, is that by its origin the enzyme carries the risk of transmitting viruses, BSE (bovine spongioform encephalopathy) and other infectious agents. Another disadvantage is that during the production of hyaluronidase, the animal tissue must be subjected to a very expensive purification in order to remove impurities and foreign enzymes. In order to obtain a sufficient amount of hyaluronidase, a very large amount of starting material must be collected under conditions that exclude deterioration.

Hyaluronidase obtained from mammalian testes - due to its non-specific nature - also hydrolyzes sulfated glycosaminoglycans found in the vascular wall. Dermatan sulfate or chondroitin sulfate, heparin and heparin sulfate, on the other hand, are assumed to inhibit the deposition of blood thrombus on the inner wall of the vessel due to their anticoagulant properties. Therefore, there is a possibility that the hydrolysis of these compounds enhances the secondary formation of the blood clot next to the intima, i.e. testicular hyaluronidase may also have an adverse side effect.

Sawyer et al. (EU 193330 B1) describe the production of a special endo-β-glucuronidase enzyme with a molecular weight of 28,500 obtained from leech and its use - in eye diseases - to stimulate the flow of physiological fluids. This enzyme is highly specific for hyaluronic acid and differs from the known endo-β-glucuronidase obtained from leech (Hirudo medicinalis) in that it is more stable at elevated temperatures and extreme pH values.

The aim of the invention is to eliminate the disadvantages of the use of animal-derived hyaluronidases as plaque-dissolving enzymes, which have been proposed so far, by using enzymes obtained from microorganisms in the form of a suitable pharmaceutical preparation for the treatment and prevention of atherosclerotic vascular wall changes. Microbial enzymes can be produced in a simple manner in principle due to the possibility of biotechnical production.

A further object of the invention is to prepare enzyme fragments with enzyme activity and plaque-dissolving activity from a suitable holoenzyme of microbial origin in a simple manner and then use them in the form of a pharmaceutical preparation. The use of the fragments is expected to result in greater stability in the form of a preparation, less immunogenicity, and better penetration.

A further aspect is that the enzymes according to the invention - in case of administration of higher enzyme activity over a longer period - should not have a detrimental effect on the living mammalian organism. The enzymes should only be able to break down sulfated glycosaminoglycans to a limited extent, because on the one hand they must break them down in order to release them from the animal testicles.

Similar to hyaluronidase derived from the human body, it dissolves plaques and incipient blood clots, but on the other hand, the highly limited breakdown of sulfated glycosaminoglycans is good for preventing so many sulfated glycosaminoglycans from being released from the intima that their absence would increase the formation of secondary blood clots.

The aim of the invention was therefore to find plaque-dissolving enzymes derived from microorganisms, which, as components of pharmaceutical preparations, have an effect similar to or exceeding that of hyaluronidase derived from bovine testicles in terms of dissolving plaques, reducing the area occupied by plaques in the inner vessel wall, thrombi and blood clots, which have a positive effect on the course of vascular diseases, are non-toxic and have no negative effects when used as an injection preparation on humans or animals.

Part of the task that ultimately led to the invention is to find a smaller protein or fragment with hyaluronate-binding activity and a method for its preparation. The enzymatic activity and properties of the fragment should be similar to or identical to the activity of the holoenzyme.

The present invention therefore relates to pharmaceutical compositions for the treatment of vascular diseases occurring directly or as a consequence of atheromatous plaque formation. The invention further relates to a novel enzyme fragment, its preparation and use.

We have found that pharmaceutical compositions containing one or more hyaluronate lyases or enzyme fragments produced from these hyaluronate lyases secreted into the submerged culture medium during fermentation by microorganisms belonging to the genus Streptococcus, in particular the commonly available Streptococcus agalactiae or Streptococcus equisimilis, possessing hyaluronate lyase activity (E.C. 4.2.2.1.), have the ability to inhibit the growth of bacteria, in vitro.

51 ^L í· ⁵ dissolves substances from plaques isolated from atheromatous tissue of mammalian and human arteries and dissolves thin plaque slices.

We have also found that in living Watanabe rabbits with a large plaque deposit in the aorta due to a genetic defect, frequent repeated administration of a pharmaceutical preparation containing hyaluronate lyase and/or the enzyme fragment with relatively high enzyme activity remarkably reduced the area of plaque deposit. While the inside of the vessels of untreated animals was covered with a thin, white plaque layer and thicker plaques were also present, the inside of the vessels of treated animals was free of the thin plaque layer and showed a deep red color, the color of the inner surface of healthy vessels. The area of the thicker plaques was also reduced.

It is surprising that this effect is triggered not only by the holoenzyme with a molecular weight of 114,000-124,000 in the pharmaceutical preparation, but also by a highly active, stable fragment of the holoenzyme, for example with a molecular weight of 84,000-86,000.

It has also been found that the fragments of the invention can be prepared by proteolytic digestion of bacterial hyaluronate lyases with proteases, preferably a protease that cleaves the C-terminal peptide bond of aromatic amino acids. In a preferred example, a hyaluronate lyase with a molecular weight of 116,000 from the microorganism Streptococcus agalactiae is cleaved into a hyaluronate lyase fragment with enzymatic activity. The molecular weight of the fragment is 84,000-86,000. There is no difference in the specificity of cleavage between the undigested hyaluronate lyase (holoenzyme) and the fragment. However, compared to the holoenzyme, the fragment is significantly more stable in aqueous solutions and against denaturing agents. The specific activity of the fragment is 400,000-800,000 IU/mg, the specific activity of the holoenzyme is 400,000 IU/mg, so the activity of the fragment is the same, or even higher.

The invention therefore relates to a pharmaceutical composition which contains highly purified microbial hyaluronate lyase in the form of a holoenzyme, the hyaluronate lyase

hyaluronate-degrading fragment or a mixture of these two forms together with at least one stabilizer and/or a pharmaceutically acceptable carrier or excipient, and optionally an additional pharmaceutically acceptable diluent.

The pharmaceutical compositions of the invention can be used in both humans and animals for the treatment of vascular diseases primarily attributable to the presence of atheromatous plaques in the blood vessels, such as cardiac arrhythmias, atherosclerosis, cerebral infarction, cerebral thrombosis, coronary artery thrombosis and myocardial infarction.

The compositions of the invention are preferably injectable compositions for intravenous or intraarterial administration. The compositions are isotonic, sterile, aqueous solutions containing highly purified enzyme protein and/or fragment of the holoenzyme or a defined mixture of these two active forms, which contain 20,000-4,000,000 IU of enzyme activity per milliliter. The enzyme protein is preferably used as a freeze-dried solid, usually containing a stabilizing additive. Examples of stabilizing additives include sodium chloride, glucose, magnesium salts, polyvinylpyrrolidone, amino acids, albumin, particularly preferably ovalbumin and its hydrolysates, or proteins of plant origin and their hydrolysates. The amount of hyaluronate lyase and/or its fragment used in an injection is 200-12,000 IU/kg body weight, based on the body weight of the mammal or human being treated.

The enzyme or enzyme fragment used according to the invention did not have any negative effect on the health of the experimental animals. It is obvious and within the scope of the invention that the effect of the proposed enzyme and/or its fragment is not limited to the removal of current deposits, but also has a beneficial effect on circulatory diseases, such as myocardial ischemia, myocardial infarction, coronary infarction, cardiac arrhythmia, atherosclerosis and similar phenomena, both in terms of prevention and therapy.

Compared to the holoenzyme, the fragment has the advantage of being able to penetrate the solid layers of plaques faster than the holoenzyme, due to its smaller size, and thus break down the plaques more quickly.

Without limiting the invention to a hyaluronate lyase or a fragment thereof from a specific microorganism, the invention is described by way of example with the commonly available enzyme Streptococcus agalactiae. The hyaluronate lyase formed has an isoelectric point IP of about 8.6, a molecular weight of about 116,000, and an endoglycan effect. The enzyme has the advantageous property that sulfated glycosaminoglycans, such as sulfated hyaluronic acid, inhibit the enzyme. By the purification methods detailed below, the hyaluronate lyase is obtained as a highly purified enzyme for injection purposes, or the purified hyaluronate lyase is converted into a fragment with a specifically acting protease. As a specifically acting protease, for example, the acid metalloprotease MO/2 (DD 270924) can be used, 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 and 4.0.

The following detailed description of the preparation of both the holoenzyme and the fragment, in particular the sequence of purification steps, is exemplary and does not limit the scope of the invention. The preparation of highly purified hyaluronate lyase and highly purified fragment, their incorporation into an injectable preparation, in terms of the sequence and selection of purification steps, can be carried out, for example - without limiting the scope of the invention - as follows.

The microorganism, for example one of the above-mentioned, is fermented in a fermenter equipped with a stirrer at a constant pH value, preferably between 6.5 and 7.5. The lactic acid formed during the fermentation is neutralized by adding a dilute sodium hydroxide solution. The medium contains inorganic salts, yeast hydrolysate, 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 ultrafiltration module is between 30 and 50 kD. A prepurified enzyme solution is obtained, which must be subjected to further purification steps before it can be used as an injection preparation. The purification steps and the use of pyrogen-free excipients ensure the pyrogen-free nature of the injection preparation. The ionic strength is increased by adding ammonium sulfate to 40% saturation, and the enzyme is then adsorbed on phenylsepharose (Sepharose). A neutral, buffered aqueous solution containing 25% ammonium sulfate based on 100% saturation is used for desorption. After a dialysis step, Q-Sepharose (Pharmacia) is added to the solution to remove impurities. This is followed by specific adsorption on a dye, e.g. aminophenyloxamic acid. As a final purification and at the same time for determining the molecular weight, molecular weight chromatography can be performed on Superdex (Pharmacia).

If the enzyme-producing microorganism Streptococcus equisimilis is used, the purification step consisting of adsorption onto the dye is omitted. This enzyme acts as an exo-glycanase, its isoelectric point is between pH 4.5 and 4.8, and it is only slightly inhibited by sulfated glycosaminoglycans.

The partial digestion of the holoenzyme - resulting in its fragment - is carried out with immobilized or non-immobilized specific protease. The reaction with immobilized protease has the advantage that the digestion can be carried out in a controlled manner, the protease does not contaminate the enzyme. If the protease is added directly, the inactivation of the protease, the removal of the protease protein and multi-step purification are required after digestion. The enzyme fraction can be used as follows to prepare the fragment: the specifically degrading protease MO/2 is bound to sepharose in a known manner, for example. The immobilized protease is mixed with an aqueous hyaluronate lyase solution at a pH of 7.5 and a temperature of 37 °C. After the digestion is complete, the fragment is precipitated with ammonium sulfate and purified by molecular weight chromatography. The highly purified enzyme or enzyme fragment, when immunized with a domestic rabbit, shows only specific precipitation. All detectable bands in the blot are stained with monoclonal antibodies. The specific activity of the holoenzyme is about 400,000 IU/mg, and the specific activity of the fragment is about. 400,000 IU/mg to 800,000 IU/mg. For stabilization, at least one stabilizer, such as albumin, preferably ovalbumin, and inorganic salts, is added to the enzyme or fragment.

Without limiting the invention, a method for preparing the fragment is described below by way of example. For the digestion, according to the invention, a protease is used which cleaves the C-terminal peptide bond of aromatic amino acids. As a preferred embodiment, the metalloprotease MO/2 is used, which is obtained from the filtrate of a culture of Streptomyces hygroscopicus (strain AP40). The metalloenzyme MO/2 contains Co ⁺⁺ in the active center (DD 270 924). The proteases are preferably used in a purified state. The purification of the protease MO/2 is carried out, for example, by chromatography on phenylsepharose, DEAE-sepharose, Q-sepharose or Sephacryl S 100, the swelling factor is 20. The specific activity is 0.25 Kunitz units/mg (U/mg), the reaction maximum is at pH 4.2 and the reaction temperature maximum is 65 °C. The molecular weight of the enzyme, determined by SDS-gel electrophoresis and molecular weight chromatography using Sephades G50 superfine, is 14,000-15,000 D. The MO/2 protease is an endopeptidase with an isoelectric point IP = 3.85 and an I-point of 3.92. The enzyme hydrolyzes natural polypeptides such as casein, hemoglobin, bovine serum, albumin and ovalbumin.

The advantage of using MO/2 protease is that the enzyme breaks down proteins very specifically and its general digestibility towards proteins is very low. The enzyme almost exclusively breaks down peptide bonds of the C-terminal residues of aromatic amino acids. It has esterolytic activity towards N-benzoyl-L-proline-nitroanilide and has pronounced milk precipitating properties. The milk precipitation detectable on the basis of the formation of a stable casein gel over a long period of time (similar to the gel precipitated from milk with the very specific foot enzyme) is a further indication that the activity of MO/2 protease is advantageously limited for the purposes of the invention. i.e. it breaks down the bonds of the holoenzyme specifically but does not break down the fragments further.

According to a further embodiment of the invention, the MO/2 protease is optionally bound to a suitable insoluble immobilization carrier and used in this form for the degradation of the holoenzyme. This has the advantage that dissolved protease cannot enter the solution of the fragments.

According to the invention, the fragment is produced by partial digestion or proteolytic partial cleavage of the holoenzyme with a specific cleavage protease that mainly cleaves the peptide bond at the C-terminal end of aromatic amino acids.

The highly purified enzyme solutions or fragment-containing fractions can be used as injection preparations after concentration and partial dehydration, optionally adding stabilizers, excipients or pharmaceutically active substances, and then sterile filtering the solution. The highly purified enzyme or fragment solution can be freeze-dried after sterile filtering and adding, for example, 5 mM magnesium chloride and 1% egg albumin. The freeze-dried enzyme or fragment is dissolved in saline to obtain an isotonic injection preparation.

The invention also relates to the use of microbial hyaluronate lyase, its hyaluronate-degrading fragment or a mixture of the two forms for the preparation of pharmaceutical compositions suitable for the treatment and prevention of vascular diseases attributable to the presence of atheromatous plaques in the blood vessels, such as cardiac arrhythmias, atherosclerosis, cerebral infarction, cerebral thrombosis, coronary artery thrombosis and myocardial infarction.

A further object of the invention is hyaluronate lyases from bacteria and fragments with hyaluronate lyase activity.

The following examples illustrate the invention in more detail, but do not limit it.

Experimental part

Example 1

The fermentation is carried out in a fermenter with a gross filling volume of 30 liters and equipped with a stirrer. The actual filling volume is 20 liters. A commonly available strain of Streptococcus agalactiae is used for inoculation, which is deposited in the German Collection of Microorganisms and Cell Cultures under the number DSM 2134 (= ATCC 13813 = NOT 8181). The strain is preserved as a cryopreserva (Mast-Diagnostica). A ball covered with the strain material is placed in a slanted agar tube filled with agar for counting and brought into contact with the agar surface by moving the tube (“A” culture). The rolled ball is then placed in 3 ml of heart-brain broth for sterility control (“B” culture), and both cultures are incubated as standing cultures at 37 °C for 24 hours.

Preculture 1. 5050 ml of a medium containing 1 g/1 casein peptone and 10 g/1 yeast extract (Difco) are filled into 2 100 ml culture bottles and sterilized at pH 7.0. 5 ml of Eagle medium are added to the 50 ml medium - shortly before inoculation. Inoculation is carried out by washing the material from the agar surface of culture “A” into the bottles with a solution of culture “B”. The culture is cultured for 24 hours at 37 °C with shaking (150 min' ¹ ).

2. preculture g/yeast extract and 10 g/1 pancreatic peptone, 1.75 g Na-acetate, 7 g/1 Na-bicarbonate (dissolved separately), 7 g/1 di-Na-hydrogen phosphate dihydrate and

2x1 liter of medium containing 3.5 g/l Na-dihydrogen phosphate is adjusted to pH 6.2 with 20% sulfuric acid before sterilization. After autoclaving, the pH value is about 7.0. A solution containing 6.0 g/l glucose is treated separately in an autoclave. 100 ml of this solution is added to the 2nd preculture before inoculation. The medium is inoculated by adding 50 ml of the 1st preculture (5% by volume). The culture is carried out with slow shaking (about 45 min' ¹ ) at 32 °C for 24 hours.

Main crop

The composition of the main culture medium is as follows: 20 g/l yeast extract, 10 g/l pancreatic peptone and 25 g/l glucose. The glucose is sterilized separately. The medium is inoculated with 1 l of the 2nd preculture. Fermentation parameters: 34 °C, pH 7.0, aeration in the headspace 25 l air/min, stirring speed: 150 min' ¹ . The pH value is kept constant by adding 40% sodium hydroxide solution. The glucose is added manually at such a rate that the glucose concentration does not fall below 5 g/l. This culture is terminated after twenty hours.

Example 2

50 liters of the culture broth of the Streptococcus agalactiae microorganism fermentation with an activity of 400 IU/ml are filtered through a 0.2 pm filter to remove live cells. The pure culture liquid is then subjected to ultrafiltration with a molecular weight cut-off of 60,000, thus obtaining 2 liters of concentrate. 480 g of solid ammonium sulfate are added to this. The proteins precipitated at this concentration are removed by filtration using a filter aid and the pure concentrate is applied to a 2.5x20 cm phenyl sepharose column previously equilibrated with a 40% ammonium sulfate solution at a pH of 6.5, buffered with phosphate. The column is washed with phosphate-buffered 35% ammonium sulfate solution, pH 6.5, then the hyaluronate lyase is eluted with phosphate-buffered 25% ammonium sulfate solution, pH 6.5. The eluted fractions are combined and adjusted to 80% saturation by adding solid ammonium sulfate. The precipitate that forms is collected, 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 passed through a 2.2x15 cm equilibrated Q-Sepharose column. The hyaluronate lyase is in the flow-through. Its saturation is adjusted to 80% by adding solid ammonium sulfate to the liquid. The precipitated protein is collected, dissolved in 0.05 M Tris buffer at pH 8.7 and freed from ammonium sulfate on a Sephadex G 10 column. The resulting solution containing hyaluronate lyase is applied to a suitably equilibrated 0.8x10 cm affinity column [N-(p-aminophenyl)-oxamic acid agarose] and then eluted with 0.1 M NaCOa solution at pH 9.7.

The eluted hyaluronate-lyase-containing fractions are combined and precipitated by adding ammonium sulfate to 80% saturation. The precipitate is dissolved in 1 ml of 0.1 M Tris buffer, pH 7.5 and the solution is applied to a 16x60 Superdex 200 column. The hyaluronate-lyase protein band appearing at 116,000 D is collected and dialyzed against 0.02 M Tris buffer, pH 7.5 containing 0.005 M MgCl. 10 mg of hyaluronate-lyase is obtained, the specific activity of which is 400,000 IU/mg. Ovalbumin is added to the dialyzed hyaluronate-lyase solution in an amount of 1% and the solution is lyophilized.

Example 3

According to Examples 1 and 2, the Streptococcus equisimilis microorganism is cultured, or the superculture is fermented and the enzyme is recovered in the thiostate form. The purification step with N-(p-aminophenyl)-oxamic acid-agarose is omitted.

Example 4 mg of purified protease labeled MO/2 is dissolved in 2 ml of 0.1 M sodium bicarbonate solution. 1 ml of cyanogen bromide sepharose (Pharmacia) is added to the solution and the mixture is shaken at 4 °C for 24 hours. The unbound MO/2 solution is separated from the formed ΜΟ/2 protease sepharose on a glass filter. Any binding sites on the cyanogen bromide sepharose that may not yet be saturated are blocked with 2 ml of 0.1 M glycine solution. After blocking, the

The MO/2-Sepharose was washed with 0.1 M acetic acid pH 4.5 followed by 0.1 M sodium bicarbonate solution.

10 mg of hyaluronate lyase from Streptococcus agalactiae microorganism is dissolved in 1 ml of 0.05 M phosphate buffer pH 7.0 and the solution is heated to 37 °C in a thermostat. 0.25 g of fixed ΜΟ/2 protease sepharose (proteolytic activity 0.1 Kunitz units/g) is added to the solution. After fifteen minutes, the protease sepharose is separated and the solution is saturated with ammonium sulfate to 80% to precipitate the formed fragment. The precipitate is separated after 18 hours and dissolved in 0.05 M Tris buffer pH 7.5. The fragment is purified by molecular weight chromatography on Superdex 200 adsorbent. The active fragment fractions are combined, dialyzed against 0.05 M Tris buffer, and then lyophilized with the addition of albumin. The specific activity of the fragment is 600,000 IU/mg.

Example 5

4 mg of hyaluronate lyase from Streptococcus agalactiae is dissolved in 1 ml of 0.05 M Tis-HCl buffer at pH 7.8 and the solution is heated to 37 °C in a thermostat. 80 pg of 0.1 Kunitz unit/mg activity of ΜΟ/2 protease is added to the solution and the mixture is digested at 37 °C for 30 minutes. 10 μΐ of 0.1 M EDTA at pH 7.0 is added to stop the reaction. The mixture is then applied to a 3 ml affinity column [N-(p-aminophenyl)oxamic acid agarose] and eluted with 0.05 M sodium carbonate solution at pH 9.7. The solution is saturated with ammonium sulfate to 80%, the formed fragment is precipitated, collected, and then purified by molecular weight chromatography. The active fragment fractions are combined, dialyzed against 0.05 M Tris buffer, and then lyophilized with the addition of albumin. The specific activity of the fragment is 460,000 IU/mg.

Example 6 • · · · · · · • · ··*· · ··· ··· ···** ·

0.5 ml of the hyaluronate lyase fragment solution of Example 4 was added to 0.5 ml of 0.1 M acetate buffer, pH 6.0, and the solution was diluted in a geometric progression. To each dilution, 0.5 ml of a hyaluronic acid solution containing 0.2 mg of hyaluronic acid in 1 ml of 0.1 M acetate buffer, pH 6.0, was added. The mixtures were incubated at 37 °C for 30 minutes. To stop the enzyme reaction, 2 ml of 2.5% cetyltrimethylammonium bromide solution (prepared with 2% sodium hydroxide solution) was added to each dilution. The dilutions are allowed to stand at room temperature for 20 minutes, then the turbidity of the dilutions is measured in 1 cm cuvettes at a wavelength of 600 nm and the amount of hyaluronic acid broken down is determined using a calibration curve. 5 IU (international units) of hyaluronate lyase fragment breaks down 16 pg of hyaluronic acid per minute.

Example 7

The holoenzyme and the fragment of Example 4, 20,000 - 20,000 IU, were dissolved in 3 ml of distilled water. Both solutions were stored at room temperature for 24 hours, after which the enzyme activity was measured. The activity of the holoenzyme solution decreased to 60%, while the activity of the fragment solution was 95% of the initial activity.

Example 8

Determination of hyaluronate-Haz and fragment activity

1. Optical test at 232 nm

Stock solutions of hyaluronic acid and sulfated hyaluronic acid are prepared and stored at 4 °C. The measurement is performed at a wavelength of 232 nm and at 26 °C. The following solution is prepared in 3 ml quartz cuvettes (d = 1 cm): 200 µl of hyaluronic stock solution is added to 1.8 ml of 0.1 M acetate buffer with a pH of 6.0 and the reaction is started with 2-4 IU/ml (50 ml) of hyaluronate lyase. The final volume is 2050 µl.

For the inhibition experiments, the above-described composition is modified as follows: 300-500 μΐ sulfated hyaluronic acid and 200 μΐ hyaluronic acid are added to 1.5-1.8 ml of buffer and the reaction is initiated by the addition of hyaluronate lyase. Hyaluronate lyase will be partially inhibited non-competitively. The Ki value is 5.5 x 10 ⁴ mg/ml.

Hyaluronate lyase shows a linear increase in extinction at 232 nm, the activity of which can be calculated from the tangent of the extinction coefficient of the resulting Á ^4,5 -unsaturated uronide, ε = 6.0 x 10 ³ Ixmof'cm' ¹ (J. Ludowig: The mechanism of hyaloronidases. JBC 236, 333-339 (1991)).

2. Optical experiment on microplates at 600 nm

Determination of lyase activity according to D. Gerlach and W. Köhler “Hyaluronatelyase von Streptococcus pyogenes. II. Charakterisierung der Hyaluronatelyase” (E C. 4.2.2.1.) (Zbl. Bakt. Hyg, I. Abt. Őrig. A 221, 296-302 (1972))

A stock solution of 50,000 U/ml activity is diluted 1:50 to prepare a hyaluronate lyase solution and 0.5 ml of the hyaluronate lyase solution is added to 0.5 ml of 0.1 M acetate buffer, pH 6.0. The resulting solution is diluted geometrically. To each dilution, 0.5 ml of a hyaluronic acid solution containing 0.2 mg of hyaluronic acid in 1 ml of 0.1 M acetate buffer, pH 6.0 is added. The mixtures are incubated at 37 °C for 30 minutes. To stop the enzyme reaction, 2 ml of 2.5% cetyltrimethylammonium bromide solution (prepared with 2% sodium hydroxide solution) is added to each dilution. The dilutions are allowed to stand at room temperature for 20 minutes, then the turbidity of the dilutions is measured in 1 cm cuvettes at a wavelength of 600 nm and the amount of hyaluronic acid broken down is determined using a calibration curve. 5 IU (international units) of hyaluronate lyase breaks down 16 μg of hyaluronic acid per minute.

Example 9 mg of hyaluronate lyase (holoenzyme) is dissolved in 1 ml of 0.05 M phosphate buffer at pH 7.0 and the solution is kept at 37 °C in a thermostat. To this solution is added protease sepharose with a specific proteolytic activity of 0.1 Kunitz units/mg. After fifteen minutes, the insoluble protease sepharose is separated and the resulting fragment is precipitated by saturating the solution with ammonium sulfate to 80%. 18 hours later, the precipitate is separated and dissolved in 0.05 M Tris buffer at pH 7.5. The fragment is purified by molecular weight chromatography with Superdex 200 adsorbent. The active fractions are combined, dialyzed against 0.05 M Tris buffer and lyophilized while albumin is added.

Example 10

To demonstrate the plaque-dissolving activity of hyaluronate lyase, whitish-yellowish tissue pieces (plaque pieces) of approximately 0.3 mm in size, taken from the neointima of the human carotid artery, are suspended in hyaluronate lyase solution (approximately 20,000 IU/1 ml Tris buffer; 10 mM, pH 7.0) and the suspension is incubated at room temperature for 12 hours. Partial dissolution of the originally sharp phase boundaries of the plaque pieces and the release of finely distributed suspended material can be observed. In parallel, plaque pieces were treated with Tris buffer only, and no dissolution phenomena occurred here.

Example 11

Experiment with a fragment of hyaluronate lyase to dissolve human atheromatous vascular deposits

Whitish-yellowish tissue fragments (plaque fragments) of approximately 0.3 mm in size taken from the neointima of the human internal carotid artery are suspended in the fragment solution (approximately 15,000 IU/1 ml Tris buffer; 10 mM, pH 7.0) and the suspension is incubated at room temperature for 12 hours. The originally sharp phase boundaries of the plaque fragments are largely dissolved and finely dispersed suspended material is released.

'J? .:. ^:, r --. In parallel, plaque pieces were treated with Tris buffer only, no dissolution phenomena occurred here.

Example 12

The influence of hyaluronate lyase on atheromatous vascular deposits in the Watanabe heritable hyperlipidemic (IbnrWHHL) rabbit

The domestic rabbit mentioned has been bred by Y. Watanabe (Kobe University, Japan) since 1981; it was transferred to Charles River Swine Farm as a breeding stock in 1992 and has been bred by the Broekman Institute in Somerset, the Netherlands, since 1994. The Watanabe rabbit is genetically hyperlipidemic due to an inherited defect in the LDL receptors. As a result of hypercholesterolemia (> 400 mg/100 ml plasma), the animals develop aortic arteriosclerosis early.

Ten 9-11 month old Wtanabe rabbits (Ibm. WHHL; 4 females, 6 males) were purchased from the Broekman Institute (Someren, The Netherlands) 14 days before the start of the experiment. The animals were housed in individual cages (0.5 x 1 m) at room temperature (20 ± 2 °C) and were given standard diet (ssniff rabbit diet) and drinking water ad libitum. Before the start of the experiment, i.e. before the 1st clinical-chemical examination, the 10 rabbits were randomly assigned by sex and then numbered from 1 to 10.

Three times a week (Monday, Wednesday and Friday) they received an infusion. The four control rabbits (1-4) received 20 ml of pyrogen-free 0.9% NaCl solution intravenously over one hour. The six experimental rabbits (5-10) received 30,000 IU of highly purified hyaluronate in 20 ml of pyrogen-free 0.9% NaCl solution over one hour. This corresponds to a dose of 10,000 IU/kg body weight.

During the experiment, general behavior, body weight, body temperature, and clinical-chemical parameters (cholesterol, triglycerides, amylase) were monitored.

The general behavior of the animals was not noticeably affected by either the 0.9% NaCl solution or the hyaluronate lyase treatment. The body weight of adult Watanabe rabbits (control and treated) changed only insignificantly during the duration of the experiment, i.e. from 18.11.1996 to 03.02.1997.

Body temperature, measured before and after each infusion, fluctuated on average between -0.3 and + 1.2 °C. No significant differences were observed between the control and treated animals. The serum cholesterol values of the control animals did not change significantly from the 0.9% NaCl solution. The serum cholesterol of one of the four control animals decreased by half. Treatment with hyaluronate lyase also did not result in a significant shift in serum cholesterol concentration. During the infusion treatment, the serum triglyceride values, which were partly very high, decreased in all animals. After the 20th infusion, the values of the control were 570-740 mg/100 ml, and the values of the animals treated with hyaluronate lyase were 200-290 mg/100 ml.

In 2/4 control animals, the aortic arches were highly atheromatous, in another 2/4 animals, the atheromatosis was moderate to severe. In 2/6 rabbits treated with hyaluronate lyase, the aortic arches were moderately atheromatous, and in 4/6 animals, the finding was mild to moderate.

In 2/4 control animals, A. thoracica showed large and 2/4 additional animals showed moderate atheromatous deposits. In animals treated with hyaluronate lyase, A. thoracica showed small and barely noticeable deposits in 4/6 animals.

In 2/4 control animals, the abdominal aorta showed extensive atheromatous deposits, and in another 2/4 animals, slight to moderate atheromatous deposits. In the animals treated with hyaluronate lyase, the abdominal aorta showed moderate deposits in 2/6 cases, weak or barely noticeable deposits in 4/6 cases.

• · · · · ♦ · • · »«*♦ · ·«· ··· * ·

In 2/4 control animals, the pulmonary aorta was heavily covered with plaques, in another 2/4 animals it was slightly or moderately covered. In 1/6 animals of animals treated with hyaluronate lyase, the A. pulmonary showed heavy deposits, in 2/6 the deposits were slightly or moderately covered, and in 3/6 animals there was no plaque. There were no noticeable differences in general behavior, body weight development, body temperature before and after infusion, and clinical-chemical parameters between Watanabe rabbits treated with 0.9% NaCl solution and hyaluronate lyase during the two treatment cycles consisting of 10 infusions each, so it can be concluded that the infusion fluid is well tolerated.

Based on the subjective macroscopic assessment of the aortic material, it can be said that the atheromatous deposits appeared larger in the animals treated with 0.9% NaCl solution than in the 6 animals treated with microbial hyaluronate lyase.

Example 13

A highly purified concentrated enzyme solution is diluted with enough NaCl solution to produce a solution containing 0.9% sodium chloride and 10,000 IU/ml hyaluronate lyase. The solution is sterile filtered through a sterile filter with a pore size of 0.2 pm. 1 ml of the solution is filled into ampoules under sterile conditions. The ampoules are lyophilized under sterile conditions and closed under sterile conditions. Before injection, 1 ml of sterile distilled water is added to the contents of the ampoule.

Example 14

A highly purified enzyme solution is diluted with a solution of magnesium nicotinate and magnesium adipate (?agnesiom compositum “Scharfifenberg”) so that the resulting solution contains 25 mg magnesium nicotinate, 88 mg magnesium adipate and 10,000 IU hyaluronate lyase in 1 milliliter. The solution is ampoulated according to Example 13.

Example 15

...· .:. ··»’ ±.

A solution of highly purified hyaluronate lyase is diluted with a solution of sodium chloride and egg albumin so that the solution contains 0.9% NaCl, 1% egg albumin and 40,000 IU of enzyme per milliliter. The solution is prepared according to Example 13.

Example 16

A highly purified hyaluronate lyase solution is diluted with sodium chloride solution and soy protein hydrolysate so that the resulting solution contains 0.9% sodium chloride, 1% soy protein hydrolysate and 40,000 IU of enzyme per ml. The solution is prepared according to Example 13.

Example 17

A solution of a fragment obtained from the holoenzyme of Streptococcus agalactiae is diluted with sodium chloride solution and egg albumin so that the resulting solution contains 0.9% sodium chloride, 1% egg albumin and 40,000 IU of enzyme activity in 1 ml. The solution is packaged in ampoules as described in Example 13.

Example 18

The fragment solution obtained from the holoenzyme of Streptococcus agalactiae is diluted with sodium chloride solution and magnesium chloride solution so that the finished solution contains 0.9% sodium chloride and 100,000 IU/ml of lyase activity. The solution is sterile filtered through a sterile filter with a pore size of 0.2 μηι. 1 ml of the solution is filled into ampoules under sterile conditions. The ampoules are lyophilized under sterile conditions and closed under sterile conditions. Before injection, 1 ml of sterile distilled water is added to the contents of the ampoule.

Claims (22)

...* *'f ·**» Patent claims 1. A pharmaceutical composition comprising a bacterial hyaluronate lyase in the form of a holoenzyme, a hyaluronate-degrading fragment thereof, or a mixture of these two forms, together with at least one stabilizer and/or a pharmaceutically acceptable carrier or excipient. 2. Pharmaceutical composition according to claim 1, characterized in that the hyaluronate lyase is derived from a microorganism of the genus Streptococcus. 3. Pharmaceutical composition according to claim 2, characterized in that the hyaluronate lyase is derived from the microorganism Streptococcus agalactiae. 4. Pharmaceutical composition according to claim 1, characterized in that the bacterial hyaluronate lyase in its holoenzyme form has an isoelectric point IP = 8.6 and a molecular weight of 116 kD. 5. The pharmaceutical composition according to claim 1, characterized in that the molecular weight of the hyaluronate lyase fragment is between 84 and 86 kD. 6. A pharmaceutical composition according to claim 1 for the treatment of vascular diseases, which vascular diseases are selected from the following group: cardiac arrhythmia, atherosclerosis, cerebral infarction, cerebral thrombosis, coronary artery thrombosis, myocardial infarction. 7. The pharmaceutical composition according to claim 1, wherein the hyaluronate lyase has a hyaluronate-degrading fragment that can be produced from bacterial hyaluronate lyase by partial hydrolysis with a protease capable of cleaving the peptide bond at the C-terminal end of aromatic amino acids. 8. The pharmaceutical composition according to claim 1, characterized in that it is an injection solution for intravenous or intraarterial injection. 9. The pharmaceutical composition according to claim 1, characterized in that the activity of the microbial hyaluronate lyase and/or its fragment is between 20,000 and 4,000,000 IU/ml. 10. The pharmaceutical composition of claim 1, which is a purified aqueous injection solution containing hyaluronate lyase and/or a fragment thereof and about 1% by weight of albumin and/or hydrolysate. 11. A pharmaceutical composition according to claim 1, which is a purified aqueous injection solution containing hyaluronate lyase and/or a fragment thereof and a magnesium salt. 12. The pharmaceutical composition according to claim 1, which is a purified aqueous injection solution containing hyaluronate lyase and/or a fragment thereof and glucose. 13. An enzyme fragment of hyaluronate lyase of bacterial origin, characterized in that it has hyaluronate lyase activity. 14. The enzyme fragment according to claim 13, characterized in that the hyaluronate lyase is derived from a microorganism of the genus Streptococcus. 15. The fragment according to claim 13, characterized in that the hyaluronate lyase is derived from the microorganism Streptococcus agalactiae. 16. The enzyme fragment according to claim 13, characterized in that it is prepared from hyaluronate lyase of bacterial origin by partial hydrolysis with a protease capable of cleaving the peptide bond of the C-terminal end of aromatic amino acids. 17. The enzyme fragment according to claim 13, characterized in that during its preparation, hyaluronate lyase of bacterial origin is subjected to enzymatic partial hydrolysis with a protease immobilized on a carrier. 18. The enzyme fragment according to claim 13, characterized in that during its preparation it is subjected to enzymatic partial hydrolysis with MO/2 metalloprotease immobilized on a bacterial hyaluronate lyase carrier. 28 ;-<· .: i » ...· ,h ··*· A. 19. The enzyme fragment according to claim 13, characterized in that during its preparation, it is subjected to enzymatic partial hydrolysis with MO/2 metalloprotease immobilized on a Streptococcus-derived hyaluronate lyase support at a pH of 6.5-8.0 and a temperature between 25 °C and 45 °C. 20. The enzyme fragment according to claim 13, characterized in that during its preparation, it is subjected to partial hydrolysis with hyaluronate lyase enzyme having a molecular weight between about 115,000 and 120,000 derived from the microorganism Streptococcus agalactiae. 21. The enzyme fragment of claim 13, characterized in that it has a molecular weight of between about 84,000 and 86,000. 22. The enzyme fragment according to claim 13, characterized in that its specific activity is between 400,000 IU/mg and 800,000 IU/mg. The authorized person: