KR20010041235A - Pharmaceutical formulation containing a microbial enzyme which digests hyaluronic acid - Google Patents

Abstract

The present invention includes pharmaceutical preparations which are particularly suitable as injectable preparations for use in the treatment of vascular diseases caused by atheromatic precipitation of arterial linings. The injectable preparation contains a bacterial hyaluronate lyase or a hyaluronate-digestive enzyme fragment prepared from the enzyme. The invention also includes hyaluronate-digesting enzyme fragments of bacterial hyaluronate lyase. It can be prepared by treating proenzymes with specifically digested proteases.

Description

PHARMACEUTICAL FORMULATION CONTAINING A MICROBIAL ENZYME WHICH DIGESTS HYALURONIC ACID}

Most microbial hyaluronate lyase is a high molecular protein having a molecular weight of 116,000 to 120,000 daltons. Such high molecular weight hyaluronate lyases are generally relatively sensitive to denaturing agents and extreme physiological conditions. Especially during the manufacture of pharmaceutical and veterinary preparations, a clear loss of activity occurs due to the denaturation process. In addition, the dispersibility of relatively large enzymes is significantly less than that of smaller molecules. In theory, the transport of hyaluronate lyase, for example, due to the dispersion of high molecular weight hyaluronate lyase (preenzyme) and in addition to penetration and / or dispersion into tissues and even plaques is consequently limited. Less immunogenic effects are possible.

For sequencing, it is generally known in the art to degrade proteins with proteases during the enzymatic process to obtain protein fragments that are generally not enzymatically active. Thus, it should be theoretically possible to produce smaller enzymes (preenzymes) formed by microorganisms and partially digested by specific active proteases. However, to date no enzyme fragment, in particular any enzymatic active fragment of a microbial hyaluronate lyase, has been known for which the hyaluronate lyase origin can be identified by the same region of the amino acid sequence or produced from the hyaluronate lyase. In addition, no method of consequently enzymatically active fragments can be produced from hyaluronate lyase is known.

Hyaluronidase from bovine testes is used to make the animal or human tissue more osmotic. It acts as a penetration or absorption enhancer for drugs administered subcutaneously or intramuscularly, or facilitates perfusion of larger amounts of liquid and functions for faster degeneration of edema. In intravenous treatment and dermatology of the heel, high levels of bovine testicular hilase "Dessau" administered intravenously in case of acute tendovaginitis and Paratenonitis crepitans. It was already known in the 1960s that a good effect in dosage was shown. For example, in the case of progressive scleroderma, limited sclerosis and keloids, the signs after hyaluronidase treatment are significantly reduced.

In the 1970s, attempts were made to affect the late sequelae of animal myocardial infarction with hyaluronidase. The purpose of the study was to reduce cardial necrosis areas and to improve peripheral blood circulation with accelerated formation of new blood vessels after infarction.

Since the 1960s, there has been literature on the use of bovine testicular hyaluronidase to treat human arterial vascular disorders, particularly to improve blood circulation in peripheral blood vessels (eg Thurnherr and Koch, Quoted by Wolff: Results of Treatment with Intravenous Magnesium Compound / Hillase Injection in the Case of Pelvic Type II Arteriopathies (Z. arzl. Fortbild., 1972, 66, 446-447). For example, Wolff improves blood circulation in blood vessels in 90% of patients with pelvic-type stage II arteriosclerosis examined when intravenous administration of hyaluronidase from bovine testis with magnesium. Reported. Intravenous administration of Grosshennig (magnesium compound "Scharffenberg" and hyaluronidase resulted in the treatment of lower terminal degenerative arterial vascular disease. Ges.-wesen, 1965, 21, 869-872) Similar mixed treatments have been performed with "impressive success" for patients with high-grade degenerative vascular occlusion as well as for lower terminal degenerative vascular disease. He administered 300 IU three times a week for about five weeks. No worsening was detected during the 6-8 week follow-up.

Hyaluronidase available from bovine testes is certainly not very beneficial as it can only be used with relatively low activity and therefore only low enzyme activity. For example, administration of a very low hyaluronidase activity of 300 IU in hypercholesterinemic rabbits for one month is i.v., i.p. Or s.c. had no effect on the vascular changes of atherosclerosis. Repeated doses of this amount for up to three months lowered the amount of blood cholesterol and plasma fibrin, increased plasma free heparin, increased vascular osmoticity in the direction of tissues, atheromatous) caused a marked decrease in vascular plaques (AIPersovskii, SI, Koval'chuk, VABaronenko, RNGold'man, S. Ya. Guz and TL Fonbarova (1973): in the course of experimental atherosclerosis Effect of hyaluronidase.Kardiologija, 13, 37-40).

An enzyme called hyaluronate lyase and "hyaluronidase which is particularly effective because of its broad specificity that appears to be essentially homogeneous" is an enzyme described in Gottlieb et al. It was explained by). Such enzymes have been proposed, for example, in British Patent No. 1,098,957 and enzymes mixed with hyaluronidase of animal origin have been proposed in British Patent No. 1,179,787 for the treatment of allergic signs. Gottlieb also treats vascular diseases of humans such as arrhythmia heart disease, thrombosis, cerebral infarction, cerebral thrombosis and myocardial infarction, and especially atherosclerosis with the hyaluronidase or hyaluronate lyase (US Pat. No. 3,708,575) Suggested. For example, 10,000 IU / mL of sterile isotonic solution is injected into a vein, artery or spinal cavity. The enzyme was obtained from animal material according to patent (US Pat. No. 3,708,575) information. It is clear from the above specification that the enzyme used was a testicular hyaluronidase. The method used to determine the activity, as well as the claimed broad specificity, the enzyme's esterase activity, which is not specific to the lyase reaction, relates to animal hyaluronidase. Thus, enzymes with degradation specificity of hyaluronate lyase from animal testes are not shown in the most recent documents (Overview: G.L. Frost, T. Csoka and R. Stern: Trends Glycosci. Glycotechnol. 8, 419-434).

According to the literature information, hyaluronidase from bovine testes is directed against the blood flow based on the fact that atheromatic precipitation and possibly the components of the coagulated blood and the blood coagulation are dissolved, degraded or osmotic. It is suitable for increasing the osmoticity of blood vessels. The component of blood thrombosis that possibly dissolves may be plaque particles that are separated from intima or where blood coagulation has formed. There is also a description that the use of hyaluronidase makes the vascular wall more osmotic in tissue as well as in adjacent tissues.

However, because of their origin, enzymes can carry viruses, bovine spongioform encephalopathy (BSE) and other infectious agents, so the use of hyaluronidase of animal origin, in particular bovine testicular hyaluronidase, is disadvantageous. There is also a drawback in the manufacture of hyaluronidase that a great deal of purification effort is required to remove constituent forms of impurities, especially foreign proteins, from animal material. In addition, in order to obtain a sufficient amount of hyaluronidase, the corresponding large amount of starting material must be collected under conditions that prevent destruction.

Because of its non-specificity, hyaluronidase from mammalian testes also hydrolyzes sulfated glycosaminoglycans in the vessel wall. However, dermatan sulfate or chondroitin sulfate, heparin sulfate and heparan sulfate are presumed to prevent blood coagulation from adhering to the inner wall of blood vessels due to their anticoagulation properties. Therefore, the hydrolysis of the compound increases the secondary formation of blood coagulated in intima, so that testicular hyaluronidase may cause undesirable side effects.

Sawyer et al. (EU 193330 B1) described the preparation and use of specific endo-β-glucuronidase having a molecular weight of 28,500 D from leeches to stimulate physiological fluid flow in the case of eye diseases. Explained. The enzyme is distinguished from the well-known endo-β-glucuronidase from Hirudo medicinalis because it is very specific for hyaluronic acid and more stable at high temperatures and extreme pH values.

In older or pathological situations, hydrophobic plaques settle in the inner wall of the arteries of humans as well as mammals. The hydrophobic plaques cause a decrease in the internal lumen due to hardening of the arterial wall, dysfunction and neointima formation in blood vessels. Pathological signs such as arrhythmia heart disease, clot formation and thrombosis, cerebral infarction, thrombosis, hypertension and decreased blood supply are closely related to vascular changes.

The present invention relates to pharmaceutical preparations having the property of reducing the area of endometrium covered with plaques or reducing their effect on blood flow when used intraarterally or intravenously.

This action is closely related to the presence of complete enzymes (preenzymes) and / or new, smaller enzyme fragments that can be prepared from the enzymes of the present invention.

The proposed proenzyme is hyaluronate lyase and the fragment is also hyaluronic acid-degrading activity. This activity results in particular in the desired decomposition of hyaluronic acid.

The present invention also relates to novel enzyme fragments produced by specific enzymatic digestion processes.

In addition to chondroitin sulfates, dermatan sulfate, heparin and heparan sulfate, hyaluronic acid belongs to glycosaminoglycan. Except for hyaluronic acid, glycosaminoglycans include sulfate groups (sulphated glycosaminoglycans). Glycosaminoglycans occur in all vertebrate tissues. Hyaluronic acid is particularly present in intracellular matrix, skin and cartilage tissue as well as body fluids such as joint fluids or eye fluids. Hyaluronic acid, along with other glycosaminoglycans, is believed to be contained in the vascular wall and also in the atheromatous precipitates or plaques of the arterial wall. Hyaluronic acid has properties such as binding with water, viscosity, formation of hydrophilic colloidal solutions, and low solubility complexes with strong base proteins. Hyaluronic acid consists of glucuronic acid and acetylated glucosamine, which are linked by β- (1-3) -glucoside bonds. The disaccharide units are in turn linked by glucoside β- (1-4) -bonds. Hyaluronic acid is degraded by enzymes bound under the collective name of hyaluronidases.

The group name hyaluronidase is inaccurate from a systematic enzymatic point of view, but three different types of active hyaluronic acid-degrading enzymes (J. Ludowieg: mechanism of hyaluronidase, JBC 236, 333-339, (1961) )) These are endohydrolases that hydrolyze the β- (1-3) -bonds by the addition of water. These include a number of hyaluronidase from higher organisms, such as hyaluronidase used medically from bovine testes. These enzymes, also called hyaluronate-glycan hydrolases (E.C. 3.2.1.35/36), hydrolyze not only hyaluronic acid but also other glycosaminoglycan bonds in a limited range. Endo-β-hyaluronidase from leeches, which break down β- (1-4) -binding very specifically, is another form. All the enzymes mentioned above are hyaluronidase with hydrolytic activity or hyaluronidase in the stricter sense.

The third type of enzyme, hyaluronate lyase (EC4.2.2.1), forms hyaluronic acid at β (1-4) -bonds according to its elimination mechanism with the formation of a double bond at the (4-5) position of glucuronic acid Decompose Thus hyaluronate lyase is not a hyaluronidase. Hyaluronate lyase is present in microorganisms. These are found, for example, in streptomycetes and other bacteria. Their unique commonality is their narrow specificity for hyaluronic acid. Other glycosaminoglycans are degraded to a general extent negligible (J.-H.Ozegowski et al .: Purification and Description of Hyaluronidase from Streptococcus agalactiae, Zbl. Bakt. 280, 497-506 (1994), A. Linker et al .: Preparation of unsaturated uronides by bacterial hyaluronidase, JBC 219 13-25, (1956), T. Ohya, Y. Kaneko: Streptomyces New hyaluronidase, BBA 198 (1970), 607-609 and K. Gase, J.-H. Ozegowski and H. Malke: Streptococcus agalac, which encodes hyaluronate lyase completion of sequence and expression analysis Tia hylB gene, BBA, 1998, 86-98).

It is an object of the present invention to overcome the shortcomings of hyaluronidase of animal origin, which has been proposed as an enzyme that breaks down plaque, by using enzymes obtained from microorganisms in suitable therapeutic pharmaceutical preparations as well as prevention of atherosclerotic vascular changes. will be. In principle, because of the possibility of producing microbial enzymes by biotechnological methods, microbial enzymes can be easily obtained.

It is also an object of the present invention to prepare enzymatically active enzyme fragments having plaque degrading activity from a suitable microbial proenzyme in a simple manner and to use them as pharmaceutical preparations. Fragments are expected to be more stable in pharmaceutical formulations, less immunogenic when used, and better to penetrate.

Furthermore, enzymes made available according to the present invention do not have a negative effect on living mammals when higher enzyme activity is applied for an extended period of time. The enzyme also degrades sulfated glycosaminoglycans only within a limited range and, on the other hand, causes the dissolution of coagulated blood components comparable to that caused by plaque and animal testicular hyaluronidase. On the other hand, due to the very limited degradation of sulfated glycosaminoglycans in intima, the risk of secondary coagulation blood formation due to the absence of sulfated glycosaminoglycans is not greatly increased.

Thus, according to the invention, as a pharmaceutical agent component which is of microbial origin and which breaks down plaque and is used in the blood vascular system, it reduces the plaque or coagulation component or the internal wall of blood vessels covered with coagulated blood to hyaluronic bovine testes Enzymes that have a comparable or better effect than nidase, have a desirable effect on the development of vascular disease, and are not toxic and have no other negative effects when used in humans or animals as injectable preparations.

Another object of the present invention is to make a fragment or smaller protein having hyaluronate lyase activity useful and to provide a method of making the same. The fragment has properties of enzymatic activity similar or identical to its preenzyme.

The present invention therefore encompasses pharmaceutical agents suitable for the treatment of vascular diseases which occur either directly or as a result of atheromatous plaque formation. The present invention also encompasses novel enzyme fragments and their preparation and use.

According to the invention, the species Streptococcus agal generally available by fermentation with the activity of hyaluronate lyase (EC4.2.2.1.), Particularly in the genus Streptococcus, preferably in a submersion medium One or more bacterial enzymes having the activity of hyaluronate lyase, excreted by microorganisms of Streptococcus agalactiae or Streptococcus aquisimilis, or enzyme fragments produced from hyaluronate lyase Pharmaceutical formulations are known which release substances in vitro or dissolve thin plaque sections from isolated plaque precipitation, which have been obtained from atheromatous blood vessels in mammals and in humans.

Furthermore, the pharmaceutical preparations of the present invention, which contain relatively high enzymatic activity and hyaluronate lyase and / or enzyme fragments, are repeatedly vened in live Watanabe rabbits with large plaque deposits in the aorta due to genetic defects. When injected by intravenous injection, the amount of plaque precipitate in the blood vessels markedly decreased after treatment. The blood vessels inside untreated animals had a thin, slightly white layered surface as well as thicker plaques. On the other hand, the blood vessels inside the treated animals had no thinner plaque deposits and had a deep red hue on the inner surface of healthy blood vessels. The area range of thicker plaque deposits has also been reduced.

Surprisingly, the effect is not limited only in the presence of pharmaceutical preparations of proenzymes in the molecular weight range of 114,000 D to 124,000 D, for example by high activity, stable fragments of preenzymes such as fragments having molecular weights of 84,000 to 86,000. Is generated.

Furthermore, it has been found that fragments of the present invention can be prepared by proteolytic digestion of bacterial hyaluronate lyase with proteases, preferably proteases that break down C-terminal peptide bonds of aromatic amino acids. In a preferred embodiment, hyaluronate lyase having a molecular weight of 116,000 Daltons from Streptococcus agalactiae is cleaved into enzymatically active hyaluronate lyase fragments. The fragment has a molecular weight of 84,000-86,000 Daltons. This is no different from undegraded hyaluronate lyase (preenzyme) in its degradation specificity. However, compared to the preenzyme, the fragments are distinguished by a significantly higher stability in the presence of aqueous solutions and denaturants. The enzyme has a specific activity between 400,000 IU / mg and 800,000 IU / mg, which is equal to or even higher than the specific activity of 400,000 IU / mg of the preenzyme.

The present invention therefore relates to hyaluronate lyases of microbial origin, hyaluronate degrading fragments thereof or mixtures of the two forms, each of which is very highly purified, and at least one stabilizer and / or pharmaceutically as proenzyme. A pharmaceutical formulation containing a stable carrier or inert substance and optionally added pharmaceutical stable diluent.

The pharmaceutical preparations of the present invention are primarily used for the treatment of vascular diseases, such as arrhythmia heart disease, arteriosclerosis, cerebral infarction, thrombosis, coronary thrombosis and myocardial infarction due to the presence of atheromatous plaques in blood vessels. Suitable for animals).

The pharmaceutical preparations prepared according to the invention consist in particular of an isotinic, sterile aqueous solution of a highly purified enzyme protein of a preenzyme and / or a fragment thereof or a defined mixture of two active enzyme species, in particular Injectable preparations for intravenous or intraarterial injection. The enzyme activity is between 20,000 and 4,000,000 IU / mL. Such enzyme proteins are generally advantageously used in the form of lyophilized solids containing 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 hydrolysates thereof. The amount of hyaluronate lyase and / or fragments thereof used for injection is between 2,000 and 12,000 IU / kg based on the weight of the mammal or human being treated.

The enzymes or fragments according to the invention do not have any negative effects on the health of the experimental animals. The effects of the enzymes and / or fragments thereof proposed in the present invention are not limited to the elimination of actual precipitation and treatment of circulatory diseases such as myocardial anemia, coronary infarction, myocardial infarction, arrhythmia heart disease, arteriosclerosis and similar signs. And it is apparent by the present invention that the preferred effect in terms of prevention, it will be considered as part of the patent protection scope. In the present invention, the fragment is advantageous over the preenzyme because of its smaller size, because it penetrates more quickly into the solid layer of the plaque and, as a result, causes its destruction more quickly.

The present invention is not limited to hyaluronate lyase or fragments thereof from certain microorganisms, and is described using the enzyme of Streptococcus agalactia, which is generally available as in the examples. The formed hyaluronate lyase has an isoelectric point of about 8.6 and a molecular weight of about 116,000 D and acts as an endoglycanase. The enzyme has the advantage that it is inhibited by sulfated glycosaminoglycans such as sulfated hyaluronic acid. By the following purification method, hyaluronate lyase is obtained as a high purity enzyme for injection or purified hyaluronate lyase is converted into fragments by a protease of specific activity. Without limiting the invention, acidic metalloprotease MO / 2 (DD 270924), which can be obtained from the microorganism Streptomyces hygroscopicus AP40, is an example of a protease of specific activity. Can be used as. The proteases are distinguished by a molecular weight of about 14,000 kD and an isoelectric point between 3.85 and 4.0.

The following detailed description of the preparation of fragments and preenzymes, in particular a series of purification steps, is given by way of example and is not intended to limit the scope of the invention. The preparation of high purity hyaluronate lyase and high purity fragments suitable for injection in pharmaceutical preparations can be carried out, for example, as follows. In view of the choice and order of purification steps, the scope of protection of the present invention is not limited to the described method.

For example, the fermentation of one of the aforementioned microorganisms takes place in a stirred fermenter in a constant pH environment with a pH acidity of 6.5 to 7.5. Lactic acid formed during fermentation is neutralized by the addition of diluted sodium hydroxide. The medium consists of inorganic salts, yeast hydrolysates, optionally casein peptone or soy peptone, and glucose. After approximately 20 hours fermentation, cells were isolated. The cell mass is discarded. The culture filtrate is concentrated and purified in a ultrafiltration unit. The cut-off of the ultrafiltration module is 30 to 50 kD. This produces pre-purification enzyme solutions, which must be further purified before they can be used as injectable preparations. The absence of pyrogen in injectable preparations is ensured by the purification step and the use of pyrogen-free inert materials.

After the ionic strength is increased by the addition of ammonium sulfate to 40% saturation, the enzyme is absorbed by phenyl sepharose. It is then desorbed by a neutral buffered aqueous solution containing 25% of the amount of ammonium sulfate required for 100% saturation. After the dialysis step to remove impurities, the solution is treated with Q-Sepharose. Thereafter, specific adsorption proceeds to dyes such as aminophenyloxamic acid. Along with the determination of the molecular weight, the final purification can consist of molecular weight chromatography on Superdex (Pharmacia).

When Streptococcus equisimilis is used for enzyme formation, the step of absorption purification with dyes can be omitted. The enzyme acts as an exoglycanase. Its isoelectric point is between pH 4.5 and 4.8 and is inhibited by sulfated glycosaminoglycans only in a very small range.

Partial digestion of proenzymes to fragments is performed by specific proteases, either immobilized or unimmobilized. Reaction with an immobilized protease is advantageous in that digestion can be performed selectively without contamination by the protease itself. In case of direct conversion to protease addition, digestion by the protease should be followed by an inactive step, protease protein isolation and high purification. Enzyme fractions can be used in the following manner for the preparation of fragments.

Specific degradation protease MO / 2 is immobilized on well known manners, for example sepharose. The immobilized protease is mixed with, for example, an aqueous solution of hyaluronate lyase at pH 7.5 and temperature 37 ° C. After digestion is complete, the fraction is precipitated with ammonium sulfate and obtained in very pure form by molecular weight chromatography. After immunization in rabbits, only high purity enzymes or enzyme fractions show specific precipitation. All recognizable bands in blots are stained by monoclonal antibodies. The proenzyme has a specific activity of about 400,000 IU / mg, and the specific activity of the fragment is between 400,000 and 800,000 IU / mg. For stabilization, albumin, preferably at least one stabilizer, such as ovalbumin, and inorganic salts are added to the enzyme or fragment.

One method of obtaining a fragment is described, but the present invention is not limited thereto. According to the present invention, proteases are used for digestion to break down the C-terminal peptide bonds of aromatic amino acids. In a preferred embodiment, metalloprotease MO / 2 produced from the culture filtrate of Streptomyuces hygroscopicus (strain AP 40) is used. MO / 2 is a metalloenzyme with Co ⁺⁺ as active center (DD 270 924). Proteases are preferably used in purified form. Protease MO / 2 is purified, for example, by chromatography on Sephacryl S100, Phenyl Sepharose, DEAE Sepharose, and Q-Sepharose with Concentration Factor 20. Specific activity has an order of 0.25 Kunitz units / mg (U / mg), reaction peak acidity is pH 4.2 and reaction peak temperature is 65 ° C. The molecular weight (Mm) of the enzyme determined by molecular weight chromatography and SDS gel electrophoresis on Sephadex G50 superfine is between 14,000 and 15,000 D. Protease MO / 2 is an endopeptidase with isoelectric points at 3.85 and 3.92. The enzyme hydrolyzes natural polypeptides such as casein, hemoglobin, bovine serum, albumin and ovalbumin.

The use of protease MO / 2, which specifically degrades enzyme proteins and has very little digestive activity in relation to proteins, is advantageous. The enzyme degrades only peptide bonds of almost aromatic amino acid C-terminal groups. With regard to N-benzoyl-L-proline nitroanilide there is esterification degradation activity and distinct milk-precipitation properties. The milk precipitation characteristic, which can be recognized by casein gel formation with extended stability formed by milk precipitation by a lab enzyme that acts very specifically, specifically cleaves coenzyme bonds without disrupting fragments in the present invention. It is evaluated by the limited but very beneficial activity of protease MO / 2.

In another embodiment of the invention, protease MO / 2 is selectively bound to a suitable insoluble immobilization support and used in this form for the degradation of the proenzyme. This method has the advantage that the dissolved protease migration to the fragment solution is inhibited.

According to the invention, the fragment is prepared from proteolytic partial disruption or partial digestion of the proenzyme by proteases of specific degradation properties, preferably at the C-terminus of the aromatic amino acid.

The high purity enzyme solution containing the fragments or fractions thereof can be concentrated by partial removal of water and used as an injectable preparation after the selective addition of stabilizers, inerts or pharmacologically active substances and sterile filtration. After sterile filtration and the addition of eg 5 mM magnesium chloride and 1% ovalbumin, high purity solutions of enzymes or fragments can also be lyophilized. The lyophilized enzyme or fragment may be dissolved in salt solution to form an injectable preparation of isotonicity.

The present invention relates to hyaluronate lyase of microbial origin for the treatment of vascular diseases such as arrhythmia heart disease, arteriosclerosis, cerebral infarction, cerebral thrombosis, coronary thrombosis and myocardial infarction, which may be due to the presence of atheromatous plaques in blood vessels, It relates to the use of hyaluronate-digested fragments or mixtures of both forms in humans and mammals.

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

The following examples provide a description of the present invention. However, it does not limit the invention in any way.

Example 1

Fermentation is carried out in a stirred fermenter with a total capacity of 30 liters. Working volume is 20L. For inoculation, a commonly available strain of Streptococcus agalactia sp. In the German group of microbial and cell culture GmbH (DSM) in Braunschweig under number 2134 ^r (= ATCC 13813 = NCT C8181) use. Stock preservation with Kryokononserve (Mast-diagnostica). A round object covered with stock material is placed in an inclined bacterial culture tube with a microbial count bacterial culturer and moved in contact with the culture medium surface (culture A). Then, for sterile check, the rolled round object is added to 3 mL of cardiac-brain bouillon (culture B). Both cultures were incubated as vertical cultures at 37 ° C. for 24 hours.

First pre-culture

Medium (2 × 50 mL) containing 5 g / L casein peptone and 10 g / L yeast extract (Difco) is charged into two 100 mL “Steilbrust” flasks and sterilized at pH 7.0. In addition to 50 mL of culture medium, 5 mL Eagle medium is added immediately in each case before inoculation. Inoculation is performed by rinsing the inclined bacterial incubator tube, Culture A with the culture solution of Culture B. The "stylebrew" flask is incubated by shaking at 150C for 24 hours (150 rpm).

Second pre-culture

20 g / L yeast extract and 10 g / L pancreatic peptone, 1.75 g / L sodium acetate, 7 g / L sodium hydrogen carbonate (dissolved each), 20% sulfuric acid prior to sterilization of the medium (2 × 1 L) containing 7 g / L of disodium hydrogen phosphate, 2H ₂ O and 3.5 g / L sodium dihydrogen phosphate To pH 6.2. After autoclaving, the pH is about 7.0. If 6.0 g / L of glucose solution is autoclaved individually, 100 mL of the solution is added to the second culture prior to inoculation performed by adding 50 mL of the first pre-culture (5% v / v inoculation). The medium is incubated at 32 ° C. for 24 hours with slow shaking at about 45 rpm.

State culture

The main culture medium consists of 20 g / L yeast extract, 10 g / L pancreatic peptone and 25 g / L glucose. The glucose is autoclaved individually. Inoculate 1 liter of the second pre-culture. The fermentation range is 34 ° C., pH 7.0, head space aeration (air 23 1 / min) and a stirring speed of 150 rpm. The pH is kept constant by titration with 40% sodium hydroxide solution. Add glucose by hand so that the glucose concentration does not drop below 5 g / L. The incubation is stopped after 20 hours.

Example 2

Fermentation culture solution (50 L) of Streptococcus agalactia spp. With hyaluronate lyase activity of 400 IU / mL is removed from live cells by sterile filtration on a 0.2 μm filter. The clean culture titration solution is then concentrated to 2 L with ultrafilteration at a cutoff of 60,000 D. Solid ammonium sulfate (480 g) is added to 2 L of culture concentrate. The protein precipitated at this concentration was removed by a clean filtration using a filter apparatus and the clean concentrate was placed on a 2.5 × 20 cm phenyl sepharose column previously equilibrated with 40% ammonium sulfate solution at pH 6.5 buffered with phosphate. Add. After washing with 35% ammonium sulfate solution at pH 6.5 buffered with phosphate, hyaluronate lyase is eluted with 25% ammonium sulfate solution at pH 6.5 buffered with phosphate. The eluted portions are combined and brought to 80% saturation by the addition of solid ammonium sulfate. The precipitate is collected and dissolved in 100 mL of 0.03 M tris buffer at pH 8.2 and dialyzed against the buffer. Dialysis crude crude hyaluronate lyase is then added to the equilibrated 2.2 × 15 cm Q-Sepharose column. The hyaluronate lyase is found in the eluate which is not absorbed and saturated at 80% with the addition of solid ammonium sulfate. Precipitated protein is collected, dissolved in 0.05 M Tris buffer, pH 8.7 and removed from ammonium sulfate on a column of sephadex G 10. The equilibrated hyaluronate lyase solution was transferred to a properly equilibrated affinity column (0.8 cm x 10 cm) of N- (p-aminophenyl) oxamic acid-agarose and 0.1 M sodium carbonate at pH 9.7 Elute with solution.

The eluted hyaluronate lyase-containing portion is combined and precipitated with 80% saturation with ammonium sulfate. The precipitate is collected, dissolved in 1 mL of 0.1 M Tris buffer at pH 7.5 and transferred to a Superdex 200 column (16 × 16). Hyaluronate lyase protein bands are collected at 116,000 D and dialyzed against 0.02 M Tris buffer, pH 7.5, containing 0.005 magnesium chloride. Hyaluronate lyase is obtained having an acid rate of 10 mg and specific activity of 400,000 IU / mg. Ovalbumin is added to the dialyzed hyaluronate lyase solution at 1% concentration and the solution is lyophilized.

Example 3

In accordance with Examples 1 and 2, microorganisms of Streptococcus equimomils species are pre-cultured and cultured to obtain enzymes in pure form. Purification using N- (p-aminophenyl) oxamic acid is omitted.

Example 4

Purified protease MO / 2 (10 mg) is dissolved in 2 mL of 0.1 M sodium carbonate solution, 1 mL of bromocyan Sepharose (Pharmacia) is added and shaken at 4 ° C. for 12 h. The uncoupled MO / 2 containing solution is then separated from the resulting M0 / 2 protease sepharose with a sintered glass filter. Thereafter, the binding site of the unsaturated bromocyan sepharose is blocked by reacting with 2 mL of 0.1M glycine solution. After the block, MO / 2 Sepharose is washed with 0.1M acetic acid and 0.1M sodium carbonate solution at pH 4.5.

Hyaluronate lyase (10 mg) from Streptococcus agalactia is dissolved in 1 mL of 0.05 M phosphate buffer at pH 7.0 and brought to 37 ° C. in a thermostat oven. An immobilized MO / 2 protease sepharose (0.25 g) combined with 0.1 Kunitz unit proteolytic activity is added to the solution. After 15 minutes, the protease sepharose is separated and the resulting fragment is 80% saturated with ammonium sulfate to precipitate from the solution. After 18 hours, the precipitate is collected and dissolved in 0.05 Tris buffer at pH 7.5. The fragment is purified by molar weight chromatography on Superdex 200. The active fragment portions are combined, dialyzed against 0.05M Tris buffer and lyophilized with albumin addition. The fragment has a specific activity of 600,000 IV / mg.

Example 5

Hyaluronate lyase (4 mg) from Streptococcus agalactia in 1 mL of Tris hydrochloride buffer at pH 7.8 is brought to 37 ° C. in a thermostat oven. To the solution, 80 μg of protease MO / 2 with 0.1 Kunitz unit specific activity per mg is added and the mixture is digested at 37 ° C. for 30 minutes. 10 μl of 0.1 M EDTA at pH 7.0 is added to stop the reaction. The mixture is then transferred to a 3 mL affinity column of N- (p-aminophenyl) oxamic acid-agarose and eluted with 0.05 M sodium carbonate at pH 9.7. The fragment was precipitated by 80% saturated with ammonium sulfate and collected and purified by molar weight chromatography. The active fragment portions were combined, dialyzed against 0.05 M Tris buffer and lyophilized by addition of albumin. The fragment has a specific activity of 460,000 IU / mg.

Example 6

Hyaluronate lyase fragment solution (0.5 mL) (Example 4) is added to 0.5 mL of 0.1 M acetate buffer at pH 6.0 and diluted in the form of a geometric series. Thereafter, 0.5 mL of a hyaluronic acid solution containing 0.2 mg of hyaluronic acid per 1 mL of 0.1 M acetate buffer at pH 6.0 is added to each dilution solution. The mixture is then incubated at 37 ° C. for 30 minutes. The enzymatic reaction was stopped when each dilute solution was treated with 2 mL of 2.5% cetyltrimethylammonium bromide in 2% sodium hydroxide solution. After 20 minutes at room temperature, the dilution of each dilution solution is measured at 600 nm in 1 cm cuvettes, and the amount of cleaved hyaluronic acid is determined by a calibration curve. Five international units (IU) of the hyaluronate lyase fragment cleave 16 μg of hyaluronic acid / min.

Example 7

Preenzyme (20,000 IU) and fragment (20,000 IU) (Example 4) are dissolved in 3 mL of distilled water. Both solutions are left at room temperature for 24 hours. Thereafter, enzyme activity is measured. The activity of the proenzyme solution drops to 60% during this period, while the activity of the fragment solution is 95% of the activity of the first solution.

Example 8

Determination of Activity of Hyaluronate Lyase or Fragment

1. Optical test at 232 nm

Stock solutions of hyaluronic acid (HA) and sulfated hyaluronic acid are prepared and maintained at 4 ° C.

Standard test formulations are performed as follows in 3 mL quartz kerbets (d = 1 cm) at a temperature of 26 ° C. and 232 nm wavelength. Add 200 μl of hyaluronic acid stock solution to 1.8 mL of 0.1 M acetate buffer at pH 6.0 and start the reaction with 2-4 U / mL (50 mL) of hyaluronate lyase. The final volume is 2050 μl.

For the reaction inhibition test, the standard test material was varied as follows. The reaction is started by addition of 1.5-1.8 mL of buffer, 300-500 μl sulfated hyaluronic acid and 200 μl hyaluronic acid and the addition of hyaluronate lyase. Hyaluronate lyase is partially inhibited uncompetitively. Ki value is 5.5 × 10 ⁻⁴ mg / mL.

Hyaluronate lyase shows an increase in extinction at 232 nm from the slope at which activity can be determined. The calculation is based on the extinction coefficient epsilon = 6.0 × 10 ⁻³ L / mole / cm for the Δ ^−1.5 unsaturated fluorides formed (L. Ludowig: mechanism of hyaluronidase, JBC 236, 333-339 (1991)).

2. Optical Testing at 600nm on Microplates

D. Determination of lyase activity by the method of Gerlach, W. Kohler, "Hyaluronate lyase of Streptococcus pyogenes. II. Properties of hyaluronate lyase" (EC4.2) .2.1.) Zbl. Bakt. Hyg, I. Abt. Orig. A 221, 296 -302 (1972)

Hyaluronate lyase solution is prepared at a 1:50 dilution of stock solution (50,000 U / mL). Hyaluronate lyase solution (0.5 mL) was added to 0.5 mL of 0.1 M acetate buffer at pH 6.0 and diluted in the form of equipotential sequence. Then, 0.5 mL of hyaluronic acid solution containing 0.2 mg / mL of 0.1 M acetate at pH 6.0 was added to each dilution solution.

The mixture was then incubated at 37 ° C. for 30 minutes. The enzymatic reaction was stopped when each dilute solution was treated with 2 mL of 2.5% cetyltrimethylammonium bromide solution in 2% sodium hydroxide solution. After 20 minutes at room temperature, the dilution of each dilution solution was measured at 600 nm in 1 cm cuvettes, and the amount of digested hyaluronic acid was determined by a calibration curve. Five international units (IU) of hyaluronate lyase digested 16 μg hyaluronic acid / min.

Example 9

Hyaluronate lyase (preenzyme, 10 mg) was dissolved in 1 mL of 0.05 M phosphate buffer at pH 7.0 and the temperature was 37 ° C. in a thermostat oven. To the solution was added 0.25 g of a specific degradation protease sepharose having a proteolytic activity of 0.1 Kunitz unit / mg. After 15 minutes, the insoluble protease sepharose was removed and the formed fragments were precipitated from solution by saturation with 80% ammonium sulfate. After 18 hours, the precipitate was removed and dissolved in 0.05 Tris buffer at pH 7.5. The fragment was purified by molar weight chromatography on Superdex 200. The active portion was purified, dialyzed against 0.05M Tris buffer and lyophilized by addition of albumin.

Example 10

For detection of plaque-lysing activity of hyaluronate lyase, about 0.3 mm pale yellow tissue pieces (plaque flakes) from neointima of human Arteria carotis interna were removed. It was suspended in hyaluronate lyase solution (about 20,000 IU at 1 mL of Tris buffer, 10 mM, pH 7.0) and incubated at room temperature for 12 hours. The previous clear phase boundary of the plaque flakes partially released, releasing suspended and finely diffused material. In the parallel test, plaque pieces were treated only with Tris buffer. Later, there was no sign of division.

Example 11

Attempts to cleave atheromatous vascular sedimentation of human origin with hyaluronate lyase fragments

About 0.3 mm pale yellow tissue pieces (plaque pieces) from neoinima of human Arteria carotis interna were collected in fragment solution (about 20,000 at 1 mL of Tris buffer, 10 mM, pH 7.0). IU) and incubated for 12 hours at room temperature. The previous clear phase boundary of the plaque flakes released extensively split, suspended and finely diffused material. In the parallel test, plaque pieces were treated only with Tris buffer. Later, there was no sign of division.

Example 12

Effect of hyaluronate lyase on atheromatous vasodilation on Watanabe rabbit Ibm: WHHL (Watanabe hereditary hyperlipidemia). Raised rabbits since 1981 by Y. Watanabe, Kobe University, Japan, and as breeding ancestors in Charles River Savo in 1992 and more since 1994 by the Broekman Institute in Someren, The Netherlands. Breeding.

Watanabe rabbits have hereditary adiposity hyperplasia due to genetic defects in the LDL receptors. As a result of hypercholesterolemia (> plasma 400 mg / 100 mL), all animals develop aortic immature atherosclerosis.

14 days before the experiment, 10 Watanabe rabbits (Ibm: WHHL; 4 females and 6 males), aged 9-11 months, were taken from the Broekman Institute in Someren, The Netherlands. The animals were placed in cages (0.5 × 1 m) at room temperature of 20 ± 2 ° C. respectively and provided standard food (ssniff Kanichen-Haltung) and beverage ad libitum. Prior to the start of the trial, ie before the first clinical chemistry investigation, ten rabbits were randomized and numbered from 1 to 10 according to gender.

Three injections a week (Monday, Wednesday and Friday) were given. Four control rabbits (1-4) each received 20 mL of pyrogen-removed 0.9% sodium chloride solution within 1 hour when administered intravenously. Six experimental animals (5-10) were each injected intravenously with an injection volume of 20 mL of pyrogen-removed 0.9% sodium chloride solution of 30,000 IU of high purity hyaluronate lyase per hour. This corresponds to a dose of 10,000 IU / kg body weight.

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

The general behavior of the animals was not significantly affected by treatment with 0.9% sodium chloride solution or hyaluronate lyase. The weight of mature Watanabe rabbits treated with control or hyaluronate lyase changed slightly from 11-18-96 to 2-3-97 during the treatment period. Body temperature measured before and after each injection ranged from -0.3 ° C to + 1.2 ° C. There was no significant difference between control rabbits and rabbits treated with hyaluronate lyase. Serum cholesterol levels in control animals were not significantly changed by 0.9% sodium chloride infusion. For one of the four animals, the cholesterol concentration in the serum was halved. Hyaluronate lyase treatment also did not cause any significant changes in serum cholesterol concentrations. Under infusion treatment, sometimes very high serum glyceride levels were reduced in all animals. After the 20th infusion, the values for the controls were between 570 and 740 mg / dL and the hyaluronate lyase treated rabbit was between 200 and 290 mg / dL.

In two of the four control rabbits, the aortal arches were severe atheromatous and in two of the four, moderate to severe atherosclerosis. In two of the six hyaluronate lyase-treated rabbits, the aortic arch was moderate atheromatic and in 4 of 6 rats between about to moderate atheromatic. In two of the four control rabbits, A. thoracia was severe atheromatous and two of four were moderate atheromatic. In four of the six hyaluronate lyase rabbits, A. thoracia was weak atheromatic and in two of the six coatings could hardly be detected. In two of the four control rabbits, the abdominal aorta was severe atheromatous and in two of the four, moderate atheroma. Very weak to light coatings were detected in two of the six hyaluronate lyase rabbits and four of the six. In 2 of 4 control rabbits, Aa. Pulmonalis was severely covered with plaque and between 2 and 4 between about moderate to moderate. In one of the six hyaluronate lyase rabbits, aorta pulmonalis was severely coated with plaque and from two out of six to about to moderately coated with plaque, whereas in three out of six plaques Did not exist.

The general behavior, weight development, body temperature before and after injection, and clinical-chemical variables of Watanabe rabbits treated with 0.9% sodium chloride solution and hyaluronate lyase were each injected 10 times and there were no significant differences during the 2 treatment cycles. It can be concluded that there is resistance.

Subjective evaluation of the visual aortic material shows that the coverage of atherosclerosis is more severe in four Watanabe rabbits treated with 0.9% sodium chloride solution than in six Watanabe rabbits treated with microbial hyaluronate lyase.

Example 13

The high purity concentrated enzyme solution is diluted with sufficient salt solution so that the resulting solution contains 0.9% salt and 10,000 IU / mL hyaluronate lyase. The solution was aseptically filtered with a sterile filler having a pore width of 0.2 μm. The ampoules were filled in 1 mL aliquots aseptically in the solution. The ampoules were lyophilized under sterile conditions and closed under sterile conditions. Prior to injection, 1 mL sterile distilled water was added.

Example 14

The high purity concentrated enzyme solution was diluted with magnesium nicotinate and magnesium adipate (magnesium compound "Schaffenberg"), resulting in 25 mg magnesium nicotinate and 88 mg and 10,000 IU / mL magnesium nicotinate. Hyaluronate lyase. The solution was filled in the ampoule of Example 13.

Example 15

The high purity hyaluronate lyase solution is diluted with salt and ovalbumin solution, so that the solution contains 40,000 IU / mL, 0.9% salt and 1% ovalbumin. The solution was subjected to the same process as in Example 13.

Example 16

Diluting the high purity hyaluronate lyase solution with the salt and soy protein hydrolyzate solution, the solution contains 40,000 IU / mL, 0.9% salt and 1% hydrolyzate prepared from the soy protein hydrolyzate. . The solution was subjected to the same process as in Example 13.

Example 17

The high purity fragment solution from the preenzyme of Streptococcus agalactia was diluted with salt and ovalbumin solution, so the solution contained 40,000 IU / mL, 0.9% salt and 1% ovalbumin. The solution was subjected to the same process as in Example 13.

Example 18

The high purity concentrated solution of the fragment from Streptococcus agalactia was diluted with sufficient saline solution and magnesium chloride so that the resulting solution contained 100,000 IU / mL of lyase activity. The solution was sterile filtered with a sterile filter having a pore width of 0.2 μm. In the solution, the ampoules were filled in 1 mL aliquots. The ampoules were lyophilized under sterile conditions and closed under sterile conditions. Prior to injection, 1 mL sterile distilled water was added.

Included in the above.

Claims (22)

Pharmaceutically containing a proenzyme, a hyaluronate lyase-digesting fragment thereof or a hyaluronate lyase of bacterial origin present as a mixture of both forms, and at least one stabilizer and / or a pharmaceutically stable carrier or inert material Formulation. The pharmaceutical preparation of claim 1, wherein the hyaluronate lyase is derived from a microorganism of the genus Streptococcus. The pharmaceutical formulation of claim 1, wherein the hyaluronate lyase is derived from the microorganism Streptococcus agalactia. The pharmaceutical formulation of claim 1, wherein the bacterial hyaluronate lyase in the preenzyme form has an isoelectric point in the 8.6 region and a molecular weight in the 116 kD region. The pharmaceutical formulation of claim 1, wherein the fragment of hyaluronate lyase has a molecular weight ranging from 84 kD to 86 kD. The pharmaceutical preparation of claim 1, wherein the pharmaceutical agent is an agent for treating a vascular disease selected from the group consisting of arrhythmia heart disease, arteriosclerosis, cerebral infarction, thrombosis, coronary thrombosis, and myocardial infarction. The hyaluronate lyase of hyaluronate lyase according to claim 1, wherein the pharmaceutical agent is partially hydrolyzed by a protease that degrades the C-terminal group of an aromatic amino acid, thereby obtaining from the bacterial hyaluronate lyase. Pharmaceutical formulations characterized in that they contain decomposed fragments. The pharmaceutical formulation of claim 1, wherein the formulation is an injectable formulation for intravenous or intraarterial injection. The pharmaceutical formulation of claim 1, wherein the activity of the hyaluronate lyase and / or fragment thereof of microbial origin is between 20,000 and 4,000,000 IU / mL. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a hyaluronate lyase and / or a fragment thereof and a purified aqueous injection solution containing about 1% (w / w) albumin and / or a hydrolyzate thereof. Preparations. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a purified aqueous injection solution containing hyaluronate lyase and / or fragments thereof and magnesium salts. The pharmaceutical formulation of claim 1, wherein the pharmaceutical formulation comprises a purified aqueous injection solution containing hyaluronate lyase and / or fragments thereof and glucose. Enzymatic fragment of bacterial hyaluronate lyase with hyaluronate lyase activity. The enzyme fragment according to claim 13, wherein the hyaluronate lyase is derived from a microorganism of the genus Streptococcus. The enzyme fragment of claim 13, wherein the hyaluronate lyase is from microorganism Streptococcus agalactia. The enzyme fragment of claim 13, wherein the enzyme fragment is prepared by partial enzymatic hydrolysis by a protease that cleaves C-terminal groups at aromatic amino acids from bacterial hyaluronate lyase. The enzyme fragment of claim 13, wherein the enzyme fragment is prepared by partially enzymatic hydrolysis of the bacterial hyaluronate lyase by a protease in a carrier-immobilized form. The enzyme fragment of claim 13, wherein the enzyme fragment is prepared by partial enzymatic hydrolysis of the bacterial hyaluronate lyase by metalloprotease MO / 2 in a carrier-immobilized form. The method of claim 13, wherein the enzyme fragment is prepared by partial enzymatic hydrolysis of hyaluronate lyase from streptococci by metalloprotease MO / 2 at an acidity in the range of pH 6.5 to 8.0 and a temperature of 25 to 45 ° C. Characterized by an enzyme fragment. The enzyme fragment of claim 13, wherein the enzyme fragment is prepared by partially enzymatic hydrolysis of hyaluronate lyase from Streptococcus agalactia having a molecular weight of about 115,000 to 120,000 D. 15. The enzyme fragment of claim 13, wherein the enzyme fragment has a molecular weight of about 84,000 to 86,000 D. 15. The enzyme fragment of claim 13, wherein the enzyme fragment has a specific activity of 400,000 to 800,000 IU / mg.