Desulfatohirudine, process for its preparation and pharmaceutical agent

The invention relates to new, biologically active polypeptides derived from hirudin and to processes for their preparation, pharmaceutical compositions containing them and their use, in particular for inhibiting blood coagulation.

Hirudin, from which the compounds according to the invention are derived structurally, is a polypeptide natural product which is produced in the organism by medicinal leeches (Hirudo medicinalis) and which keeps the blood taken up by the leech in an unclotted state. Its isolation, cleaning, chemical composition and broad biological and medical use as an anticoagulant are known and in review articles, eg P. walsmann and F.Markwardt, Pharmazie 36, 653-660 (1981), summarized and discussed in detail. Recently, the complete amino acid sequence of hirudin was finally elucidated, thereby also creating the first theoretical basis for experiments on its synthetic production. The primary structure of hirudin corresponds to the formula:<img file="EP0142860A2_D0001.tif" />(Which specific cysteine ​​residues pair with each other through disulfide bridges remains to be determined; This structural detail is of little importance for the synthesis planning.) The structure is characterized by a characteristic accumulation of hydrophobic amino acids against the amino end and of polar amino acids against the carboxyl end of the peptide and, as a special feature, by the strongly acidic sulfate monoester group on the phenolic hydroxyl of the tyrosine residue in position 63 according to the partial formula<chemistry id="chem0001" num="0001"><img file="EP0142860A2_D0002.tif" /></chemistry>

So far there have been no clear ideas about the biological function of the sulfate residue in proteins in general and in this active ingredient in particular. The following hypotheses were discussed:<ul id="ul0001" list-style="none"><li>(1) importance for the biological activity of the protein;</li><li>(2) involvement in regulatory cell processes (analogous to that known for reversible phosphorylation);</li><li>(3) stimulation of secretion, ie sulfation serves as a marker for recognition as a secretory protein: all sulfated proteins discovered to date are secretory or transmembrane proteins. In any case, the sulfate residue is one of the most striking structural features of hirudin.</li></ul>

Biologically, Hirudin belongs to the K_i-Value of about 6,₁₀^-11M one of the most effective thrombin inhibitors; it acts specifically against thrombin and does not inhibit any other proteinases in the blood coagulation cascade. In contrast to heparin, hirudin exerts its inhibitory influence on thrombin directly and does not act like that through antithrombin III. The only pharmacologically detectable effect of purified hirudin is the inhibition of blood coagulation or the prophylaxis of thrombosis. When administered intravenously to dogs, there was no influence on heart rate, respiration, blood pressure, platelet count, fibrinogen and hemoglobin even at unusually high doses. In experiments on rats, pigs and dogs, hirudin was found to be effective in experimental thrombosis (caused either by stasis or by injection of thrombin), endotoxin shock and DIC (disseminated intravascular coagulation). In direct comparison tests with heparin, wherever they were carried out, hirudin was superior to it.

Although known for a long time, hirudin has not yet achieved the broad therapeutic application that one could rightly expect due to its excellent biological properties. Indeed, its extremely limited accessibility is a major disadvantage of its widespread use in medicine. Until now, hirudin preparations have only been available from expensive and difficult to access natural material - the medical leeches - through complex isolation and cleaning processes. The relatively long sequence of 65 amino acids offers little hope of practical success for a synthetic approach using classic peptide synthesis. The alternative, biosynthetic approach, which could arise through the synthesis of a suitable polynucleotide and its integration into the genetic code of a production microorganism in accordance with the general working methods of gene manipulation, did not seem to be promising either. It was to be expected that the necessary introduction of the 0-sulfonated tyrosine residue would cause almost insurmountable difficulties for direct biosynthesis.

Surprisingly, it has now been found that, contrary to the theoretical ideas above, the favorable biological properties of hirudin also then _er_halremain when the characteristic sulfate monoester group is derived from the phenolic hydroxyl of [Tyr⁶³] Remnants is removed. The resulting degradation products, the desulfatohirudins of the formula I and II, are on the Tyr except for the absence of the sulfate ester group⁶³ characterized by all other structural features of hirudin, with the desulfatohirudin of formula I, which is the actual desulfatohirudin in the narrower sense of the word, the unabridged amino acid sequence, while in the analog, effectively equivalent hexacontatripeptide, referred to as desulfatohirudin of formula II, the two additionally C-terminal amino acids Leu and Glu are missing. Surprisingly, however, these compounds are at least equal to hirudin in biological terms, in particular in their anticoagulant activity, in terms of quality and quantity.

This finding is of great importance with regard to the possibilities of a conventional biotechnological synthesis of this peptide: while the presence of the unusual hydroxysulfonyl group in hirudin practically excludes its direct biosynthesis, the lack of desulfatohirudin means that the structural conditions for successful biotechnological synthesis are considerably more favorable . If the biological effectiveness is the same, desulfatohirudin is clearly superior to hirudin from a technical and economic point of view due to the much better accessibility by biotechnological means.

According to the invention, the desulfatohirudins of the formula I and II are obtainable in a manner known per se. For example, they can be prepared by using the hexacontapentapeptide hirudin of the above formula (_A) which releases the phenolic hydroxyl group of the tyrosine residue in position 63 as the sulfuric acid monoester.

The release of this group according to the scheme<chemistry id="chem0002" num="0002"><img file="EP0142860A2_D0003.tif" /></chemistry>(in which Pept represents the remaining part of hirudin) can be carried out in a manner known per se, for example by hydrolysis, both chemical and biological methods being applicable for this purpose.

In the case of a chemical release, the work is preferably carried out under the general conditions of acid-catalyzed hydrolysis, such as under the action of a dilute, for example an approximately 2 to about 4N aqueous hydrochloric acid solution, advantageously in trifluoroacetic acid as the reaction medium, or with water-containing trifluoroacetic acid as the sole reaction and solvent. In order to keep the risk of hydrolytic cleavage of peptide bonds to a minimum, it is advisable to work under mild reaction conditions, e.g. at temperatures which do not exceed room temperature, and to monitor the progress of the hydrolysis analytically, e.g. by thin-layer chromatography.

However, the hydrolysis takes place primarily by biological means, in particular by the use of specific enzymes, aryl sulfatases, which cleave the phenolic sulfate ester groups to free phenolic groups under mild conditions. The biological release of the sulfated hydroxyl group can take place with the aid of a suitable enzyme preparation with enriched active ingredient or an isolated enzyme, or a suitable enzyme system can be used in situ, ie as is directly present in a living or killed biological material, for example a growing or resting microorganism, a cell culture, a cell homogenate or an autolysate. One of the greatest advantages of biological hydrolysis is its high selectivity, which only brings about the desired cleavage of the monosulfate ester bond without attacking the other functional groups, especially the peptide bonds, in the sensitive starting material. The compounds according to the invention are primarily prepared by mixing hirudin in an aqueous, preferably buffered solution or suspension with an individual arylsulfatase preparation, e.g. B. the aryl sulfatase from Helix pomatia, treated at a temperature customary for enzymatic processes, such as about 20 to 45 and preferably 25 to 30 ° C. In the case of a weakly acidic reaction, ie at a pH of about 4 to about 7, especially from about 5 to about 6, which uses a buffer such as an about 0.03 to about 0.3 molar solution of an organic carboxylic acid salt with an alkali metal or with a organic base, for example with sodium acetate or in particular pyridine acetate (with a pH of about 5.4). The ratio of the enzyme used to the substrate (hirudin) generally depends on the activity of the particular preparation and is usually from about 1: 2 to about 1: 100, in particular from about 1: 5 to about 1:20; it is advantageous to use enzyme preparations of the highest possible degree of purity and activity. Since the aryl sulfatases not only catalyze the cleavage but also the introduction of the sulfate group and bring about an equilibrium of the starting and end substances, it is advantageous to determine the optimal concentration, the ratio of the amount to the substrate and the time course of the desulfation for each enzyme preparation . As a rule, however, the reaction ends after a few minutes; the quality of the reaction product is not impaired even after prolonged (up to about 4 hours) contact with active enzyme (eg when the reaction mixture is standing).

The course of the enzymatic desulfation can be followed bioanalytically on the samples taken. B. so that the enzyme activity is destroyed by briefly (about 3 minutes) heating the sample to about 100 ° C. and the substrate is treated with a carboxypeptidase Y. (The carboxypeptidase Y degrades the peptide chain from the carboxy side by splitting off the amino acids one after the other by cleaving the respective amide bonds.) Usually the degradation of the peptide chain has progressed after about 15 minutes so that the sulfated and / or free 63- permanent amino acid (Tyr⁶³) is completely split off and is therefore accessible for determination in a conventional amino acid analyzer.

The desulfatohirudin of formula II is formed by splitting off the two C-terminal amino acid building blocks Leu and Gln during the hydrolysis of hirudin. The resulting mixture can be separated, for example, by preparative HPLC chromatography. The desulfatohirudin of formula II has the same biological properties as the desulfatohirudin of formula I.

The desulfatohirudins according to the invention can be present not only in the free form but also in the form of their salts. Since they contain free amino groups or amidino groups in several amino acid residues, the compounds according to the invention can be present in the form of acid addition salts. Suitable acid addition salts are, in particular, physiologically tolerable salts with customary, therapeutically applicable acids; inorganic acids include hydrohalic acids (such as hydrochloric acid), but also sulfuric acid and phosphoric or pyrophosphoric acid; as organic acids are primarily_Arensulfonic acids (such as benzene or p-toluenesulfonic acid or lower alkanesulfonic acids, such as methanesulfonic acid) and carboxylic acids such as acetic acid, lactic acid, palmitic and stearic acid, malic acid, tartaric acid, ascorbic acid and citric acid. However, since the desulfatohirudins also contain free carboxyl groups in several amino acid residues which give the whole peptide an acid character, they can also be present as a salt, for example Sodium, potassium, calcium or magnesium salt, or also as ammonium salt, derived from ammonia or a physiologically compatible, organic, nitrogenous base. However, since they also contain free carboxyl groups and free amino (and amino) groups, they can also be present as an inner salt.

Depending on the procedure, the compounds according to the invention are obtained in free form or in the form of acid addition salts, internal salts or salts with bases.

The free compound can be obtained in a manner known per se from the acid addition salts. The latter, in turn, can be obtained by reaction with acids, for example with those which form the salts mentioned above, and evaporation or lyophilization, therapeutically applicable acid addition salts. The inner salts can be obtained by adjusting the pH to a suitable neutral point.

The invention also relates to pharmaceutical compositions which contain at least one of the compounds according to the invention or their pharmaceutically usable salts, optionally together with a pharmaceutical carrier and / or auxiliaries.

These compositions can be used in particular in the indications given above, for example if they are administered parenterally (such as intravenously, intrautaneously, intramuscularly or subcutaneously), orally or topically. The_Do-sierung depends primarily on the specific form of administration and the purpose of the therapy .. or. Prophylaxis. The size of the individual doses and the administration schedule can best be determined on the basis of an individual assessment of the respective disease case; the methods required for determining relevant blood factors are familiar to the person skilled in the art. In the normal case, the therapeutically effective amount of the compounds according to the invention in the case of an injection is in the dose range from about 0.005 to about 0.1 mg / kg of body weight. The range from about 0.01 to about 0.05 mg / kg of body weight is preferred. Administration is carried out by intravenous, intramuscular or subcutaneous injection._jection. Accordingly, pharmaceutical preparations for parenteral administration in single dose form, depending on the type of application, contain about 0.4 to about 7.5 mg of the compound according to the invention per dose. In addition to the active ingredient, these pharmaceutical compositions usually also contain a buffer, for example a phosphate buffer which is intended to maintain the pH between about 3.5 and 7, and also sodium chloride, mannitol or sorbitol to adjust the isotonicity. They can be in freeze-dried or dissolved form, with solutions advantageously containing an antibacterial preservative, for example 0.2 to 0.3% 4-hydroxybenzoic acid methyl ester or ethyl ester.

A preparation for topical application can be in the form of an aqueous solution, lotion or jelly, oily solution or suspension, or fatty or, in particular, emulsion ointment. A preparation in the form of an aqueous solution is obtained, for example, by dissolving the active compounds according to the invention or a therapeutically applicable salt thereof in an aqueous buffer solution of pH 4 to 6.5 and, if desired, a further active compound, for example an anti-inflammatory agent and / or a polymeric adhesive, for example polyvinylpyrrolidone, and / or a preservative. The concentration of the active ingredient is about 0.08 to about 1.5 mg, preferably 0.25 to 1.0 mg, in about 10 ml of a solution or 10 g of a jelly.

An oily form of application for topical administration is obtained, for example, by suspending the active compounds according to the invention or a therapeutically applicable salt thereof in an oil, optionally with the addition of swelling agents, such as aluminum stearate, and / or surface-active agents (surfactants) whose HLB value ("hydrophilic -lipophilic balance ") is below 10, such as fatty acid monoesters of polyhydric alcohols, for example Glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A fatty ointment is obtained, for example, by suspending the active compounds according to the invention or their salts in a spreadable fat base, optionally with the addition of a surfactant with an HLB value below 10. An emulsion ointment is obtained by triturating an aqueous solution of the active compounds according to the invention or their salts in a soft, spreadable fat base with the addition of a surfactant whose HLB value is below 10. All of these topical application forms can also contain preservatives. The concentration of the active ingredient is about 0.08 to about 1.5 mg, preferably 0.25 to 1.0 mg, in about 10 g of the basic mass.

In addition to the pharmaceutical compositions described above and analogous to them, which are intended for direct medical use in the human or mammalian body, the present invention also relates to pharmaceutical compositions and preparations for medical use outside the human or mammalian living body. Such compositions and preparations are used primarily as an anticoagulant additive to blood which is subjected to circulation or treatment (for example dialysis in artificial kidneys), preservation or modification (for example haemose separation) outside the body. The composition of such preparations, such as stock solutions or preparations in single dose form, are similar to the injection preparations described above; However, the amount or concentration of active ingredient is expediently based on the volume of the blood to be treated. or, more precisely, based on its thrombin content. It should be noted that the active compounds according to the invention (in free form)<ul id="ul0002" list-style="none"><li>(a) completely deactivate about 5 times the amount by weight of thrombin;</li><li>(b) are physiologically harmless even in large quantities; and</li><li>(c) are excreted from the circulating blood very quickly, even in high concentrations, so that there is no risk of overdosing, even in the case of transfusions, for example. Depending on the specific purpose, the suitable dose is approximately 0.01 to approximately 1.0 mg of active substance / l blood, the upper limit being able to be exceeded without any danger.</li></ul>

The subject of the present invention also includes the bioanalytical use of the compounds according to the invention and their salts for the determination of thrombin, as well as the preparations used for this purpose and containing the active compounds according to the invention, for example solid mixtures and, above all, solutions, in particular aqueous solutions; in addition to an exact quantity or Concentration of the active compounds according to the invention (also in the form of a salt) expediently also contain inert auxiliaries, for example those mentioned above for injection preparations, which have, for example, a stabilizing and / or preserving task. These preparations are used in bioanalyses in an analogous, known manner to the hirudin preparations, for example for the determination of thrombin.

In the present description and in the claims, the short designations for amino acids and their residues are used in accordance with the generally accepted nomenclature rules and relate to a-amino acids and their residues of the naturally occurring L series.

The following examples serve to illustrate the invention.

example 1

Material: hirudin, activity 630 IU / mg

The biological activity is determined via the inhibitory action against thrombin, the enzymatic activity of which in turn against the chromogenic substrate Chromozym TH (a product of Boehringer, Mannheim, FRG, for thrombin or hirudin determination) according to known, supplied with the test preparation Regulation is determined.

Arylsulfatase (ARS) from Helix pomatia (a product from Boehringer, Mannheim, Germany), 5 U / mg.

The enzymatic activity is determined according to the known method of Leon et al., Biochem. J. 75, 612-617, using the chromogenic substrate p-nitrophenol sulfate (1.8 mmol / 1 in the batch).

Desulfation

(1) Preliminary test (to determine the optimal ARS concentration)

(a) The following stock solutions are prepared:

<ul id="ul0003" list-style="none"><li>(A) Hirudin solution of concentration 2 mg / ml obtained by dissolving hirudin in solution (C).</li><li>(B) Arylsulfatase solution of concentration 1.25 mg / ml: by mixing 25 parts of the commercially available suspension with 100 parts of the solution (C).</li><li>(C) Buffer solution: 0.1 M aqueous pyridine acetate solution, pH 5.4.</li></ul>

(b) Procedure:

A series of samples is obtained by mixing the following components: each sample contains 15_/ul of solution A (corresponding to 30_/μg of hirudin) and 10 μl of solution B (corresponding to 12.5_/µg arylsulfatase) or a solution in which the concentration of the enzyme is brought to 1/2, 1/4, 1/8, 1/16 or 1/32 of the original by diluting solution B with buffer C. The samples of 25 each_/ul are incubated for 60 min at 25 ° C, then heated for 3 min at 100 ° C for the purpose of denaturing the sulfatase, cooled rapidly and analyzed for free and sulfated tyrosine (according to the method described below).

(2) Preparative procedure

15 parts by volume of solution A and 10 parts by volume of a dilute solution B are mixed, the respective optimum, lowest possible concentration of which was determined in the preliminary test and is adjusted by diluting the stock solution B with the buffer solution C. The mixture is incubated at 25 ° C. for about 30 to 60 minutes, briefly (for example under conditions of "flash sterilization") heated to 100 ° C. and immediately cooled in order to denature the desulfurizing enzyme. The reaction mixture is, if necessary after concentration in vacuo at or below room temperature, on a column of Sephadex^(R) G50 or G75, CM-Sephadex (^R), Woiatit^(R)CP, Amberlite^(R)IRC or another equivalent cation exchanger separated. If necessary, this separation is repeated until desulfatohirudin of the desired purity (determined, for example, by the inhibition test with thrombin and / or the amino acid analysis, see below) results. The product in solid form is obtained by lyophilizing the corresponding solutions (eluates). According to the amino acid analysis (after C-terminal proteolysis), pure product should be free of

Be tyrosine-0-sulfate and have the full activity of hirudin in the inhibitory activity test against thrombin (eg using Chromozym TH - see above).

Analytical control of desulfation

is done by gradually proteolytically degrading hirudin (as the starting material), samples from the desulfation process and desulfatohirudin of the formula I (as the end product) from the carboxyl end with carboxypeptidase Y and quantitatively determining the amino acid residues split off with the aid of a conventional amino acid analyzer.

(a) The following stock solutions are prepared:

(Aa) Hirudin solution of concentration 0.806 mg / ml is obtained by dissolving 0.250 parts by weight of hirudin in 310 parts by volume of Ca buffer solution (see below).

(Ba) CPY solution with a concentration of 2 mg / ml is obtained by dissolving 2 parts by weight of carboxypeptidase Y (CPY) in 1000 parts by volume of Ca buffer solution.

(Ca) buffer solution: 0.1 M aqueous pyridine acetate solution, pH 5.4.

(b) Procedure:

275_/ul solution Aa, corresponding to 222_/ug hirudin is added with 8_/ul solution Ba, corresponding to 16_/ug CPY,

ie in a ratio to hirudin of 1: 4 (w / w) or 1: 125 (mol / mol), and incubated for 30 min at 25 ° C. A 30 µl sample is taken from the mixture, 5 µl of trifluoroacetic acid is added and the mixture is centrifuged to remove the CPY precipitate; the supernatant solution is dried and the amino acids remaining in the residue are taken up in a buffer solution intended for the amino acid analysis and quantified using a conventional amino acid analyzer. Desulfatohirudin of the formula I is also analyzed in the same way. (The result can also be expressed as a molar ratio of the amino acids, in particular the tyrosine 0-sulfate or free tyrosine, to hirudin) (for the control samples, both hirudin and CPY are each subjected to the same procedure alone.)

In the same way, samples of 25µl from the desulfation preliminary test after destruction of the ARS activity by brief heating are used with 2_/µg of CPY (in the form of the solution Ba) were added and incubated at 25 ° C. for 30 min. After adding 5µl trifluoroacetic acid, centrifuging and lyophilizing the supernatant, released amino acids are determined quantitatively in the analyzer. - (Control approaches are carried out individually with: (1) hirudin, (2) CPY, (3) ARS, (4) hirudin + CPY, (5) hirudin + ARS, (6) CPY + ARS.)

Example 2

material

^{Hirudin: 1.5 mg hiru}din (cleaned by HPLC).

Arylsulfatase (from Helix pomatia): Boehringer suspension: 5 mg / ml = 5 U / mg. The aryl sulfatase (ARS) is desalted on a PD 10 column before the experiment. 100_/ul suspension are added to 2.5 ml of buffer (0.1 M NH₄Ac, pH 5.5), added to the PD 10 column and eluted with 3 ml of buffer. The extinction of the eluted solution is E₂₈₀ = 0,139, 100_/ul of which corresponded to 16_/ug ARS.

method

Hirudin becomes 2 µg / µl buffer (0.1 M NH₄Ac, pH 5.5) dissolved. To 20_/ug hirudin (= 10_/ul) turn 100_/ul of the ARS solution added to buffer.

Weight ratio of enzyme (ARS): substrate (hirudin) = 1: 1.25.

A preparation is carried out on a preparative scale for 22 h at 25 ° C. The implementation is monitored by means of HPLC analysis, 2.7 per determination_/ug inhibitor can be used.

It follows that the hirudin is 90% desulfated to desulfatohirudin of the formula (I) after 6 hours. After 22 h, desulfatohirudin of formula (I) is present in a mixture with desulfatohirudin of formula (II) (I: II = 55:45). This mixture can be separated using the chromatography method given in Example 1.

The protein chemical characterization of the end products is carried out using the Danayl chloride method (N-terminal determination), by degradation with carboxypeptidase Y (C-terminal determination) and by 24- or. 48 hour amino acid analysis.