IE920531A1 - Combination of active substances comprising a sydnone imine¹and a hirudin - Google Patents

Abstract

The present invention relates to a combination of active ingredients composed of a sydnone imine and of a hirudin as antithrombotic agent.

Description

The present invention relates to a combination of active substances comprising a sydnone imine and a hirudin and to the use thereof.

Sydnone imines are compounds whose pharmacology has now been known for a long time (Chemie in unserer Zeit, volume 13, page 51 (1984)), and besides the vasodilating, in particular also the antithrombotic effects have been described (J. Cardiovasc.

Pharmacol. 1989, 14 (Suppl. 11), page 129). Sydnone imines can be used as agents to prevent and treat disorders of the cardiovascular system and are commercially available as such.

Hirudin is a polypeptide which can be isolated from the medicinal leech (Hirudo medicinalis) and is known as thrombin inhibitor (Die Pharmazie 36, 653 (1981)). It has recently become possible to prepare by genetic engineering processes adequate amounts of recombinant hirudin (r-hirudin) (Munch med. Wschr. 127. 16 (1985)). However, a large number of synthetic hirudins is also known.

It has now been found, surprisingly, that there is mutual enhancement of the antithrombotic effects of sydnone imines and hirudins.

The present invention accordingly relates to a combination of active substances comprising a sydnone imine and a hirudin.

The term sydnone imine means all pharmacologically active sydnone imines. In particular, these are the sydnone imines embraced in the following publications: German Offenlegungsschrift 1,620,501; German Offenlegungsschrift 1,670,127; German Offenlegungsschrift 1,695,897; EP-A-23,343; EP-A-59,356; EP-A-76,952; EP-A-210,474; EP-A-276,710; EP-A-312,773; EP-A-324,408; EP-A-346,684; EP-A-346,694; EP-A-367,036; EP-A-406,659 and EP-A-406,661.

Preferred sydnone imines are those of the general - lA(I) formula I o or a pharmacologically acceptable salt thereof, where R1 denotes an amino group of the formula R2 denotes hydrogen, alkyl, cyeloalkyl, arylalkyl, aryl-X-alkyl, alkoxyalkyl, alkylthioalkyl or alkenylthioalkyl; R3 denotes hydrogen or the group -COR7; R* denotes alkyl, cyeloalkyl, alkyl-X-alkyl, arylalkyl, hydroxy10 alkyl; R5 denotes hydrogen or has one of the meanings of RA; R6 denotes hydrogen or methyl; R7 denotes aryl, aryl radical mono-, di- or trisubstituted by 1 to 3 halogen atoms and/or 1 to 3 alkyl radicals and/or 1 to 3 alkoxy radicals and/or 1 or 2 nitro groups, or OR2, or has one of the meanings of R2; X denotes NR4, NSO2R8, NC02alkyl, S(O)n, 0, CH2 or a single bond; n is 0, 1 or 2, and R8 denotes alkyl, aryl, alkylaryl or dialkylamino.

Alkyl radicals and alkenyl radicals can be straight-chain or branched and preferably have 1 to 6 C atoms, particularly preferably 1-4 C atoms. This also applies when they are present in conjunction with other groups, for example as alkoxy, arylalkyl etc.

Examples of alkyl radicals are methyl, ethyl, propyl, isopropyl, butyl, sec.-butyl, and tert.-butyl.

Cyeloalkyl preferably has 5 to 7 C atoms and particularly preferably denotes cyclopentyl and cyclohexyl.

Aryl preferably has 6 to 10 C atoms and particularly preferably denotes a- or ^-naphthyl or phenyl.

Arylalkyl is preferably benzyl and phenylethyl.

Aryl-X-alkyl is preferably phenoxymethyl and phenoxyethyl.

The aryl radicals representing R7 can be mono-, di- or trisubstituted, but it is possible for a maximum of only 2 nitro groups to be present even in the case of trisubstitution, such as, for example, 2-methyl-4,6-dinitrophenyl and 2-chloro10 6-methyl-4-nitrophenyl. Examples of suitable halogen substituents for the aryl radicals are chlorine and bromine atoms. Aryl radicals representing R7 which should be particularly mentioned are: methylphenyl (= tolyl), nitrophenyl and chlorophenyl, especially 4-nitrophenyl and 4-chlorophenyl.

Preferred R1 radicals are morpholino, 3,3-dimethyl-thiomorpholino, cis-2,6-dimethylpiperidino and 1,l-dioxo-3,3-dimethyl thiomorpholino.

Rz preferably denotes hydrogen.

R3 preferably denotes hydrogen, ethoxycarbonyl, propionyl or p-anisoyl.

The sydnone imines according to the claims form acid addition salts with inorganic or organic acids. Examples of suitable acids are hydrogen chloride, hydrogen bromide, naphthalenedisulphonic acids, especially naphthalene-1,5-di25 sulphonic acid, phosphoric, nitric, sulphuric, oxalic, lactic, tartaric, acetic, salicylic, benzoic, formic, propionic, pivalic, diethylacetic, malonic, succinic, pimelic, fumaric, maleic, malic, sulphamic, phenylpropionic, gluconic, ascorbic, isonicotinic, methanesulphonic, p-toluenesulphonic, citric or adipic acid. Pharmacologically acceptable addition salts are preferred. The hydrochlorides are particular preferred.

Examples of sydnone imines according to the claims are: N-ethoxycarbonyl-3-morpholino-sydnone imine (molsidomine); 3-morpholino-sydnone imine hydrochloride (SIN-1); N-(4-methoxy35 benzoyl)-3-(cis-2,6-dimethylpiperidino)sydnone imine; N-propionyl-3-(3,3-dimethyl-thiomorpholino)sydnone imine; 3-(1,l-dioxo-3,3-dimethyl-thiomorpholino)sydnone imine hydrochloride; 3-(3,3-dimethyl-thiomorpholino)sydnone imine hydrochloride; 3-(cis-2,6-dimethylpiperidino)sydnone imine - 3 IE 920531 hydrochloride; 3-(tert.-butyl-2-hydroxyethyl-amino)sydnone imine hydrochloride; 3-( tert.-butyl-(2-diisopropylaminoethyl)amino)sydnone imine dihydrochloride; 3-(tert.-butyl(2-morpholinoethyl)amino)sydnone imine hydrochloride; 3-(3,3-dimethylmorpholino)sydnone imine hydrochloride; 3-(2,2-dimethylpiperidino)sydnone imine hydrochloride; 3-(4-isopropyl-2,2-dimethyl-piperazino)sydnone imine dihydrochloride; N-p-anisoyl-3-(4-isopropyl-2,2-dimethylpiperazino)sydnone imine dihydrochloride; 3-(2,2,6,6-tetramethyl 4-isopropyl-piperazino)sydnone imine dihydrochloride; 3- (2,6-dimethylpiperidino)-4-hexyl-sydnone imine hydrochloride; 4- benzy1-3-(2,6-dimethylpiperidino)sydnone imine hydrochloride; 3- (2,6-dimethylpiperidino)-4-(2-phenylethyl)sydnone imine hydrochloride; 3-(2,6-dimethylpiperidino)-4-phenylthiomethyl-sydnone imine hydrochloride; N-benzoyl-4-benzylthiomethyl-3-(2,6-dimethylpiperidino) -sydnone imine ; 3-(2,6-dimethylpiperidino)4- phenoxymethylsydnone imine hydrochloride; 3-(2-hydroxyethylamino)-sydnone imine hydrochloride; 3-ethylamino-sydnone imine hydrochloride; 3-cyclohexylamino-sydnone imine hydrochloride; 3-(2-hydroxycyclohexyl-amino)sydnone imine hydrochloride; 3- (3,3-dimethyl-1,4-tetrahydrothiazin-4-yl 1,1-dioxide)sydnone imine hydrochloride; 3-(3,3-dimethyl-perhydro-l-oxo-l,4-thiazin4- yl)sydnone imine hydrochloride; N-p-anisoyl-3-dicyclohexylamino-sydnone imine; N-pivaloyl-3-dicyclohexylamino-sydnone imine; 3-(2-carboxypiperidino)sydnone imine hydrochloride; 3- (2,2-dimethyl-4-methanesulphonyl-piperazino)sydnone imine hydrochloride; 3-(2,2-dimethyl-4-toluenesulphonylpiperazino)sydnone imine hydrochloride; 3-(2,2-dimethyl4- dimethylaminosulphonyl-piperazino)sydnone imine hydrochloride; 30 N-isobutyroyl-3-(2,2-dimethyl-4-methanesulphonylpiperaz ino) sydnone imine; 3-(N-tert.-butyl-N-methylamino)sydnone imine hydrochloride; 3-(N-tert.-butyl-N-butylamino)sydnone imine hydrochloride.

The term hirudins means all pharmacologically active 35 hirudins. These can be of natural or synthetic, but also of genetic engineering origin. Compounds with N- or C-terminal extensions beyond the active amino-acid sequence are embraced just as much as pharmacologically active part-sequences and fragments. Compared with the natural sequence, it is also possible for one or more amino acids to be modified or replaced by other amino acids, specifically those occurring naturally as well as those not occurring naturally.

Covered by the term hirudins are, in particular, the 5 compounds specified in the following publications: DE-A-3,445,532 ; EP-A-142,860; EP-A-158,564; EP-A-158,986; EP-A-168,342; EP-A-171,024; EP-A-173,619; EP-A-193,175; EP-A-200,655; EP-A-209,061; EP-A-225,633; EP-A-227,938; EP-A-236,330; EP-A-252,854; EP-A-273,800; EP-A-324,712; EP-A-345,616; EP-A-364,942.

Particularly preferred hirudins are recombinant desulphato-hirudins (r-hirudin) which are obtained from E. coli, yeast of B. subtilis and differ from natural hirudin in 2 amino acids. One example of an r-hirudin of this type is HBW 023 (r-desulphato-Ile^Th^-hirudin165; Drugs of the Future 15, 267-280 (1980)).

The ratio by weight between sydnone imine and hirudin in the combination of active substances according to the invention can vary within wide limits.

In particular, the sydnone imine : hirudin ratio by weight is 1:(0.025 to 40), preferably 1:(0.1 to 10).

The combination of active substances according to the invention can be prepared by mixing the individual active substances in the specified ratios by weight.

It is normally expedient to administer the combination of active substances in the form of a pharmaceutical preparation. These preparations can be administered orally, for example in the form of pills, tablets, lacquered tablets, sugar-coated tablets, hard and soft gelatin capsules, solutions, syrups, emulsions or suspensions or aerosol mixtures. However, administration can also take place rectally, for example in the form of suppositories, or parenterally, for example in the form of injection solutions, or percutaneously, for example in the form of ointments or tinctures.

Pharmaceutically inert inorganic or organic vehicles can be used to produce the pharmaceutical preparations. Examples which can be used for the production of pills, tablets, sugarcoated tablets and hard gelatin capsules are lactose, maize starch or derivatives thereof, talc, stearic acid or salts thereof, etc. Examples of vehicles for soft gelatin capsules and suppositories are fats, waxes, semi-solid and liquid polyols, natural or hardened oils, etc. Suitable examples of vehicles for the production of solutions and syrups are water, sucrose, invert sugar, glucose, polyols, etc. Suitable examples of vehicles for the production of injection solutions are water, alcohols, glycerol, polyols or vegetable oils.

The pharmaceutical preparations can contain, besides the active substances and vehicles, also additives such as, for example, fillers, extenders, disintegrants, binders, lubricants, wetting agents, stabilisers, emulsifiers, preservatives, sweeteners, colorants, flavourings or aromatising agents, buffer substances, also solvents or solubilisers or agents to achieve a depot effect, as well as salts for altering the osmotic pressure, coating agents, or antioxidants. They can also in each case contain two or more sydnone imines and hirudins as well as other therapeutically active substances.

Examples of other therapeutically active substances of this type are: ^-receptor blockers such as, for example, pro20 pranolol, pindolol, metoprolol; vasodilators such as, for example, carbochromen; sedatives such as, for example, barbituric acid derivatives, 1,4-benzodiazepines and meprobamate; diuretics such as, for example, chlorothiazide; cardiac tonics such as, for example, digitalis products; agents lowering blood pressure, such as, for example, hydralazine, dihydralazine, ramipril, prazosin, clonidine, rauwolfia alkaloids; agents which lower the level of fatty acids in the blood, such as, for example, bezafibrate, fenofibrate; agents for thrombosis prophylaxis, such as, for example, phenprocoumon.

In a particular embodiment of the present invention, the combination of active substances according to the invention is administered not in the form of a mixture, that is to say in a pharmaceutical preparation, but separately from one another, that is to say in two separate pharmaceutical preparations, simul35 taneously or in quick succession.

In this case, the pharmaceutical preparations contain either sydnone imine or hirudin, but otherwise correspond to the preparations described above.

In a further embodiment of the present invention, a preparation which contains sydnone imine and hirudin is administered and, simultaneously or shortly thereafter, a preparation which contains sydnone imine or hirudin. The preparations correspond to those described above in this case too.

The combinations of active substances according to the invention can be used in humans to control or prevent disorders of the cardiovascular system, in particular as antithrombotic medicines.

The dosage can vary within wide limits and should be 10 suited to the individual circumstances in each individual case.

In general, on oral administration, a daily dose of about 0.5 to 100 mg, preferably 1 to 20 mg, of combination of active substances is appropriate per human individual. Because of the good absorption of the active substances, the daily dose with other types of administration is also in similar ranges of amounts, that is to say generally likewise 0.5 to 100 mg/person. The daily dose is normally divided into several, for example 2 to 4, part administrations .

Surprisingly, there is mutual enhancement of the anti20 thrombotic effects of sydnone imine and hirudin in the combinations of active substances according to the invention. It is thus possible to achieve a very strong effect even at dosages at which neither sydnone imine nor hirudin alone has an effect.

This effect has been determined in the following test: A coronary thrombosis was produced by the method of Folts et al. (Circulation 1976, 54; 365-70) in male domestic pigs about 4 months old. The animals were deeply anaesthetised and the thorax was opened; the left descending coronary artery was exposed and provided with an electromagnetic blood flow sensing element. The coronary vessel was damaged distal of the sensing element by brief (1 s) pinching with a vessel clamp. A plastic constrictor (internal diameter 1.7-2.8 mm, length 2 mm) which stenosed the vessel was placed at this point. These experimental conditions resulted in the occurrence of regular cyclic changes in blood flow (CFVs), caused by the repeated formation and embolisation of thrombi at the damaged stenosed site. The thrombi were quantified by determining the frequency (number per 60 min) of the cyclic changes in blood flow. After cycles had occurred regularly for 60 min, the animals received one of the following Group 2 (η = 6) substances : Group 1 (n = 6) N-ethoxycarbonyl-3-morpholino-sydnone imine (molsidomine), 0.1 mg/kg intravenously as bolus injection; recombinant desulphato-hirudin (r-hirudin) in a total dose of 0.2 mg/kg divided into intravenous bolus injection of 0.1 mg/kg followed by a continuous intravenous infusion of 16.67 pg/kg min (= 1 mg/kg 60 min); molsidomine + r-hirudin, i.e. 0.1 mg.kg molsidomine i.v. together with 0.1 mg/kg r-hirudin i.v., followed by 0.1 mg/kg·60 min r-hirudin infusion.

In preliminary experiments, these single doses of molsidomine and r-hirudin were found to be just without antithrombotic effect.

The experiment was terminated 1 hour after the end of the infusion or 2 hours after bolus injection of the substances. The frequencies of the cyclic changes in blood flow in the hour before and in the first and second hour after injection of the test substances were averaged for each experimental group. The average frequencies of the cycles before and after administration of the substances were compared statistically (Student's t test for connected samples). A p value of < 0.05 was regarded as a significant difference. The results are to be found in the following table: Experimental group Number of thrombotic flow cycles (n/60 min) Group 3 (η = 6) 1st hour preliminary period 1st hour 2nd hour after injection of substances (molsidomine) (r-hirudin) + 2 + 3 ★ ± 1 + 1 + 2 ± 1 + 2* no effect; number of cycles could not be determined because continuous occlusion occurred in some cases p < 0.01 compared with preliminary phase. - 8 IE 920531 In addition, the systolic and diastolic peripheral arterial blood pressure (mm Hg) of all the animals was measured.

In group 2, r-hirudin infusion did not change the blood pressure.

Molsidomine (group 1) at the dose administered lowered the systolic blood pressure by a maximum of 18 + 5 mm Hg (p < 0.05) and the diastolic blood pressure not significantly by 10 ± 5 mm Hg. In combination with r-hirudin (group 3), molsidomine lowered the systolic blood pressure by 21 + 4 (p < 0.05) and the diastolic blood pressure not significantly by 11 ± 3 mm Hg. This lowering effect of molsidomine on blood pressure was the same in both experimental groups.

The results show that the combination of active substances according to the invention in these doses leads to an almost complete inhibition of experimental thrombi. The anti15 thrombotic effects of these two substances with a different mechanism of action accordingly potentiate each other without leading to an increase in the haemodynamic effect.

The advantage of the combination of active substances according to the invention is that low doses of both substances are possible. This reduces the potential for side effects and the combination can also be used in those patients whose cardiovascular status does not permit a lowering of blood pressure. Another advantage of the combination according to the invention is that the simultaneous influencing of two independent mechan25 isms of thrombosis means that a particularly effective treatment and prophylaxis of thrombo-embolic disorders is possible. On the one hand, the effect of thrombin is abolished by the hirudin.

This inhibits fibrin formation and platelet activation. Thrombin is regarded as the principal platelet activator in arteries too (Proc. Natl. Acad. Sci. USA 1988; 85: 3184-88). On the other hand, the sydnone imine activates one of the two endogenous counter-regulations of platelet stimulation by NO-mediated increase in cyclic guanosine monophosphate. This results in inhibition of platelet adhesion and aggregation (Circulation 1989; 79: 657-65). The two mechanisms together potentiate one another in the antithrombotic effect.

The following examples illustrate the combination of active substances according to the invention: - 9 IE 920531 Example 1 N-Ethoxycarbonyl-3-morpholino-sydnone imine (molsidomine) and r-hirudin in the ratio 1:2 by weight.

Example 2 3-Morpholino-sydnone imine hydrochloride (SIN-1) and r-hirudin in the ratio of 1:2 by weight.

Example 3 N-(4-Methoxybenzoyl)-3-(cis-2,6-dimethylpiperidino)sydnone imine and r-hirudin in the ratio of 5:2 by weight.

Example 4 N-Propionyl-3-(3,3-dimethylthiomorpholino)sydnone imine and r-hirudin in the ratio of 1:2 by weight.

Example 5 3-(1,1-Dioxo-dimethyl-thiomorpholino)sydnone imine hydrochloride and r-hirudin in the ratio of 1:1 by weight.

Example 6 3-(3,3-Dimethyl-thiomorpholino)sydnone imine hydrochloride and r-hirudin in the ratio of 1:3 by weight.

Example 7 3-(cis-2,6-Dimethylpiperidino)sydnone imine hydrochloride and r-hirudin in the ratio of 1:1 by weight.

Claims (12)

1. PATENT CLAIMS

1. Combination of active substances comprising a sydnone imine and a hirudin.

2. Combination of active substances according to Claim 1, characterised in that it contains a sydnone imine of the general formula I -N-|-R N \ / C=N-R 3 (I) or a pharmacologically acceptable salt thereof, where R 1 denotes an amino group of the formula \ r> ;/ N— X N- A .CH CH. X N—

3. c „ 3 CH_ or R 2 denotes hydrogen, alkyl, cycloalkyl, arylalkyl, aryl-X-alkyl, alkoxyalkyl, alkylthioalkyl or alkenylthioalkyl; R 3 denotes hydrogen or the group -COR 7 ; R* denotes alkyl, cycloalkyl, alkyl-X-alkyl, arylalkyl or hydroxyalkyl; R 5 denotes hydrogen or has one of the meanings of R 4 ; R 6 denotes hydrogen or methyl; R 7 denotes aryl, aryl radical mono-, di- or trisubstituted by 1 to 3 halogen atoms and/or 1 to 3 alkyl radicals and/or 1 to 3 alkoxy radicals and/or 1 or 2 nitro groups, or OR 2 , or has one of the meanings of R 2 ; X denotes NR 4 , NSO 2 R 8 , NC0 2 alkyl, S(O)„, 0, CH 2 or a single bond; n is 0, 1 or 2, and R 8 denotes alkyl, aryl, alkylaryl or dialkylamino. - 12 3. Combination of active substances according to Claim 2, characterised in that R 1 denotes morpholino, 3,3-dimethyl-thiomorpholino, cis-2,6-dimethylpiperidino or 1,1-dioxo3,3-dimethylthiomorpholino.

4. Combination of active substances according to Claim 2 and/or 3, characterised in that R 2 denotes hydrogen.

5. Combination of active substances according to one or more of Claims 2 to 4, characterised in that R 3 denotes hydrogen, ethoxycarbonyl, propionyl or p-anisoyl.

6. Combination of active substances according to one or more of Claims 1 to 5, characterised in that a recombinant desulphato-hirudin (r-hirudin) is used as hirudin.

7. Use of a combination of active substances according to one or more of Claims 1 to 6 as antithrombotic agent.

8. Use according to Claim 7, characterised in that sydnone imine and hirudin are administered simultaneously or in quick succession.

9. Pharmaceutical product characterised in that it contains a combination of active substances according to one or more of Claims 1 to 6 together with pharmaceutically acceptable vehicles and additives and, where appropriate, also one or more other pharmacological active substances.

10. A combination of active substances, substantially as hereinbefore described.

11. Use according to claim 7, substantially as hereinbefore described .

12. A pharmaceutical product according to claim 9, substantially as hereinbefore described.