CA2288415A1 - Pharmaceutical compositions containing hydroximic acid derivatives - Google Patents

Abstract

The invention refers to pharmaceutical compositions suitable for the protection of the mitochondrial genome and/or mitochondrium from damages or for the treatment of diseases connected with such damages, said compositions comprising a hydroximic acid derivative of formula (I) or a pharmaceutically acceptable acid addition salt thereof.

Description

PHARMACEUTICAL COMPOSITIONS CONTAINING
HYDROXIMIC ACID DERIVATIVES
The invention refers to pharmaceutical compositions suitable for the protection of the mitochondrial genom and/or mitochondrium from damages or for the treatment of diseases connected with such damages, said compositions comprising a hydroximic acid derivative of the formula wherein R' represents a hydrogen or a C1_5 alkyl group, Rz stands for a hydrogen, as C,_5 alkyl group, a C3_g cycloalkyl group or a phenyl group optionally substituted by a hydroxy or a phenyl group, or Rl and RZ together with the nitrogen atom they are attached to form a to 8 membered ring optionally containing one or more further nitrogen, oxygen or sulfur atoms) and said ring can be condensed with another alicyclic or heterocyclic ring, preferably a benzene, naphthalene, quinoline, isoquinoline, pyridine or pyrazoline ring, furthermore, if desired and chemically possible, the nitrogen and/or sulfur heteroatom(s) are present in the form of an oxide or dioxide, ,~ '.r ~ ~~ ~~_ R3 means a hydrogen, a phenyl group, a naphthyl group or a pyridyl group wherein said groups can be substituted by one or more halo atoms) or C1~ alkoxy group(s), Y is a hydrogen, a hydroxy group, a C ~-24 alkoxy group optionally substituted by an amino group, a CZ_24 polyalkenyloxy group containing 1 to 6 double bond(s), a C,_~s alkanoyl group, a C3_9 alkenoyl group or a group of the formula R'-COO-, wherein R' represents a CZ_30 polyalkenyl group containing 1 to 6 double bond(s), X stands for a halo, an amino group, a C1_4 alkoxy group, or X forms with B an oxygen atom, or X and Y together with the carbon atoms they are attached to and the -NR-OCHZ group being between said carbon atoms form a ring of the formula /Z -CH
-C CHZ a ~N O/
wherein Z represents an oxygen or a nitrogen, R stands for a hydrogen or R forms with B a chemical bond, A is a C1_4 alkylene group or a chemical bond or a group of the formula Ra Rs _~C~m _ ~C~n_ b wherein Ra represents a hydrogen, a C1_s alkyl group, a C3_g cycloalkyl group or a phenyl group optionally substituted by a halo, a C1~ alkoxy group or a C1_s group, Rs stands for a hydrogen, a C1_a alkyl group or a phenyl group, m has a value of 0, 1 or 2, n has a value of 0, 1 or 2, with the proviso that Y is other than hydorxy when X is an amino group, or a pharmaceutically acceptable acid addition salt thereof as the active ingredient.
HLT-P No. 177 578 and its equivalent US-P No. 4,308,399 describe hydroximic acid derivatives suitable for the treatment of diabetic angiopathy.
HL1-P No. 207 988 and its equivalent E-P No. 417 210 also describe hydroximic acid halogenides within the formula I having a selective beta-blocking effect, thus, being suitable for the treatment of diabetic antiopathy.
HU-P Application No. 2385/92 published under No. T/66350 describes further hydroximic acid derivatives within the formula I.
These known compounds can be used in the treatment of vascular deformations, mainly in the therapy of diabetes mellitus.

It is well-lalown that the nuclear genom of a human cell encodes about 100 000 genes, but in the cytoplast there is also a small, independent mitochondrial genom /Wellace, D.C., Science, 256, 628-632 ( 1992)/.
The mitochondrial genom codes only for 13 genes /Clayton, D.A., Cell, 28, 693-705 (1982)/, but without them the cell is unable to consume the oxygen, therefore, as an effect of the damages in the mitochondrial genom, the cell becomes anaerobic. Unlike the nuclear genom, the mitochondria) genom does not have a DNA repair capacity and the mitochondria) DNA (mtDNA) is not surrounded by histons which makes the mitochondria) genes much more vulnerable than the nuclear encoded genes /Tzagoloff, A., Myer, A.M., Annu.
Rev. Biochem., 55, 249-285 (1986)/. More than 90 % of the oxygen consumption of a cell takes place in the mitochondria) inner membrane where besides normal oxidation also oxygen free radicals are formed /Stryer, L., Biochemistry, 4th edition, W.H. Freeman and Co., New York, 1995/. Such free radicals can easily modify the mitochondria) DNA in the immediate vicinity of their formation. The formation of the reactive oxygen free radicals significantly increases e.g. during the reoxigenation following an ischaemia which increased free radical concentration may cause considerable and irreversible damages to the mitochondria) DNA /Marklund, S.L., J. Mol. Cell.
Cardiol., 20, (Supplement II), 23-30 (1988)/. Even under normal circumstances, free radicals cause minor but accumulative damages to the mtDNA. Therefore it is understandable that the damages of mtDNA increase by age /Wellace, D.C., Annu. Rev. Biochem., 61, 1175-1212 ( 1992)/, although the level of such damages depends on the individual, and that such damages of mtDNA may well cause the development of cardiomyopathy and neurodegenerative diseases in elderly people /Cortopassi, G.A., Arnheim, N., Nucleic Acids Res., 18, 6027-6033 ( 1990)/.
Through damages of the energy metabolism of a cell, the damages of the mitochondria) genom can cause severe illnesses such as myopathy /Loft, R., Proc. Natl. Acad. Sci. USA, 91, 8731-8738 (1994)/, dilatative or hypertrofic cardiomyopathy /Ozawa, T. et al., Biochem. Biophys. Res. Common., 170, 830-836 ( 1990)/, fi~rthermore may have a role in the aggravation by age of a number of neurodegenerative diseases (such as Parkinson's disease, Huntington's disease, Alzheimer's disease) and of the severe symptoms of diabetes /Loft, R, cited publication/.
In a number of the above diseases (e.g. the myopathy), a treatment with antioxidants was applied (treatment with coenzyme Q
and vitamin C) /Shoffner, J.M., Wallace, D.C., Adv. Hum. Genet., 19, 267-330 (1990)/. These treatments bring results only occasionally.
Further test treatments were made to avoid damages of after-ischaemia reoxidation applying antioxidant and metabolic therapy, using lipoamid. Lipoamid corrects the damages to the heart caused by the ischaemia on one hand by its antioxidant effect, on the other hand by its positive influence on the mitochondria) metabolism /Siimegi, Balazs et al., Biochem. J., 297, 109-113 (1994)/. Without a profound knowledge of the damaging process, no breakthrough therapy has been developed yet.
Based on the above, there is a need for the development of a pharmaceutical product which can protect the mitochondria) genom from damages or also prevent such damages.

It was found that the compounds of the formula I are able to protect the mitochondria) genom from damages, thus, they are suitable for the protection of the mitochondria) genom and/or mitochondrium from damages or for the treatment of diseases connected with such damages. Examples of diseases of mitochondria) origin:
KSS (Kearns-Sayre's syndrome), MERRF (myoclonus epilepsy and ragged red fibers syndrome), LHON (Leber's hereditary optic neuropathy), MELAS (mitochondria) myopathy, encephalopathy, lactic acidosis and stroke-like episodes), Leigh disease, CPEO (chronic progressive external phthalmoplegia), Alper's syndrome.
Examples of age-dependent degenerative diseases where the mitochondria) genom has been damaged:
Neurodegenerative diseases:
Alzheimer's disease, Parkinson's disease, ALS (amyotrophic lateral sclerosis), HD (Huntington's disease), Cardiomyopathies and other myopathies.
Thus, the invention refers to pharmaceutical compositions comprising 0.1 to 95 % by mass of a hydroximic acid derivative of the formula I or a pharmaceutically acceptable acid addition salt thereof as the active ingredient in admixture with one or more conventional carrier(s).

In the specification and Claims, a C1_5 alkyl group is, for example, a methyl, ethyl, n-propyl, isopropyl, n-butyl or n-pentyl group, peferably a methyl or an ethyl group.
A C3_8 cycloalkyl group is, for example, a cyclopropyl, cyclopentyl, cyclohexyl, cycloheptyl or cyclooctyl group, preferably a cyclopentyl or a cyclohexyl group.
A 5 to 8 membered ring containing one or more heteroatom(s) can be, for example a pyrrole, pyrazole, imidazole, oxazole, thiazole, pyridine, pyridazine, pyrimidine, piperazine, morpholine, indole, quinoline etc. ring.
A C1_za alkoxy group is, for example, a methoxy, ethoxy, n-propoxy, tert.-butoxy, n-pentoxy, decyloxy, dodecyloxy, octadecyloxy etc. group.
A C1_ZS alkanoyl group is, for example, a formyl, acetyl, propionyl, butiryl, caproyl, palmityl, stearyl etc. group.
A C3_9 alkenoyl group is, for example, an acryloyl, pentenoyl, hexenoyl, heptenoyl, octenoyl etc. group.
A C 1_4 alkylene group is, for example, a methylene, ethylene, propylene or butylene group.
A halo atom is, for example, a ffuoro, chloro, bromo or iodo atom, preferably a chloro or a bromo atom.
If Y stands for a group of the formula R'-COO-, it can represent, for example, a linolenoyl, linoloyl, docosahexanoyl, eicosapentanoyl, arachidonoyl etc. group.
The pharmaceutically acceptable acid addition salts of the compounds of the formula I are the acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, g sulfuric acid etc. or with pharmaceutically acceptable organic acids such as acetic acid, fumaric acid, lactic acid etc.
A preferred subgroup of the compounds of the formula I
consists of the hydroximic acid derivatives of the formula Ra Rs R3_(C~m_(C~~_C_X
R' N-O-CHZ-CH-CHZ-N II
I
Y ~ Rz wherein R', R2, R3, R4, R5, m and n are as stated in relation to formula I, X represents a halo atom, Y means a hydroxy group.
Especially preferred compounds of the formula II are those wherein Rl and RZ together with the nitrogen atom they are attached to form a piperidino group, R3 stands for a pyridyl group, m and n have a valaue of 0, X is as defined above. Of these compounds, preferred species are as follows:
0-(3-piperidino-2-hydroxy-1-propyl~yrid-3-ylhydroximic acid chloride (Compound "A").
A further preferred subgroup of the hydroximic acid derivatives of the formula I consists of the compounds of the formula O OH / R' R3-A-C-NH-O-CHZ-CH-CHz-N III
~RZ
wherein R', RZ, R3 and A are as stated in relation to formula I.

Another preferred subgroup of the hydroximic acid derivatives of the formula I consists of the compounds of the formula R' /CHZ-N
~Z CH\ \R' R3-A-C\ ~Hz N
/O
wherein Rl, R2, R3 and A are as stated in relation to formula I, Z
represents an oxygen or a nitrogen atom.
A still further preferred subgroup of the hydroximic acid derivatives of the formula I consists of the compounds of the formula OR6 OH /R' R3-A-C=N-O-CHZ-CH-CHz-N V
Ra wherein Rl, RZ, R3 and A are as stated in relation to formula I, R6 stands for a C,_4 alkyl group.
The compounds of the formula I can be prepared by the processes known from HU-P No. 207 988 as well as from HU-P
Application published under No. T/66350.
The pharmaceutical composition of the invention comprises 0.1 to 95 % by mass, preferably 1 to 50 % by mass, especially 5 to 30 by mass, of a hydroximic acid derivative of the formula I or a pharmaceutically acceptable acid addition salt thereof as the active ingredient and one or more conventional carner(s).

The pharmaceutical compositions of the invention are suitable for peroral, parenteral or rectal administration or for local treatment, and can be solid or liquid.
The solid pharmaceutical compositions suitable for peroral administration may be powders, capsules, tablets, film-coated tablets, microcapsules etc., and can comprise binding agents such as gelatine, sorbitol, poly(vinylpyrrolidone) etc.; filling agents such as lactose, glucose, starch, calcium phosphate etc.; auxiliary substances for tabletting such as magnesium stearate, talc, poly(ethyleneglycol), silica etc.; wetting agents such as sodium laurylsulfate etc. as the earner.
The liquid pharmaceutical comositions suitable for peroral administration may be solutions, suspensions or emulsions and can comprise e.g. suspending agents such as gelatine, carboxymethylcellulose etc.; emulsifiers such as sorbitane monooleate ete.; solvents such as water, oils, propyleneglycol, ethanol etc.;
preservatives such as methyl p-hydroxybenzoate etc. as the earner.
Pharmaceutical compositions suitable for parenteral administration consist of sterile solutions of the active ingredient, in general.
Dosage forms listed above as well as other dosage forms are known per se, see e.g. Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Co., Easton, USA (1990).
The pharmaceutical compositions of the invention contain, generally, unit dosage. A typical daily dose for adult patients amounts to 0.1 to 1000 mg of the compound of the formula I or a pharmaceutically acceptable acid additon salt thereof. The above dose can be administered in one portion or in more portions. The actual dose depends on many factors and is determined by the doctor.

The pharmaceutical compositions of the invention are prepared by admixing a compound of the formula I or a pharmaceutically acceptable acid addition salt thereof to one or more carrier(s), and converting the mixture obtained to a pharmaceutical composition in a manner known per se. Useful methods are blown from the literature, e.g. Remington's Pharmaceutical Sciences.
Another embodiment of the invention consists of a use of a compound of the formula I, wherein R, Rl, RZ, R3, X, Y, A and B are as stated in relation to formula I, or a pharmaceutically acceptable acid addition salt thereof, optionally in admixture with one or more carner(s) commonly employed in pharmaceutical compositions for the preparation of a pharmaceutical composition useful in the protection of the mitochondria) genom and/or mitochondrium from damages.
A still another embodiment of the invention consists of a use of a compound of the formula I, wherein R, Rl, RZ, R3, X, Y, A and B are as stated in relation to formula I, or a pharmaceutically acceptable acid addition salt thereof, optionally in admixture with one or more carriers) commonly employed in pharmaceutical compositions for the preparation of a pharmaceutical composition useful in the treatment of diseases connected with the damage of the mitochondria) genom and/or mitochondrium. Such diseases include especially myopathy, cardiomyopathy as well as neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease or Huntington's disease.
According to a preferred use of the invention, a hydroximic acid derivative of the formula II, wherein R', R2, R3, R4, R5, X, Y, m and n are as stated in relation to formula II, or a pharmaceutically acceptable acid addition salt thereof is employed.
In accordance with a still preferred use of the invention, a compound of the formula II, wherein Rl and RZ together with the nitrogen atom they are attached to form a piperidino group, m and n have the value of 0, X and Y are as stated in relation to formula II, or a pharmaceutically acceptable acid addition salt thereof is employed.
According to an especially preferred use of the invention, 0-(3-piperidino-2-hydroxy-1-propyl)pyrid-3-yl-hydroximic acid chloride or a pharmaceutically acceptable acid addition salt thereof is employed.
In vitro tests The mitochondria) genom protection effect of the hydroximic acid derivatives of the formula I was tested by their ability to protect the oxidative phosphorylation, in vitro. The theoretical background of the tests is that the energy needed for the cells is produced by the adenozin-triphosphate (ATP) which is synthesized in the mitochondrium. The abnormalities of the substrate transport, the citrate pathwax, the defect of the respiratory complexes and a disconnect in the oxidative phosphorylation entails a disturbed energy supply of the cell. In the test the oxidative phosphorylation was damaged by applying heat-shock on Sacharomyces cerevisiae yeast cells and K562 human eritroleukemic cells and the protective effect of the test compound was determined.
It is known that one of the damaging effects of the heat-shock that is developped immediately, i.e. within a few minutes, affects the mitochondrium by disconnecting the respiratory chain from the oxidative phosphorylation. Tests using chemical uncouplers showed that protons pumped into the space between the inner and outer mitochondrium membranes by the enzyme complexes of the respiratory chain during electron transport get back to the inner space due to the effect of the uncouplers, thus, no ATP is synthesized. Due to heat-shock, there is a similar process going on which results in a rapidly decreasing energy supply to the cells.
Materials used in testing:
Sacharomyces cerevisiae cell culture. The S288C haploid wild-type cell line was cultured on a YPG medium that contained 1 % of yeast extract, 2 % of peptone and 3 % of glycerol. The culture was shaken on a liquid medium in a water bath at 25 °C under aerobic conditions.
The K562 culture.
The K562 eritroleukemic type cell line of human chronic myeloid leukemic origin was cultured on an RPMI 1640 liquid medium in the presence of 10 % of calf serum, at a temperature of 37 °C, in a wet gas mixture containing 95 % of air and 5 % of carbon dioxide.
Oligomycin.
Carbonylcyanide m-chlorophenylhydrazone (CI-CCP) manufacturer:
Sigma Chemicals Co., St. Louis, USA).
Oxygen consumption was measured in the following way:
The cells were centrifuged during their logarithmic growth phase and, in case of the Sacharomyces cerevisiae, were taken in a tenfold amount of YPG medium containing 1 % of mannose instead of the 2 of glycerol. In case of the K 562, after the separation, the cells were taken in a 4x 106 cell/ml concentration in an RPMI 1640 medium containing 20 mM of HEPES. The oxygen consumption was measured in a 2 ml thermostated cuvet, with Clare's electrode. Details of the method are described in the following article: Patriarca, E.J.
and Maresca, B. Experimental Cell Research, 190, 57-64 (1990).
Stimulation of the respiratory rate is given in % using the formula:
r(VCl-CCP~OIig)-11 x 100 One hour before the hLeat shock, after Jthe separation, 10-5, 2.5x10-5, 5x10-5 M of the test compound and solvent (PBS i.e. physiological sodium chloride solution containing phosphate buffer), respectively, were added to the medium. The heat-shock was carned out by keeping the culture at 42 °C for 5 minutes instead of the original temperature of 25 °C. In case of the K562 cells the culture was kept at 48 °C for 10 minutes instead of the original 37 °C.
It has been noted during the experiments that the heat-shock significantly uncoupled the electron transport chain from the ATP
synthesis in both the Sacharomyces cerevisiae and K 562 cells.
Data obtained demonstrate that the application of the test compound undoubtedly provided an increased protection to the cells by preventing the uncoupling of the mitochondrial respiratory complexes.
In additon to retaining the proper cell functions, most probably the test compound indirectly prevents the formation of oxygen free radicals. From this we can conclude that the use of the test compound provides protection against damages of the mitochondria) genom.
Protection of the mitochondria from heat induced uncoupling Under normal circumstances the respiratory complexes pump out proton during the oxidation of NADH creating a proton gradient in the two sides of the inner mitochondria) membrane. This proton gradient provides energy to ATP synthesis from ADP and inorganic phosphate. The protons can only reenter the inner membrance space through F1F°ATPase utilizing the energy of proton gradient for ATP
synthesis from ADP and inorganic phosphate (PI). In the absence of ADP or Pi, the proton gradient increases and inhibits the respiratory complexes and the mitochondria) oxygen consumption. However, if there is any damage in the inner membrane, the protons can reenter the inner membrane space through the damaged region, and the energy of proton gradient is not utilized by F1F°.ATPase, and so the mitochondria) oxidation becomes ADP independent (mitochondria becomes uncoupled).
It is well known that heat-stress can induce an uncoupling of mitochondria) oxidation from mitochondria) energy (ATP) production which is the consequence of heat-stress induced mitochondria) inner membrane damage. In the damaged membrane regions, protons leak back from the intermembrane space to innermembrane compartment, thus, the mitochondria) oxidation becomes ADP independent.
For the test, mitochondria were isolated from control rats or from rats treated with 40 mg/kg of the test compound 6 hours before preparation, the preparation taking place as described by Siimegi et al., J. Biol. Chem., 259, 8748 (1984). Oxygen consumption was determined with Clark electrode in a chamber at 37 °C. The rate of oxygen consumption in the presence of SmM of ADP as well as in the absence of ADP was determined both for untreated mitochondria and mitochondria preincubated for 8 minutes at 42 °C.
Under normal conditions, the mitochondria) oxidation is approximately 6 times faster in the presence of ADP than in ADP free medium showing a good coupling between mitochondria) oxidation and ATP synthesis. However, heat-stress significantly decreases the coupling of mitochondria, and in the control cases this value decreased but in the mitochondria from animals pretreated with the test compound still preserve a relatively high coupling ratio. These data show that the compounds of the formula I protected the mitochondria) energy production (ATP synthesis) from heat-stress caused damage.
Protection of cholinergic neurons from hydrogen peroxide induced cell degeneration It is well known that hydrogen peroxide causes oxidative cell damage through generating oxygen related free radicals in cells.
Therefore, hydrogen peroxide induced cell damage can be used as a general model for neuron degeneration. SN6.10.2.2 hybrid , N18TG2 + ED15 septa) neurons cell-line /Hammond et al., Science, 234, 1237 ( 1986)/ were used to study the protecting effect of the compounds of the formula I against oxygen related free radical caused cell damage which is the main pathway in most neurodegenerative diseases.
For the test, the cells were divided into two groups on 96-wells plate. One of the groups was maintained in the medium containing the test compound (40 mg/1), another one was maintained in the base medium. The treatment was started 24 hours after dividing. Both of the groups were treated with their medium containing different concentrations of hydrogen peroxide. The survival test was performed after 48 hours' treatment periods.
Survival test:
The medium was removed from the well, the cells were rinsed with sterile PBS and then 150 ~.g of alkaline phosphatase substrate dissolved in 150 ~g of fresh diethanolamine buffer (pH 9.8) was added to each well. Plates were incubated at 30 °C and the reaction was stopped by adding 50 p.l of sodium hydroxide to each well. The absorbance was measured at 405 nm by Dynatech ELISA reader and peripheral wells of each plate containing only medium were utilized for blank background determination.
The results show that the use of compound of the formula I
tested effectively protects cholinergic neurons from hydrogen peroxide induced cell lysis. Since hydrogen peroxide kills cell by generating a large quantity of oxygen related free radical, the test compound can protect neurons in any diseases where neuronal damage is associated with oxygen related free radicals. Therefore, compounds of the formula I can be used advantageous in Parkinson's disease, Amyotrophic Lateral Sclerosis (ALS), Huntington's disease and several dementia of mixed origin /Life Sciences, 56, 1151-1171 ( 1995)/.
In vivo tests The mitochondria) genom protective effect of the hydroximic acid derivatives of the formula I was also tested in vivo, treating rats.
Vistar rats were treated daily with AZT (3'-azidothymidine, manufacturer: Sigma Chemicals Inc.) in a dose of 50 mg/kg for 14 days. Certain test goups were treated with AZT in combination with the compound "A" (daily dose of 20 mg/kg). During and after the treatment various measurements were made.
1) Schiller AT-6 ECG was used to monitor cardiac function of the animals, on all four limbs. The ECG parameters were evaluated using a standard method described in the technical literature /Kawai, C., Takatsu, T., New Engl. J. Med., 293, 592 ( 1975); Angelakos, E.T., Bernardini, P.J. Appl. Physiol., 18, 261-263 (1963)/. We have determined the RR, PR and TQ intervals and the J point depressions.
The results demonstrate that as an effect of the AZT treatment, compared to the control group, the animal heart frequency was significantly prolonged (RR) and also the PQ intervals were increased.
Furthermore, the QT value increased significantly and in leads I and VL which represent they main muscle mass of the left ventricle, significant J point depressions (over 0.1 mV) were found. These parameters characterize a developed myocardial ischaemia or a defective oxygen consumption. However, in cases when, in addition to AZT, also the test compound was administered to the rats in a daily dose of 40 mg/kg, the heart parameters returned to the normal range, that is the compound protected the heart from the abnormalities induced by AZT.
2) The respiratory activity of the animals was determined. In doing so the activities of the NADH: cytochrome C oxidoreductase, cytochrome oxidase and citrate synthase were determined with methods described in the technical literature /Siimegi, Balazs et al.;
Clip. Chim. Acta., 192, 9-18, ( 1990)/. (NADH: nicotinic acid adenine dinucleotid, reduced form). The results obtained are shown in Table 1.

Table 1 Effect of compound "A"on the AZT induced decrease in the respiratory activity Treatment Cytochrome NADH: cytochrome C Citrate oxidase oxidoreductase synthase unit/gram wet tissue Control group 14.7+1.6 11.6+0.7 292+28 AZT 8. 7+2 9. 5+0. 2 242+ 19 AZT+compound "A" 10.3+1.2 11.3+0. 5 262+47 It is well demonstrated in Table 1 that the AZT treatment significantly decreases the activity of the respiratory complexes in the mitochondria of the heart. In this was, AZT remarkably reduces the oxidative energy production in the heart which can lead to a state in which the heart is unable to properly perform its basic fimction.
Besides this, a decreased capacity of the mitochondrial respiration can lead to an abnormal mitochondrial metabolism which may cause further heart damages.
When AZT was administered to animals in combination with compound "A", its respiratory activity decreasing effect almost disappeared and the respiratory activity values stayed close to normal.
That is the tested compounds of the formula I significantly decreased the AZT induced mitochondria) membrane damages by protecting the respiratory complexes.
3) The damages to the mitochondria) genom were examined.
Damages to the mitochondria) genom were determined applying the PCR method. (PCR: polimerase chain reaction). The primers were selected by amplifying the range from the cytochrome oxidase component I to the cytochrome B in order to look for deletions. (The primers were purchased from the Ransonhill BioScience Co.). The method applied is described in the publication of Siimegi, Balazs et al.
B.B.A. ( 1996)/ which publication is being edited. Using PCR primers in amplifying the region 4929 to 16298 of the mitochondria) genom showed that 0.5 and 1.5 kb ranges significantly amplified in AZT
treated rats. At the same time, no amplificiation of such short ranges is seen on the animals of the control group. It is understandable that an undamaged genom does not amplify short DNA ranges as in these tests the primers are more than 11.3 kb from each other. The fact that such short DNA ranges are amplified in AZT treated rats shows that, due to the AZT treatment, 10 kb ranges are deleted from the mitochondria) genom and it is in such damaged genoms that the primers become as close to each other as 0.5 - 1.5 kb. As a conseqeuence, the DNA range can be amplified. The amplification of such DNA ranges shows the damage to the mitochondria) genom, that is the partial or complete deletion of the genes coding for the subgroups I, II and III of the cytochrome oxidase, of the genes encoding for ATP 6 and 8, the genes coding for Complex I or NADH: ubiquinone oxidoreductase 2, 4, 4L, 5 and 6, and coding for cytochrome B.

When AZT was administered to animals in combination with the test compound, the amplification of the above short DNA ranges have significantly decreased and certain DNA fragments could not be detected.
This means that the test compound protected the above genes from AZT ~ induced damages or at least significantly decreased those damages. It is to be noted that the AZT induced artificial damages to the above genes can also occur as an effect ischaemic cardiomyopathy or cardiomyopathy of aged people.
The effect of the use of compounds of the formula I on inherited mitochondria) cardiomyopathies.
Test were carned out using inherited mitochondria) cardiomyopathic rats that were treated with a daily dose of 40 mg/kg of the test compound for 14 days. The rat heart function was monitored by ECG.
Tests were performed on rats with inherited cardiomyopathy which have abnormal heart functions. These cardiomyopathic rats serve as a perfect model of ischaemic cardiomyopathy and cardiomyopathy of aged people. As an effect of the 14 days' treatment with the test compound, the animals' heart functions improved significantly and the ECG parameters moved back to the normal range.
The above tests show that the hydroximic derivatives of the formula I are able to protect the mitochondria) genom against various damages. In the case of the animal models used, they have virtually eliminated the AZT induced heart damages and this can bear a great significance in the human medical science considering that, at present, more than a hundred thousand people are treated with AZT
worldwide.
Furtlier important feature of the use of compounds is that in case of a developed cardiomyopathy (where the mitochondrial damages are similar to those of the ischaemic cardiomyopathy and the cardiomyopathy of aged people), they eliminate heart function abnormalities and restore the normal ECG parameters.
Based on the above tests it can be said that the pharmaceutical compositions of the invention containing as active ingredient a compound of the formula I can protect the mitochondrial genom or the mitochondrium from damages, furthermore can treat diseases with already developed damages of that kind.

Claims (23)

1. A pharmaceutical composition for the preparation of the mitochondrial genom and/or mitochondrium from damages or for the treatment of diseases connected with such damages, comprising 0.1 to 95 % by mass of a hydroximic acid derivative of the formula I, wherein R1 represents a hydrogen or a C1-5 alkyl group, R2 stands for a hydrogen, a C1-5 alkyl group, a C3-8 cycloalkyl group or a phenyl group optionally substituted by a hydroxy or a phenyl group, or R1 and R2 together with the nitrogen atom they are attached to form a 5 to 8 membered ring optionally containing one or more further nitrogen, oxygen or sulfur atom(s) and said ring can be condensed with another alicyclic or heterocyclic ring, preferably a benzene, naphthalene, quinoline, isoquinoline, pyridine or pyrazoline ring, furthermore, if desired and chemically possible, the nitrogen and/or sulfur heteroatom(s) are present in the form of an oxide or dioxide, R3 means a hydrogen, a phenyl group, a naphthyl group or a pyridyl group wherein said groups can be substituted by one or more halo atom(s) or C1-4 alkoxy group(s), Y is a hydrogen, a hydroxy group, a C1-24 alkoxy group optionally substituted by an amino group, a C2-24 polyalkenyl-oxy group containing 1 to 6 double bond(s), a C1-25 alkanoyl group, a C3-9 alkenoyl group or a group of the formula R7-COO-, wherein R7 represents a C2-30 polyalkenyl group containing 1 to 6 double bond(s), X stands for a halo, an amino group, a C1-4 alkoxy group, or X forms with B an oxygen atom, or X and Y together with the carbon atoms they are attached to and the -NR-O-CH2 group being between said carbon atoms form a ring of the formula wherein Z represents an oxygen or a nitrogen, R stands for a hydrogen or R forms with B a chemical bond, A is a C1-4 alkylene group or a chemical bond or a group of the formula wherein R4 represents a hydrogen, a C1-5 alkyl group, a C3-8 cycloalkyl group or a phenyl group optionally substituted by a halo, a C1-4 alkoxy group or a C1-5 alkyl group, R5 stands for a hydrogen, a C1-4 alkyl group or a phenyl group, m has a value of 0, 1 or 2, n has a value of 0, 1 or 2, with the proviso that Y is other than hydroxy when X is an amino group, or a pharmaceutically acceptable acid addition salt thereof as the active ingredient in admixture with one or more conventional carrier(s).

2. A pharmaceutical composition of Claim 1 in which the active ingredient is a compound of the formula II, wherein R1, R2, R3, R4, R5, m and n are as stated in Claim 1, X
represents a halo atom, Y stands for a hydroxy group, or a pharmaceutically acceptable acid addition salt thereof.

3. A pharmaceutical composition of Claim 1 in which the active ingredient is a compound of the formula III, wherein R1, R2, R3 and A are as stated in Claim 1, or a pharmaceutically acceptable acid addition salt thereof.

4. A pharmaceutical composition of Claim 1 in which the active ingredient is a compound of the formula IV, wherein R1, R2, R3 and A are as stated in Claim 1, Z represents an oxygen or a nitrogen atom, or a pharmaceutically acceptable acid addition salt thereof.

5. A pharmaceutical composition of Claim 1 in which the active ingredient is a compound of the formula V

wherein R1, R2, R3 and A are as stated in Claim 1, R6 represents a C1-4 alkyl group, or a pharmaceutically acceptable acid addition salt thereof.

6. A pharmaceutical composition of Claim 1 or 2 in which the active ingredient is a compound of the general formula II, wherein R1 and R2 together with the nitrogen atom they are attached to form a piperidino group, m and n have the value of 0, X and Y are as stated in Claim 2, or a pharmaceutically acceptable acid addition salt thereof.

7. A pharmaceutical composition of Claims 1, 2 or 6 in which the active ingredient is 0-(3-piperidino-2-hydroxy-1-propyl)pyrid-3-ylhydroximic acid chloride or a pharmaceutically acceptable acid addition salt thereof.

8. The use of a compound of the formula I wherein R, R1, R2, R3, X, Y, A and B are as stated in Claim 1, or a pharmaceutically acceptable acid addition salt thereof, optionally in admixture with one or more carrier(s) commonly used in pharmaceutical compositions for preparing a medicaments having an activity in the protection of the mitochondrial genom and/or mitochondrium from damages.

9. The use according to Claim 8 in which a compound of the formula II wherein R, R1, R2, R3, X, Y, m and n are as stated in Claim 2, or a pharmaceutically acceptable acid addition salt thereof is used.

10. The use according to Claim 8 in which a compound of the formula III, wherein R1, R2, R3 and A are as stated in Claim 3, or a pharmaceutically acceptable acid addition salt thereof is used.

11. The use according to Claim 8 in which a compound of the formula IV, wherein R1, R2, R3, A and Z are as stated in Claim 4, or a pharmaceutically acceptable acid addition salt thereof is used.

12. The use according to Claim 8 in which a compound of the formula V, wherein R1, R2, R3, R6 and A are as stated in Claim 5, or a pharmaceutically acceptable acid addition salt thereof is used.

13. The use according to Claims 8 and 9 in which the substitutents of a compound having the formula II or a pharmaceutically acceptable acid addition salt thereof are R1 and R2 which together with the nitrogen atom they are attached to form a piperidino group, m and n have the value of 0, whereas X and Y are as stated in Claim 2.

14. The use according to any of Claims 8, 9 or 13 in which the compound of Formula I or II is 0-(3-piperidino-2-hydroxy-1-propyl)pyrid-3-ylhydroximic acid chloride or a pharmaceutically acceptable acid addition salt thereof.

15. The use of a compound of the formula I, wherein R, R1, R2, R3, X, Y, A and B are as stated in Claim 1, or a pharmaceutically acceptable acid addition salt thereof, optionally in admixture with one or more carrier(s) commonly used in pharmaceutical compositions for preparing a medicament for the treatment of diseases connected with the damage of the mitochondrial genom and/or mitochondrium.

16. The use according to Claim 15 in which the medicament is used for the treatment of myopathy and cardiomyopathy.

17. The use according to Claim 15 in which the medicament is used for the treatment of neurodegenerative diseases especially Alzheimer's disease, Parkinson's disease or Huntington's disease.

18. The use according to Claims 15 to 17 in which a compound of the formula II, wherein R1, R2, R3, R4, R5, X, Y, m and n are as stated in Claim 2, or a pharmaceutically acceptable acid addition salt thereof is used.

19. The use according to Claims 15 to 17 in which a compound of the formula III, wherein R1, R2, R3 and A are as stated in Claim 3, or a pharmaceutically acceptable acid addition salt thereof is used.

20. The use according to Claims 15 to 17 in which a compound of the formula IV, wherein R1, R2, R3, A and Z are as stated in Claim 4, or a pharmaceutically acceptable acid addition salt thereof is used.

21. The use according to Claims 15 to 17 in which the substituents of the compound of formula V, or a pharmaceutically acceptable acid addition salt thereof, R1, R2, R3, R6 and A are identical with the substituents stated in Claim 5.

22. The use according to Claims 15 to 18 in which the substituents of a compound of the formula II or a pharmaceutically acceptable acid addition salt thereof are R1 and R2 which together with the nitrogen atom they are attached to form a piperidino group, m and n have the value of 0, whereas X and Y are as stated in Claim 2.

23. The use according to any of Claims 15 to 18 and 22 in which the compound of formula I or II is 0-(3-piperidino-2-hydroxy-1-propyl)pyrid-3-ylhydroximic acid chloride or a pharmaceutically acceptable acid addition salt thereof.