WO2000007580A2

WO2000007580A2 - Pharmaceutical compositions against autoimmune diseases

Info

Publication number: WO2000007580A2
Application number: PCT/HU1999/000054
Authority: WO
Inventors: Balázs SÜMEGI
Original assignee: N-Gene Kutato Kft
Current assignee: N-Gene Kutato Kft
Priority date: 1998-08-03
Filing date: 1999-08-02
Publication date: 2000-02-17
Anticipated expiration: 2001-02-03
Also published as: AU5296799A; WO2000007580A3

Abstract

The invention refers to the use of hydroximic acid derivatives of formula (I), wherein R?1, R2, R3¿, R, X, Y, A and B are as defined in the description, for the preparation of a pharmaceutical composition against autoimmune diseases.

Description

PHARMACEUTICAL COMPOSITIONS AGAINST AUTOIMMUNE

DISEASES

The invention refers to the use of hydroximic acid derivatives of the formula

X R Y

1 1 /"^Rl

R³-A-C I -N-O-CH₂-CH-CH₂-N (I)

1 \_^

B ^ R² wherein

R¹ represents a hydrogen or a C1-5 alkyl group,

R² stands for a hydrogen, a Cι_-5 alkyl group, a C_3-8 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 C_M alkoxy group(s),

Y is a hydrogen, a hydroxy group, a Cι- alkoxy group optionally substituted by an amino group, a C_2-24 polyalkenyloxy group containing 1 to 6 double bond(s), a C₁.₂₅ alkanoyl group, a C_3-9 alkenoyl group or a group of the formula R⁷-COO-, wherein R⁷ represents a C_2-30 polyalkenyl group containing 1 to 6 double bond(s), X stands for a halo, an amino group, a C_M 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 C_w alkylene group or a chemical bond or a group of the formula

R⁴ R⁵

I I

-(CH)_m - (CH)_n- wherein R⁴ represents a hydrogen, a Ci-s alkyl group, a C_3-8 cycloalkyl group or a phenyl group optionally substituted by a halo, a C_1- alkoxy group or a Cι_-5 alkyl group, R⁵ stands for a hydrogen, a C_w alkyl group or a phenyl group, m has a value of 0, 1 or 2, n has a value of 0, 1 or 2, or a pharmaceutically acceptable acid addition salt thereof for the preparation of a pharmaceutical composition against autoimmune diseases.

The hydroximic acid derivatives of the formula I are partly known from HU-P No. 177 578 and its equivalent US-P No. 4,308,399 as well as from HU-P 207 988 and its quivalent E-P No. 417 210. According to these documents the compounds are suitable for the treatment of diabetic angiopathy. In addition some of the compounds have a selective beta-blocking effect.

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 complications of various types, especially of vascular complication due to diabetes, for example in the therapy of myocardial ischemia.

According to HU-P Application No. P 94 01488 published under No. T/71409 as well as HU-P Application No. P 95 01756 published under T/47621, the compounds of the formula I have anti-ischaemic effect.

In accordance with PCT Application No. WO 97/13504 compounds of the formula I are suitable for the prevention and treatment of diseases of mitochondrial origin.

On the basis of HU-P Application No. P 95 03141 also the hydroximic acid derivatives of the formula I enhance the level of the molecular chaperone (i.e. stress protein) of the cells.

According to PCT Application No. WO 97/23132 the compounds of the formula I can be used for the promotion of plant production.

From PCT Application No. WO 97/23198 it is known that the hydroximic acid derivatives of the formula I can delay the ageing processes of skin, thus the compounds can be used as active ingredients of cosmetic compositions.

It is known that reactive oxygen species (e.g. hydroxy radical, superoxide, peroxynitrite, hydrogen peroxide) form continuously in the living organism /Richter, C, FEBS Lett., 241, 1-5 (1988)/, and in low quantity they play a role in controlling important physiological processes /Beck, K.F. et al., J. Exp. Biol., 202, 645-53 (1999); McDonald, L.J. and Murad, F., Proc. Soc. Exp. Biol. Med., 211. 1-6 (1996)/ (such as angiectasis, platelet aggregation, leukocyte adhesion). The concentration of reactive oxygen species and nitrogen oxide is significantly higher in acute and chronic inflammations, for example in the majority of autoimmune diseases /Taraza, C. et al., Rom J. Intern. Med., 35, 89-98 (1997)/. The source of the reactive oxygen species includes partly the leukocytes and macrophages that adhere to the inflamed tissue, partly the normal tissue cells (endothehum) due to the inductive effect of the inflammatory cytokines (such as tumor necrosis factor alpha). It was established that the leukocytes and monocytes of patients suffering from active rheumatic inflammation that can be classified as an autoimmune disease produce more reactive oxygen species than the cells of healthy persons by a factor often /Miesel, R et al., Free radic. Res., 25, 161-9 (1996); Kroger, H. et al., Inflammation, 20, 203-214 (1996)/. In the autoimmune diseases, the enhanced formation of reactive oxygen species is not simply the consequence of the pathological state but a significant element of the pathomechanism.

The reactive oxygen species injure, among others, the DNA. A complex defensive and repair process is initiated in the cell by the damage of DNA. An important element of this process is the activation of the enzyme poly(adenosine diphosphate ribose)polymerase (PARP). PARP is an enzyme of nuclear arrangement which is present in nearly every cell in large amount and catalyzes the transport of the andenosine diphosphate ribose unit from nicotinic acid adenine dinucleotide (NAD) to proteins and the build-up of poly(adenosine diphosphate ribose) chains. The main substrates of the enzyme include itself /Gonzalez, R. et al., Mol. Cell. Biochem., 138, 33-37 (1994)/, nuclear proteins, histones, topoisomerase I and II, transcription factors. The activity of the PARP enzyme is enhanced by a factor of about 500 in case of a break in the DNA chain (Mennisier de Murcia, J. et al., J. Mol. BioL, 210. 229-233 (1989)/. A critical lowering of the NAD concentration is caused by PARP enzyme activation owing to an extreme high DNA damage. As a consequence, the synthesis of adenosine triphosphate (ATP) is reduced in the cell and, at the same time the use of ATP becomes higher since the cell tries to restore the NAD level from adenosine diphosphate ribose and nicotinic amide by using ATP. These biochemical processes damage the energy state of the cells heavily and may lead to cellular destruction.

Thus, in the therapy of autoimmune disease there is a great significance of the inhibition of the PARP enzyme since in this way catabolism of NAD can be eliminated. By doing so, levels of nicotinic acid and adenosine diphosphate ribose are reduced in the cell and the use of adenosine triphospbate for the synthesis of NAD is inhibited. Thus, by the inhibition of the PARP enzyme, the damage or destruction of the cells mentioned above can be avoided /Szabo, C. et al., Trends Pharmacol. Sci., 19, 287-98 (1998)/.

It was found that the hydroximic acid derivatives of the formula I and the pharmaceutically acceptable acid addition salts thereof inhibit the PARP enzyme, consequently, they can be used for the effective treatment of autoimmune diseases.

Thus, the invention refers to a novel use of the known compounds mentioned above, namely the hydroximic acid derivatives of the formula I or the pharmaceutically acceptable acid addition salts thereof is employed for the preparation of a pharmaceutical composition against autoimmune diseases.

An autoimmune disease is an illness in which an immune reaction is started by the organism against a normal constituent thereof /Ring, G.H. et al., Semin. Nephrol., 19, 25-33 (1999)/; Theofilopoulos, A.N., Ann. N.Y. Acad. Sci., 841. 225-35 (1998)/. The various autoimmune diseases differ from each other in the antigene that starts the process, however, a great similarity can be established in the cell tissue destroying mechanism of the autoimmune processes developed /Szabo, C. et al., Proc. Natl. Acad. Sci. USA, 95, 3867- 3872 (1998)/.

Autoimmune diseases include in the first place the following ones:

- hormonal diseases: insulin dependent diabetes melhtus (IDDM);

- liver diseases: hepatitis; - skin disease: bullous pemphigoid lupus, pemphigus vulgaris, psoriasis, scleroderma, vitiligo;

- diseases of the blood forming organ: sarcoidosis;

- arthtopathies: rheumatoid arthritis;

- vascular diseases: vascuHtis, takayasu arteritis, polyarteritis nodosa, ankylosing spodnyUtis;

- intestinal diseases: colitis ulcerosa;

- diseases of the muscular and nervous system: sceloris multiplex, myasthenia gravis, chronic inflammatory demyelinating polyneuripathy.

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, preferably a methyl or an ethyl group.

A C_M alkyl group is, for example, a methyl, ethyl, isopropyL n-propyL n-butyl or isobutyl group.

A C_3-8 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, quinoline etc. ring.

A Cι-24 alkoxy group is, for example, a methoxy, ethoxy, n-propoxy, tert.-butoxy, n-pentoxy, decyloxy, dodecyloxy, octadecyloxy etc. group.

A Cι-25 alkanoyl group is, for example, a formyL, acetyl, propionyL, butiryl, caproyL, palmityl, stearyl etc. group.

A C_3-9 alkenoyl group is, for example, an acryloyL pentenoyL hexenoyl, heptenoyl, octenoyl etc. group.

A CM alkylene gtroup is, for example, a methylene, ethylene, propylene or butylene group. A halo atom is, for example, a fluoro, chloro, bromo or iodo atom, preferably a chloro or a bromo atom.

If Y stands for a group of the formula R⁷-COO-, it can represent, for example, a linolenoyL, linoloyl, docosahexaenoyl, eicosapentaeneoyL 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, 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

R⁴ R⁵ ³-(CH)_m-(CH)_n-C-X /R¹

N-O-CH₂-CH-CH₂-N π

1 \

Y \ _R2

wherein R¹, R², R³, R⁴, R⁵, m and an are as stated in relation to formula I, X represents a halo atom or an amino group, Y means a hydroxy group.

Especially preferred compounds of the formula II are those wherein R¹ and R² together with the nitrogen atom they are attached to form a piperidino group, R³ stands for a pyridyl group, m and n have a value of 0, X is as defined above. Of these compounds, preferred species are as follows: 0-(3-piperidmo-2-hydroxy-l-propyl)-pyrid-3-ylhydroximic acid chloride (Compound "A") and 0-(3-piρeridmo-2-hy(koxy-l-propyl)mcotinic amidoxime (Compound "B").

A further preferred subgroup of the hydroximic acid derivatives of the formula I consists of the compounds of the formula R³-A- III

wherein R¹, R², 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

.R¹

wherein R¹, 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

wherein R¹, R², R³ and A are as stated in relation to formula I, R⁶ stands for a Cι-4 alkyl group.

The compounds of the formula I can be prepared by the processes known from US-P No. 4,308,399 and E-P No. 207 988 as well as from HU-P Application No. 2385/92 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 carriers(s). The pharmaceutical compositions of the invention are suitable for peroraL parenteral or rectal aά^rninistration 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, micorcapsules etc. and can comprise binding agents such as gelatine, sorbitol, poly(vinylpyrroKdone) 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 carrier.

The liquid pharmaceutical compositions 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 etc. ; solvents such as water, oils, propyleneglycol, ethanol etc.; preservatives such as methyl p-hydroxybenzoate etc. as the carrier.

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 addition 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 carriers), and converting the mixture obtained to a pharmaceutical composition in a manner known per se. Useful methods are known from the literature, e.g. Remington's Pharmaceutical Science.

We have studied the PARP enzyme inhibitory effect of the hydroximic acid derivatives in a rat heart preparation according to Langendorffs method. The use of this model can be explained by the following facts: the postischemic necrosis of cardiac tissue is mainly due to oxidative injuries incurred during reoxigenation when the concentration of reactive oxygen species is increased.

This, in turn, can lead to single strand DNA breaks, and subsequently, to the induction of the PARP enzyme. Lindahl, T. and Co., Trends Biochem. Sci., 20, 405-411 (1995)/.

Heart Perfusion

The heart of adult male Wistar rats weighing 300-350 g was used for Langerdorffs heart perfusion experiments. All animal experiments were conducted in conformity with the guiding principles for the care and use of animals.

Rats were anaesthetized with ketamine (200mg/kg i.p.) intraperitoneally and heparinized with sodium heparin ( 100 IU/rat Lp. ). Hearts were perfused via the aorta according to the LangendorfFs method at a constant pressure of 70 mmHg, at 37 °C /Stryer L., Biochemistry, W.H Freeman and Co., New York, 1995/. The perfusion medium was a modified phosphate-free Krebs- Henseleit buffer consisting of 118 mM NaCL 5 mM KC1, 1.25 mM CaC12, 1.2 mM MgSO4, 25 mM NaHCO3, 11 mM glucose and 0.6 mM octanoic acid and in the treated group 40 mg/ml of compound B (a hydroximic acid derivative of formula I). The perfusate was bubbled with a 95% 02/ 5% CO2 through a glass oxigenator and adjusted to pH 7.40. After a washout, non- recirculating period of 15 minutes, hearts were either perfused under normoxic conditions for the given time, or were subjected to a global ischemia of 1 hour by closing the aortic influx and then reperfusing that for 15 or 30 minutes. During ischemia hearts were submerged into perfusion buffer at 37 °C. At the end of perfusion hearts were freeze clamped.

Assay of nicotinamide-adenine-dinucleotide (NAD⁺)

The concentration of NAD+ in the neutralized perchloric acid extract of the cardiac muscle was measured by using alcohol dehydrogenase reaction /Sύmegj, B. es Srere, P.A., J. Biol. Chem., 259, 15040-15045/. The freshly prepared reaction buffer contained 0.1M tris, pH 8.40, 1 mM EDTA, 4 mM L- cysteine chloride, 2% ethanol. Each cuvette contained 300 μl of the tissue extract, 650 μl of the reaction buffer and 4 units of the freshly prepared enzyme in 50 μl double distilled water. The reaction was initiated by the addition of enzyme. The exact tissue NAD⁺ was determined from a calibration curve.

Determination of DNA single-strand breaks

Single-strand DNA breaks were determined by the alkaline fluorescence analysis of DNA unwinding as described by Jevcak and Birnboim / Fleischer, S. and Kervina, M., Methods in EnzymoL, 31, 6-41 (1974)/. DNA samples were prepared from normoxic and ischemic hearts. To estimate the quantity of undamaged double stranded DNA, samples were divided into 3 sets of tubes. DNA fluorescence was determined under different conditions. At the determination of F value, DNA was kept at pH 12.40 to permit partial unwinding of DNA. At the determination of F_min, DNA was kept at pH 12.4 but at the beginning of the incubation period DNA sample was sonicated for 60 sec. At the determination of F_max DNA sample was kept at pH 11.0 which is below the pH needed to induce unwinding. Solutions were incubated for 30 minutes at 0 °C followed by 15 minutes at 15 °C. Unwinding was stopped by adjusting the pH to 11.0. Fluorescence was measured after the addition of 0.67 μg/ml of ethidium bromide with an excitation wavelength of 520 nm and an emission wavelength of 590 nm on a Perkin Elmer luminescence spectrometer. Results are expressed as D ( percent of double stranded DNA )= ( F-F_min )/( F_max-F_min ) X 100 /Miles, A.M. and Co., J. Biol. Chem., 271, 40-47 (1996)/.

Detection of Reactive Oxygen Species (ROS)

Reactive oxygen related radical formation was detected using the oxidation-sensitive non-fluorescent probe dihydrorhodamine 123 (DHR), which can be oxidized by reactive oxygen species to fluorescent rhodamine 123 /Pinson,A. and Tirosh, R.., Mol. Cell. Biochem., 160-161. 137-141 (1996)/. The detection is based upon the fact that the ROS are oxidize the non- fluorescent dihydrorhodamine to fluorescent rhodamine. Since the dihydrorhodamine can penetrate the cell membrane whereas the rhodamine remains in the mitochondrium, this reaction can be used for the determination of ROS in the Langendorffs heart perfusion system. The reactive oxygen species formation was first studied in normoxic hearts. After a 15 min washout period, DHR (5 μM) was added to the perfusate and the heart was perfused for additional 15 minutes. In cases when hearts subjected to 60 minutes ischemia and 15 minutes reperfiision, DHR (5(M) was added to the perfusate just before reperfiision. In all cases, hearts were perfused with DHR for 15 minutes, and freeze clamped at the end of the perfusion. For the extraction of rhodamine 123, 90 mg of heart pieces were homogenized in 2 ml of 20 mM tris buffer (tris(hydroxy-methyl)-amino-methane) at pH 7.40 and an equal amount of ice- cold 70 % ethanol containing 0.1 M HCl was added. The precipitated proteins were removed by centrifugating the homogenate at 3000 g for 15 minutes. The precipitate was extracted once again, and the unified supernatants were neutralized with NaHCO₃ and centrifuged at 6000 g. The rhodamine 123 content in the clear supernatant was determined using a Perkin Elmer fluorescence spectroscope at an excitation wavelength of 500 nm and an emission wavelength of 536 nm.

ROS were also detected in vitro in heart tissues following normoxic perfusion (30 minutes), and in heart tissues deriving from hearts subjected to one hour ischemia. In both cases heart pieces (50 mg ) were homogenized in 3 ml of well oxygenated buffer containing 150 mM KG, 20 mM tris, 0.5 mM EDTA (ethylene-diamine-tetraacetic acid), 1 mM MgCl₂, 5 mM glucose and 0,5 mM octanoic acid (pH 7.4), and incubated in the presence of 5 μM DHR for 30 minutes at 37 °C. The reaction was stopped by the addition of equal amount of ice cold 70 % ethanol which contained 0.1 M HCl and the formed rhodamine 123 was extracted as described in the case of in situ assays. To correct background fluorescence samples were incubated under the same conditions but without DHR, and the 5 μM DHR was given to tissue only at the end of the incubation period.

Adenosine-diphosphate (ADP) ribosylation assay

50 mg of cardiac muscle was homogenized with Ultra-Turrax in 500 μl of 50 mM tris pH 7.80 and 500 μl of 2X Laemmli sample buffer was then added, homogenized with Potter and cleared by centrifugation for 5 minutes at 10.000 rpm. In some experiments the extraction buffer contained 8 M urea, 20 mM tris and 4 mM EDTA. Samples were subjected to SDS polyacrylamid gel electrophoresis (Sambrook and Fritsch, A Laboratory Manual, 18.47-18.50) using a 10 % gel and blotted to nitrocellulose membrane for Western blot analysis. ADP-ribosylated proteins were detected by anti-ADP-ribose monoclonal antibody and anti-mouse IgG peroxidase complex and visualized by enhanced chem uminescence (ECL) method.

Of all samples 10 μg of protein for electrophoresis was used. The postischemic heart was subjected to 1 hour ischemia and a 30 minute reperfiision. The postischemic heart was pre-perfused for 10 minutes in the presence of the studied compound for the investigation of a hydroximic acid derivative of formula I, this was followed by a 1 hour ischemia and a 30 minute reperfiision. The normoxic hearts were perfused under normoxic conditions for the same period of time as the postischemic hearts.

Isolation of nuclei

The isolation of nuclei from cardiac tissue was carried out by using standard extraction procedure /Claycomb, W.C., Biochem. J., 154. 387-393 (1976)/. The purified nuclei were prepared for dot blotting using extraction buffer containing 8M urea, 20 mM tris, 4 mM EDTA, and 2X Laemmli sample buffer. The immune reaction was carried out as described in the ADP- ribosylation assay.

In vitro poly ADP-ribosylation of nuclear proteins

The inhibition of poly-ADP-ribose polymerase was measured on isolated nuclei essentially as described before /Schraufstatter, I. U. and Co., Proc. Natl. Acad. Sci. USA, 4908-4912 (1986)/. Cardiomyocytes nuclei equivalent to 1 mg protein were incubated at 25 °C in a reaction mixture containing 0.1 mM NAD⁺ (³²P-labeled, 2.10⁷ cpm) , 250 mM sucrose, 100 mM tris-HCl buffer (pH = 8.2), 2 mM dithiothreitol, 10 mM MgCl₂ , 0.5 mM EDTA and 1 mM phenylmethane sulfonyl fluoride in a final volume of 1 ml. After incubation with agitation for 10 minutes the reaction was terminated with 4% trichloroacetic acid. The precipitated proteins were washed twice with 4% trichloroacetic acid, dissolved in 10 ml of Bray" s solution, and the total amount of [H³]NAD⁺ incorporated into the protein precipitate was determined by Beckman LS-230 counter. Assessment of cell membrane integrity

The release of lactate dehydrogenase EC 1.1.1.27 (LDH), creatine kinase EC 2.7.3.2 (CK) and glutamate oxaloacetate transaminase EC 2.6.1.1 (GOT) enzymes was measured in the perfusate of Langendorffs perfused hearts under normoxic and ischemic conditions. Enzyme activities were measured by standard methods as described in /Birnboim, H.C. and Jevcak, J.J., Cancer Research, 41, 1989-1892 (1981)/ for LDH, / Schraufstatter, LU. and Co., Proc. Natl. Acad. Sci. USA, 1986, 4908-4912/ for GOT and / Pinson, A. and Tirosh, R., Mol. Cell. Biochem., 160-161, 137-141 (1996)/ for CK.

RESULTS

Release of cytoplasmic enzymes

Ischemia-reperfusion can cause serious abnormality in the energy state and ion balance of cells which, together with direct oxidative membrane damages, can lead to the release of cytoplasmic enzymes from cardiomyocytes /Fleischer, S. and Kervina, M., Methods in EnymoL, 31, 6-41 (1974)/. The release of frequently studied cytoplasmic enzymes, such as CK, LDH and GOT, is extremely low in normoxic hearts (Table 1), but ischemia- reoxigenation-induced cell damage caused a significant amount of release of these enzymes into the perfusate (Table 1). Lα those experiments when hearts were pre-perfused with hydroximic acid derivatives of the formula I (for 10 minutes) under normoxic conditions followed by 1 hour ischemia and 30 minutes reperfiision, a significantly reduced enzyme release was observed (Table 1). These data indicate that hydroximic acid derivatives of the formula I protected the integrity of cardiomyocytes against ischemia-reperfusion- induced damages. Table 1

The effect of hydroximic acid derivatives of the formula I on the intracellular release during the ischemia-reoxygenation according to Langendorff on the model of a perfused rat heart. We studied the integrity of the membrane 30 minutes following the reperfiision with the release of intracellular enzyme into the perfusate. The results show the mean value ± standard deviation.

Quantity of enzyme, mU/ml GOT LDH CK

Normoxic heart 2 + 1 1 ± 1 4 + 2

Ischemia-reperfusion 96 + 8 419± 36 148+17

Ischemia-reperfusion +

+ 40 mg/1 of compound "B" 33 + 6 125 ±10 61±_ 5

Ischemia-reperfusion +

+ 20 mg1 of compound "A" 31 ± 8 119 ± 13 59± 7

GOT =glutamate oxaloacetate transaminase LDH =lactate dehydrogenase CK =creatine kinase

Detection of reactive oxygen species in Langendorffs heart perfusion system

Several reactive oxygen species reoxidize the non-fluorescent dmydrorhodaminel23 to a fluorescent rhodaminel23. Since DHR is cell permeable and the oxidized rhodamine 123 is retained by the mitochondria, this reaction can be used for the detection of ROS in Langendorffs perfused heart. The oxidation of DHR to rhodamine 123 is well detectable in normoxic perfused hearts (Table 2), but the oxidation rate is significantly increased as a result of ischemia-reperfusion (Table 2). The pre-perfusion of hearts with hydroximic acid derivatives of the formula I only moderately decreased the ischemia-reperfusion-induced rhodamine 123 formation (Table 2), indicating that hydroximic acid derivatives of the formula I have only moderate effect on the steady state level of ROS in postischemic hearts.

Table 2

The effect of hydroximic acid derivatives of the formula I on the formation of free radicals and on the single strand DNA breaks in ischemia- reoxidation in Langendorffs perfused rat heart preparation.

The results show the mean value ± standard deviation.

Detected rhodamine Single stranded quantity in artificial units DNA as % of non-damaged DNA

normoxic heart 30.5 ±3 71 ± 7 ischemia-reoxygenation 54.2 ± 4 20 ± 6 ischemia-reoxygenation +

+ 40 mg/1 Compound "B" 49.1± 2 39 ± 5 ischemia-reoxygenation +

+ 20 mg/1 Compound "A" 43.2+ 4 37 ± 4 Single-strand DNA breaks in postischemic hearts

The concentration of reactive oxygen species is significantly increased in ischemia-reperfusion, and this can induce single-strand DNA breaks. In our experimental system most of the DNA is undamaged in normoxic hearts, but the ischemia-reperfusion induces large amount of single-strand DNA breaks (undamaged DNA < 20 %) (Table 2). The 10 minutes pre-perfusion of hearts with hydroximic acid derivatives of the formula I decreased the amount of single-strand DNA breaks and doubled the amount of undamaged DNA in postischemic heart (Table 2).

Ischemia-reperfusion induced NAD⁺ catabolism in perfused rat hearts

The NAD⁺ content was determined in normoxic, ischemic and postischemic hearts (Table 3). It was found that ischemia caused only a slight decrease in the nicotinamide-aden e-dinucleotide pool (Table 3), whereas ischemia followed by 5, 30 and 60 minute reperfiision induced a significant depletion of the intracellular NAD⁺ pool (Table 3). The pre-reperfusion of hearts partially protected the heart against ischemia-reperfusion induced loss of NAD⁺ (Table 3).

Table 3

The effect of hydroximic acid derivatives of the formula I on the decrease of NAD level caused by ischemia reoxygenation in Langendorf s rat heart in different reperfiision periods.

The results show the mean value ± standard deviation. μM NAD+/g wet tissue after 0 5 30 60 min. reperfiision

normoxic heart 0.45 + 0.06 ischemic heart 0.38 ± 0.07^xx ischemia-reoxygenation 0.29 0.26 0.24 ischemia-reoxygenation +

+40 mg/1 of compound "B" 0.39^x 0.3 l^x 0.30^x

^x There is a significant difference from the values measured without compound

"B", p < 0.02. ^** Since a significant portion of NAD⁺ was reduced to NADH, the total amount of NAD⁺ and NADH was determined under ischemic conditions.

Regulation of nuclear poly-ADP-ribose polymerase by hydroximic acid derivatives of the formula I

The nuclear poly-ADP-ribose polymerase could not be extracted by standard extraction procedure from rat heart tissues. However, when the extraction buffer contained 8 M urea, then the ADP-ribosylation of the high molecular weight nuclear poly-ADP-ribose polymerase becomes readily detectable with Western blot analysis (Table 4). The ADP-ribosylation of poly-ADP-ribose polymerase (Mw. -116 kD) was induced by ischemia- reperfusion in accordance with the observation that ischemia-reperfusion increased the single-strand DNA breaks, too. Hydroximic acid derivatives of the formula I showed clear inhibitory effects on the self ADP-ribosylation of the nuclear poly-ADP-ribose polymerase (Table 4). Table 4

The self-adenosine-diphosphate-ribosylation of PARP induced by the ischemia reperfiision

Adenosine diphosphate ribosylation of PARP enzyme with Western-blot determination

Normoxic heart 100 + 12

Postischemic heart 168 + 21

Postischemic heart +

+ 40 mg/1 of compound "B" 53 ± 14

Postischemic heart +

+ 20 mg/1 of compound "A" 61 + 13

Direct inhibition of nuclear poly-ADP-ribose polymerase

The previous in situ experiments showed that hydroximic acid derivatives of the formula I inhibit the ischemia-rep erfusion-induced ADP- ribosylation of nuclear poly-ADP-ribose polymerase. Since hydroximic acid derivatives of the formula I themselves are structural analogues of nicotinic amide, it is possible that they directly inhibit the poly-ADP-ribose polymerase. This mechanism was tested under in vitro conditions using isolated nuclei and ³²P- or ³H-labelled NAD⁺. The covalent incorporation of ³²P- or ³H-labelled NAD⁺ into the nuclear poly-ADP-ribose polymerase was measured. Using this method it was found that hydroximic acid derivatives of the formula I inhibit the poly-ADP-ribose polymerase reaction (Table 5), and the I0.5 for hydroximic acid derivatives of the formula I under our experimental conditions was found to be 17 mg/1 of compound "B". These data show that hydroximic acid derivatives of the formula I have a direct inhibitory effect on the nuclear poly-ADP-ribose polymerase which can explain the inhibitory effect seen in the in situ Langendorffs heart perfusion model system (see Table 5).

Table 5

The effects of hydroximic acid derivatives of the formula I on nuclear PARP enzyme.

The studied compounds and their concentration Enzyme activity

1742 ± 154

Nicotinic amide 5mM 138 ± 79

Compound "B" lOmg/1 1260 + 132

20mg/l 703 ± 120

40 mg/1 219 ± 87

Compound "A" 20 mg/1 627 ± 210

Investigation of the effect against autoimmune diseases

investigation of the prevention of the streptozotocin-induced autoimmune type I diabetes mellitus on mice.

Insulin, which is the main regulator of the carbohydrate metabolism in the body, is produced and transferred to the blood stream by the cells of the Langerhans islet of the pancreas. Damage or destruction of the β-cells causes the decrease of cease or insulin production which leads to the development of the type I diabetes mellitus (insulin-dependent diabetes mellitus = IDDM). ^β- cells are especially sensitive to ROS and to the toxic effect of NO. The study of DNA damage, caused by NO led to the assumption that the excessive activation of the PARP enzyme and the decrease of NAD level are responsible for the death of β-cells /Heller, B. and Co., J. Biol. Chem., 270, 176-180 (1995)/. With a similar mechanism, streptozotocin /2-deoxy-2-(3-methyl-3- nitrosoureido)-D-glucopyranose/ (STZ) is damaging the insulin producing ^β- cells, which is offering the model of the type I diabetes when used in animal experiments /Yamamoto,H. and Co., Nature, 294, 284-286 (1981)/. DNA is damaged by alkylating streptozotocin and by the formation of NO, which causes activation of the PARP enzyme as mentioned above.

In the course of the experiments we have studied wether a single dose of a hydroximic acid derivative protects against the effect of streptozotocin which increases the blood sugar level.

In our experiments CD-I female mice (17-19 g b.w.; breeder: Charles River, Hungary) were involved in three experimental groups. 10 animals were in each group. Animals of Group 1 serving as control were given physiological saline i.p. Animals of Group 2 were exposed to the single i.p. injection of 160 mg/kg dose of STZ, whereas those of Group 3 were given single dose of compound "B" (200 mg/kg. p.o.) and 160 mg/kg of STZ, i.p.

Pretreatment with compound "B" was carried out for 15 rnin before STZ administration. Blood glucose level was deteπnined on the second and fifth day after treatment. Blood glucose values are demonstrated in Table 6.

Table 6

Change of blood glucose level Groups Blood glucose level mean±SD (mM) on post-treatment day

Control 10.07 12.26

±1.76 ±3.99

STZ 17.37 22.91

±4.26 ±3.72

Compound "B" + STZ 12.46 14.33

+1.89 +5.22

Blood sugar level of STZ-treated animals significantly increased in comparison with the control. The blood sugar level of animals, treated with both STZ + compound „B" remained close to the value of the control, and differed significantly from the values of animals treated exclusively with STZ.

Conclusively, ROS has protecting effect on the development of insuline-dependent diabetes. This protecting mechanism becomes effective through the inhibition of the PARP enzyme, since the hydroximic acid derivatives of the formula I effectively inhibit the PARP enzyme as shown in Tables 1-5.

Based on the result of the above experiment the hydroximic acid derivatives of the formula I or pharmaceutically acceptable acid addition salts thereof are used preferably for preparing a pharmaceutical composition against insulin-dependent diabetes mellitus.

Claims

Claims:

1. Use of a hydroximic acid derivative of the formula

wherein

R¹ represents a hydrogen or a Ci.5 alkyl group,

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 C_1-4 alkoxy group(s),

Y is a hydrogen, a hydroxy group, a C_1-24 alkoxy group optionally substituted by an amino group, a C_2-24 polyalkenyloxy group containing 1 to 6 double bond(s), a Cι_-25 alkanoyl group, a C3-9 alkenoyl group or a group of the formula R⁷-COO-, wherein R represents a C_2-3o polyalkenyl group containing 1 to 6 double bond(s),

X stands for a halo, an amino group, a C_M 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 C_1-4 alkylene group or a chemical bond or a group of the formula

R⁴ R⁵ i i

-(CH)_ra - (CH)_n- b wherein

R⁴ represents a hydrogen, a Cι_-5 alkyl group, a C_3-8 cycloalkyl group or a phenyl group optionally substituted by a halo, a C_M alkoxy group or a C₁.₅ alkyl group, R⁵ stands for a hydrogen, a C_1-4 alkyl group or a phenyl group, m has a value of 0, 1 or 2, n has a value of 0, 1 or 2, or a pharmaceutically acceptable acid addition salt thereof for the preparation of a pharmaceutical composition against autoimmune diseases.

2. The use of Claim 1 in which the hydroximic acid derivative is a compound of the formula R⁴ R⁵

R³-(CH)_m-(CH)_n-C-X /R¹ ii ^

N-O-CH₂-CH-CH₂-N π

Y R²

wherein R¹, R², R³, R⁴, R⁵, m and n are as stated in Claim 1, X represents a halo atom or an amino group, Y means a hydroxy group, or a pharmaceutically acceptable acid addition salt thereof.

3. The use of Claim 1 in which the hydroximic acid derivative is a compound of the formula

wherein R¹, R², R³ and A are as stated in Claim 1, or a pharmaceutically acceptable acid addition salt thereof.

4. The use of Claim 1 in which the hydroximic acid derivative is a compound of the formula

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. The use of Claim 1 in which the hydroximic acid derivative is a compound of the formula

OR⁶ OH .R¹

R³-A-C=N-O-CH₂-CH-CH₂-N V

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

6. The use of Claim 1 or 2 in which the hydroximic acid derivative is a compound of the formula π, 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. The use of Claims 1, 2 or 6 in which the hydroximic acid derivative is 0-(3-piperidmo-2-hydroxy-l-propyl)-pyrid-3-ylhydroximic acid chloride or a pharmaceutically acceptable acid addition salt thereof.

8. The use of Claims 1, 2 or 6 in which the hydroximic acid derivative is 0-(3-piperidmo-2-hydroxy-l-propyl)nicotinic amidoxime or a pharmaceutically acceptable acid addition salt thereof.

9. The use of any of Claims 1 to 8 in which the autoimmune disease is insulin dependent diabetes.

10. A method for the treatment of autoimmune diseases which comprises administering an effective non-toxic dose of a hydroximic acid derivative of the formula I, wherein R¹, R², R³, R, X, Y, A and B are as defined in Claim 1, or a pharmaceutically acceptable acid addition salt thereof to a patient suffering from said diseases.