RU2674286C1 - Agent for treatment of disease caused by oxidative stress - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to the field of pharmaceutical industry, in particular to the use of a therapeutically effective amount of a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid of the formula:

for treatment of disease caused by oxidative stress and selected from the group that includes Parkinson’s disease, Alzheimer's disease, Huntington’s chorea, retinitis pigmentosa, mitochondrial encephalomyopathy, multiple sclerosis, stroke, Crohn's disease, ulcerative colitis, rheumatoid arthritis, psoriasis.

EFFECT: invention provides an expansion of the means for treatment of diseases caused by oxidative stress, and increasing the effectiveness of such treatment.

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Description

The invention relates to the field of medicine and the pharmaceutical industry, and in particular to the creation of an agent for the treatment of diseases caused by oxidative stress.

Oxidative (oxidative) stress is the result, as a rule, as a result of inflammation and / or hypoxic circumstances, in which there is a violation in the metabolic processes of the human body, which provokes the accumulation of free radicals (active agents) that cause damage to cells and also trigger primary mechanisms of the damaging effect in the cells, which in turn causes various pathological conditions and diseases.

Currently, mitochondrial dysfunction is considered a key element in oxidative damage to cells. Human mitochondria contain proteins encoded by mitochondrial DNA (m-DNA), the main role of which is to build the respiratory chain and ensure the energy needs of the cell. It is well known that in the process of aging <accumulation of m-DNA mutations occurs, which violates the structure and functions of mitochondria, leading to an imbalance between the production of free radicals and the protective capabilities of the body [Chunyan Guo et al. Oxidative stress, mitochondrial damage and neurodegenerative diseases / Neural Regeneration Research, 2013, V. 8, N. 21, pp. 2003-2014].

In addition to mitochondrial dysfunction, one of the mechanisms for the development of oxidative damage to cells can be a violation of glutathione metabolism. The ratio of reduced and oxidized glutathione reflects the redox potential of the cell, and a decrease in this potential is another trigger for the development of oxidative stress [Ondrej Zitka et al. Redox status expressed as GSH: GSSG ratio as a marker for oxidative stress in pediatric tumor patients / Oncology Letters, 2012, V. 4, No. 6, pp. 1247-1253].

Another trigger of oxidative stress is the accumulation of iron in the basal ganglia in a number of neurodegenerative diseases [Jinze Xu et al. Iron Accumulation with Age, Oxidative Stress and Functional Decline / PLoS ONE, 2008, V.3, N 8, pp. e2865 (l-8)].

Oxidative stress is the cause or an important component of serious diseases, which at present can also be called mitochondrial diseases, for example, such as Parkinson's disease, Alzheimer's disease, Huntington’s chorea [Vasenina E.E. et al. Oxidative stress in the pathogenesis of neurodegenerative diseases: possibilities of therapy / Neuroprotective therapy, 2013, No. 3-4, p. 39-46] and others.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases that are clinically characterized by progressive hypokinesia, muscle rigidity, and resting tremor. BP is based on the death of dopaminergic neurons of the substantia nigra, while Levi bodies are found in the surviving cells, and dopamine levels are reduced in the striatum [Jenner P. et al. Oxidative stress in Parkinson's disease / Annals of neurology, 2003, V. 53, No. 3, pp. S26-S38].

Alzheimer's disease (AD) is a neurodegenerative disease of predominantly late age, characterized by progressive cognitive decline. The disease is based on irreversible cell death, especially in the cerebral cortex and hippocampus. Key features of the disease are the accumulation of amyloid plaques and neurofibrillary tangles in brain tissue. In AD, oxidative stress is one of the key links in pathogenesis. It is oxidative stress that can lead to a violation of the metabolism of the protein - the precursor of amyloid [Dubinina E.E. et al. Oxidative stress and its effect on the functional activity of cells in Alzheimer's disease / Biomedical Chemistry, 2015, V. 61, No. 1, p. 57-69].

Huntington’s disease (BG) (or Huntington’s syndrome, or Huntington’s chorea, or Huntington’s chorea) is an autosomal dominant inherited disease associated with unstable expansion of the polyglutamine site at the N-terminal end of a protein called huntingtin. BG is characterized by the death of neurons and progressive atrophy of the subcortical structures, especially the caudate nucleus. Various evidence indicates that a key consequence of changes in the structure of the protein is a defect in the mitochondrial chains, which as a result leads to a violation of the energy supply of the cell. Violation of the structure of mitochondria and dysfunction of the respiratory chain in turn leads to excessive production of free radicals, which is likely to cause cell damage [Browne S.E. et al. Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia / Annals of neurology, 1997, V. 41, N. 5, pp. 646-653].

There is also evidence in the literature that retinitis pigmentosa is also a disease due to oxidative stress [Maria Miranda et al. Antioxidant therapy in retinitis pigmentosa / Novel Aspects of Neuroprotection, 2010: ISBN: 978-81-308-0394-4, 15 pages], mitochondrial encephalomyopathy [Genki Hayashi et al. Oxidative Stress in Inherited Mitochondrial Diseases / HHS Public Access, 2015, V. 88, pp. 10-17], multiple sclerosis [Krotenko N.V. and other Oxidative stress - a characteristic feature of the pathogenesis of multiple sclerosis / Bulletin of Siberian medicine, 2008, No. 5, p. 208-214], stroke [Lutsky M.A. et al. Formation of oxidative stress, one of the links in the complex pathogenesis of socially significant diseases of the nervous system — stroke and multiple sclerosis / Fundamental Research, 2014, No. 10 (part 5), p. 924-929], Crohn’s disease [Iborra M. et al. Role of oxidative stress and antioxidant enzymes in Crohn's disease / Biochemical Society Transactions, 2011, V. 39, N. 4, pp. 1102-1106], ulcerative colitis [Rana S.V. et al. Role of oxidative stress & antioxidant defense in ulcerative colitis patients from north India / Indian Journal of Medical Research, 2014, V. 139, N. 4, pp. 568-571], rheumatoid arthritis [Celia Maria Quinonez-Flores et al. Oxidative Stress Relevance in the Pathogenesis of the Rheumatoid Arthritis: A Systematic Review / BioMed Research International, 2016, V. 2016, 14 pages], psoriasis [Qiang Zhou et al. Oxidative stress in the pathogenesis of psoriasis / Free Radical Biology & Medicine, 2009, V. 47, pp. 891-905].

The prior art [patent RU 2218918 C1, publ. December 20, 2003] it is known that dialkyl ethers and monoalkyl ethers of fumaric acid in the form of a free acid or its salts can be used to treat a disease caused by impaired oxidative phosphorylation and selected from the group including Parkinson's syndrome, Alzheimer's disease, Huntington’s chorea, retinitis pigmentosa and mitochondrial encephalomyopathy.

However, according to the data presented in this document, the stimulating effect of various fumaric acid derivatives on the enzymatic activity of succinate dehydrogenase varies greatly and is not always high. Some derivatives of fumaric acid exhibit moderate or even weak effects. What this may be connected with is not discussed in the document.

The present invention solves the problem of expanding the arsenal of tools effective for the treatment of diseases caused by oxidative stress.

As such a tool, the present invention proposes to use a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid.

In the document [patent RU 2365582 C1, publ. August 27, 2009] a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid of the following formula is disclosed:

,

,

which has metabolic and cardioprotective activity. The specified salt is obtained by the interaction of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid at a molar ratio of 2: 1 in a solvent (ethanol) environment.

However, this document does not discuss or even mention the possibility of using this salt to treat diseases caused by oxidative stress.

The technical result of the present invention is to create a new effective tool for the treatment of diseases caused by oxidative stress, and to increase the effectiveness of such treatment.

The specified task and technical result is achieved by using a therapeutically effective amount of a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid of the formula (I):

for the treatment of a disease caused by oxidative stress and selected from the group including Parkinson's disease, Alzheimer's disease, Huntington’s chorea, retinitis pigmentosa, mitochondrial encephalomyopathy, multiple sclerosis, stroke, Crohn’s disease, ulcerative colitis, rheumatoid arthritis, psoriasis.

A therapeutically effective amount of an active agent, a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid, is preferably 100-300 mg.

The specified salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid can be administered to a patient in need thereof, both in the form of a dosage form for oral administration and as a dosage form for parenteral administration.

Moreover, such dosage forms may further include pharmaceutically acceptable carriers, excipients or excipients.

As pharmaceutically acceptable carriers, excipients, excipients, various additives can be used that are used in the manufacture of finished dosage forms that are compatible with the active agent and other excipients and do not adversely affect the patient, for example, lubricants, diluents, binders, stabilizers, disintegrants or suspending agents, etc.

In addition to the foregoing, additional excipients may provide for controlled, prolonged, immediate or modified release of the active agent.

An oral dosage form can be selected from a tablet, capsule, powder, granules, dragee, gel, jelly, orodispersible form, buccal or sublingual form in the form of a plate or tablet, solution, syrup or suspension.

Tablets and capsules may contain standard excipients, necessary both for the technology of their manufacture, and to achieve the necessary qualitative and quantitative indicators that meet the requirements of the corresponding pharmacopeia. Examples of fillers may be lactose, mannitol, starches, modified starches, cellulose and its derivatives, gelatin, stearic acid or its salts, moisturizers, perfumes, colorants, etc.

Oral liquid preparations can be made either in the form of solutions (aqueous or oily suspensions, solutions, emulsions, syrups, etc.), or in the form of dry products that must be diluted with water or other solvents before direct use. For example, suspensions and solutions may contain emulsifying and suspending agents.

Dosage form for parenteral administration can be selected from a solution for injection, powder for the preparation of a solution for injection, gel, infusion solution, depot form. This form must be made under sterile conditions and may contain various preservatives, buffering agents, isotonic agents.

The following examples illustrate the present invention.

Example 1. Obtaining a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid.

In contrast to the technical solution disclosed in the document [patent RU 2365582 C1, publ. August 27, 2009], in the present invention, the salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid was obtained at a molar ratio of the starting reagents (2-ethyl-6-methyl-3-hydroxypyridine and fumaric acid) equal to 1: 1 .

Method A. 50 ml of isopropyl alcohol and 15.4 g (0.133 mol) of fumaric acid were charged into a 250 ml reaction flask. The suspension was boiled with stirring for 30 minutes until completely dissolved. In parallel, 18.2 g (0.133 mol) of 2-ethyl-6-methyl-3-hydroxypyridine was dissolved in 50 ml of isopropyl alcohol with heating. The resulting solutions were mixed and boiled with stirring for 1 hour. The resulting product was crystallized at a temperature of from 0 to + 5 ° C for 12 hours. After filtration, washing and drying, 32.5 g of the expected product (salts of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid of formula (I)) was obtained in the form of white crystals with a melting point of 93-95 ° C. Yield 96.6%. The preparation of the desired product was confirmed by high performance liquid chromatography (HPLC) —the chromatogram of an aqueous solution of 2-ethyl-6-methyl-3-hydroxypyridine salt with fumaric acid obtained by Method A is shown in FIG. 1 (a).

Method B. 300 ml of acetone, 15.4 g (0.133 mol) of fumaric acid and 18.2 g (0.133 mol) of 2-ethyl-6-methyl-3-hydroxypyridine were charged into a 500 ml reaction flask. The suspension was boiled with stirring for 2 hours, cooled to room temperature and crystallized at a temperature of 0 to + 5 ° C for 6 hours, after filtration, washing and drying, 31.4 g of the expected product (2-ethyl-6-methyl- 3-hydroxypyridine with fumaric acid of the formula (I)) in the form of white crystals with a melting point of 94-97 ° C. Yield 93.3%. The preparation of the target product was confirmed by high performance liquid chromatography (HPLC) —the chromatogram of an aqueous solution of 2-ethyl-6-methyl-3-hydroxypyridine salt with fumaric acid obtained by method B is shown in FIG. 1 (b).

The following are examples that confirm the antioxidant effect of the salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid of the formula (I) (EMHP fumarate) obtained in Example 1 (both method A - EMHP fumarate ^A , and method B - EMGP fumarate ^B ).

Example 2. Evaluation of the antioxidant properties of a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid in vitro and in vivo.

Evaluation of the antioxidant properties of EMHP fumarate was carried out in two stages according to the methods described in [Grebenyuk A.N. et al. Evaluation of the antioxidant properties of domestic synthetic genistein on in vitro and in vivo models / Experimental Studies, 2013, V. 2, No. 42, p. 83-87].

At the first stage, in order to study the antioxidant activity (AOA) of EMHP fumarate in an in vitro model, the chemiluminescent reaction of riboflavin in the presence of ferrous ions and hydrogen peroxide was used [F. Putilina. Workshop on free radical oxidation / educational-methodical manual. - Publishing House of St. Petersburg State University, 2006. - S. 82-83]. To create a model system, 710 μl of potassium phosphate buffer pH 9.0 was added to the measuring cuvette of the biochemiluminometer; 40 μl of 10 mM riboflavin; 100 μl of distilled water; 50 μl of 25 mM FeSO ₄ × 7H ₂ O. The oxidation process was initiated by introducing 100 μl of a 0.1% hydrogen peroxide solution. Instead of 100 μl of distilled water, 100 μg EMHP fumarate was added to the test system, which was dissolved in water to an initial concentration of 1000 μg / ml. Then, a series of serial dilutions of the preparation was prepared in concentrations from 0.78 to 100 μg / ml. Light sum measurements were carried out for 60 s at room temperature. The AOA value was calculated as the ratio of the light sums of the experimental and model systems in percent. The concentration of the compound was determined graphically, which reduced the chemiluminescence intensity of the model by 50%.

According to studies, it was found that the average effective dose for EMHP fumarate ^A and EMHP fumarate ^B in vitro is 6.3 μg / ml and 6.2 μg / ml, respectively. For comparison, the value of this indicator for quercetin and ascorbic acid is 3.9 μg / ml and 12.5 μg / ml, respectively (according to [Grebenyuk AN et al. Assessment of the antioxidant properties of domestic synthetic genistein in in vitro and in models vivo / Experimental studies, 2013, T. 2, No. 42, pp. 83-87]). In FIG. Figure 2 shows the dependence of AOA EMGP of fumarate ^A , quercetin and ascorbic acid on their concentrations (the dependence for EMGP fumarate ^{B is} not shown in Fig. 2, since the dependence for EMGP of fumarate ^{A is} almost the same).

Studies aimed at studying the antioxidant properties of EMHP fumarate in vivo were performed on 52 white outbred male rats weighing 160-220 g. During the study, the requirements of regulatory legal acts on the procedure for experimental work with animals, including the humane attitude to them, were fulfilled .

Before the introduction of EMHP, the fumarate was previously dissolved in water for injection. Solutions were prepared immediately before their administration to rats. The drug at a dose of 200 mg / kg was administered intraperitoneally. The volume of fumarate EMGP solution administered to the animals was 0.4 ml per 100 g of animal body weight. The control group was injected with water in the same manner and in the same volume.

Studies of indicators of the glutathione system and lipid peroxidation (POL) were performed 1, 4, 24, and 72 hours after drug administration. Blood for research was taken from animals after decapitation, stabilized with 4% sodium citrate solution, cooled and immediately used in the studies. Red blood cells were isolated by centrifugation, then hemolysed with 5 mM TRIS-Hcl buffer with a pH of 7.6 in a 1: 9 ratio. The resulting hemolysate was used to determine the following biochemical parameters.

The concentration of reduced glutathione (HD), malondialdehyde (MDA), the activity of glutathione reductase (GR), glutathione S-transferase (GT), glucose-6-phosphate dehydrogenase (G-6-FDG) in erythrocyte hemolysate was evaluated using conventional methods [Harutyunyan A.V. Methods for assessing free radical oxidation and the antioxidant system of the body: guidelines. - St. Petersburg: Tome, 2000. - 104 p.]. The calculation of enzyme activity was performed per 1 g of hemoglobin. The hemoglobin concentration was determined by the hemiglobin cyanide method and expressed in g / l.

Statistical processing of the obtained results was carried out on a personal computer using the Statistica 12.0 application package. In each group, average values were calculated. The results obtained are summarized in table 1.

It was shown that in the peripheral blood erythrocytes of rats 4 hours after the administration of EMGP fumarate ^A and EMGP fumarate ^{B to} animals, the concentration of GH increased by 45%, and after 1 day the value of this parameter exceeded the control values by more than 55%. The inducing effect of EMHP of the fumarate on the activity of enzymes of the glutathione system, such as GR and HT, was also revealed. So, 4 hours after the administration of EMHP of the fumarate, there was a tendency to increase the activity of GR by more than 1.5 times for, and after 24 hours - by 2 times compared with the control. It was also noted that the introduction of EMHP fumarate was characterized by an increase in HT activity after 24 hours by ~ 1.2 times compared with the control level. One hour after the administration of EMHP of fumarate, the level of MDA decreased by 1.3 times compared with the control. Thus, the presence of antioxidant properties of the salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid was reliably confirmed.

Example 3. The effect of salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid on the dynamics of oxidative stress when exposed to reactive oxygen species (ROS).

The example also shows for comparison the results of the antioxidant activity of dimethyl fumarate (DMF), which, in accordance with the data given in [patent RU 2218918 C1, publ. December 20, 2003], showed the highest stimulating effect on the enzymatic activity of succinate dehydrogenase in comparison with other studied derivatives of fumaric acid. The results were compared with the corresponding data for ethylmethylhydroxypyridine succinate (EMHP succinate) [Solovyova A.G. et al. Effect of Mexidol on the lipid peroxidation system of canned blood when exposed to reactive oxygen species in an in vitro experiment / Bioradicals and Antioxidants, 2016, Vol. 3, No. 3, p. 98-100].

The experiment was performed on blood samples. Each sample was divided into 9 samples: sample 1 - control (initial blood); sample 2 - blood sparged with oxygen-ozone mixture (O ₃ ); sample 3 - blood exposed to O ₃ and EMHP of fumarate ^A ; sample 4 - blood exposed to O ₃ and EMHP of fumarate ^B ; sample 5 - blood exposed to O ₃ and DMF; sample 6 - blood treated with a gas stream containing singlet oxygen (SC); sample 7 - blood treated with SC and EMHP with fumarate ^A ; sample 8 - blood treated with SC and EMHP with fumarate ^B ; sample 9 - blood treated with SC and DMF.

The intensity of lipid peroxidation (POL) was determined by the level of the content of the secondary product of free radical oxidation (CPO) - malondialdehyde (MDA) in plasma and red blood cells by the method of [Uchiyama M. et al. Determination of malonaldehyde precursor in tissues by thiobarbituric acid test / Analytical Biochemistry, 1978, V. 86, p. 271]. The activity of superoxide dismutase (SOD) was determined in the hemolysate of washed red blood cells (1:10) by inhibiting the formation of the product of the autooxidation of adrenaline [Sirota T.V. A new approach to the study of the process of autooxidation of adrenaline and its use for measuring the activity of superoxide dismutase / Questions of medical chemistry, 1999, V. 45, No. 3, p. 109-116]. Protein concentration was evaluated by the Lowry method in modification [Dawson J.M. et al. Lowry method of protein quantification Evidence for Photosensitivity / Analytical Biochemistry, 1984, V. 140, N. 2, pp. 391-393]. The obtained experimental data are summarized in table 2.

The data on the decrease in lipid peroxidation under experimental oxidative stress and an increase in the activity of superoxide dismutase reliably demonstrate the antioxidant effectiveness of ethyl methyl hydroxypyridine fumarate. Moreover, the test compound is characterized by higher antioxidant properties compared with dimethyl fumarate and ethyl methyl hydroxypyridine succinate.

* values of indicators are taken from [Solovyova A.G. et al. Effect of Mexidol on the lipid peroxidation system of canned blood when exposed to reactive oxygen species in an in vitro experiment / Bioradicals and Antioxidants, 2016, Vol. 3, No. 3, p. 98-100].

Claims (9)

1. The use of a therapeutically effective amount of a salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid of the formula (I):

for the treatment of a disease caused by oxidative stress and selected from the group including Parkinson's disease, Alzheimer's disease, Huntington’s chorea, retinitis pigmentosa, mitochondrial encephalomyopathy, multiple sclerosis, stroke, Crohn’s disease, ulcerative colitis, rheumatoid arthritis, psoriasis. 2. The use according to claim 1, characterized in that the therapeutically effective amount of said salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid is 100-300 mg. 3. The use according to claim 1, characterized in that said salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid is used in the form of a dosage form for oral administration. 4. The use according to claim 3, characterized in that the dosage form for oral administration can be selected from a tablet, capsule, powder, granules, dragee, gel, jelly, orodispersible form, buccal or sublingual form in the form of a plate or tablet , solution, syrup or suspension. 5. The use according to claim 1, characterized in that the said salt of 2-ethyl-6-methyl-3-hydroxypyridine with fumaric acid is used in the form of a dosage form for parenteral administration. 6. The use according to claim 5, characterized in that the dosage form for parenteral administration can be selected from a solution for injection, powder for the preparation of a solution for injection, gel, infusion solution, depot form. 7. The use of PP. 3-6, characterized in that the dosage form may further include pharmaceutically acceptable carriers, excipients or excipients.

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