RU2627610C2 - Amino acids composition for methanol poisoning prevention and treatment - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: amino acid composition from a group of natural or synthetic hydrophobic compounds that are alcohol dehydrogenase (ADH) inhibitors and contain D- and L-amino acids in the following component ratio, wt %: tyrosine 10-19.5, valine 13-22, leucine 14-24, alanine 4.0-23.3, tryptophan 1.7-36, proline 9.2-19 as active ingredients for the detoxification of methanol.

EFFECT: composition acts as an antidote that completely removes acute methanol poisoning, possesses protective properties, exhibiting a pronounced inhibitory effect of ADH activity on methanol oxidation, and significantly reduces the amount of highly toxic secondary metabolites.

7 cl, 4 ex, 1 tbl

Description

The invention relates to biotechnology and the production of biologically active substances and / or drugs for preventive and emergency medical care in the field of toxicology, which studies the production risks of poisoning with methanol, methanol vapors, solutions containing methanol and alcohol substitutes.

The amino acid composition proposed in the application contains natural or synthetic amino acids. It is recommended for the medical prevention of methanol poisoning, as an antidote for relieving acute poisoning and subsequent treatment of poisoning by deactivating the liver enzyme alcohol dehydrogenase (ADH), which is responsible for the oxidation of methanol with the formation of toxic aldehyde - formaldehyde.

Inhibitors of ADH activity obtained by chemical synthesis are known: 4-methylpyrazole, fomepizole, isovalerianic acid amide, benzamide, sulfides, oximes. Known drugs have their own residual toxicity and require additional research in rehabilitation therapy and treatment of the possible negative effects of methanol poisoning.

In medical practice, ethanol is used to relieve acute methyl alcohol poisoning. Ethanol is a competitive methanol inhibitor for its interaction with ADH. Ethanol and methanol serve as substrates for ADH, but unlike ethanol, methyl alcohol oxidizes ADH much more slowly. Up to 20% of the taken methyl alcohol is excreted through the kidneys in the form of formic acid, which is determined in the urine up to 5-7 days from the moment of methanol intake. Formic acid is a highly toxic product of the oxidation of formaldehyde by the liver enzyme acetaldehyde dehydrogenase (AcDG).

Ethanol is oxidized 9 times faster than methanol by ADH, which can significantly reduce the manifestation of acute methanol poisoning. At the same time, the use of ethanol in the treatment of methanol poisoning is accompanied by the manifestation of complications, which initiates subsequent poisoning pathological processes in the body. Ethyl alcohol reduces the formation of highly toxic oxidation products - formaldehyde and formic acid in the liver and can relieve an acute attack of poisoning, but reduces the effectiveness of subsequent treatment in cases of manifestations of complications during rehabilitation therapy.

Since methanol is sequentially oxidized by the liver enzymes ADH and AcDG with the formation of highly toxic products, the first step in the conventional treatment of methanol poisoning is the relief of acute poisoning by inhibiting the activity of the ADH enzyme.

For the treatment of acute intoxication with methanol, it seems justified to use drugs that can suppress the toxicity of poisoning products, but devoid of ethanol deficiencies. Therefore, the use of ethanol as an antidote can stimulate an increase in the rate of absorption of methanol from the stomach, which in turn can lead to an increase in the concentration of poison in the tissues and accelerate its biotransformation. On the other hand, acute methanol intoxication is accompanied by the development of toxic gastritis, which can also significantly affect the amount of ethanol absorbed by oral administration. This is indicated in well-known sources - (Mashkovsky M. MD Medicines, 2001, Moscow, edition, volume 2, p. 351; Strelchuk IV Treatment of alcoholics with antabuse, “Neurology and Psychiatry”, 1952, No. 4, p. 43-50).

Used in practical medicine for the treatment of methanol poisoning 4-methylpyrazole can reduce the metabolic component of the toxic effects of methanol on the body after its oxidation by the liver to aldehydes and acids. 4-methylpyrazole has fewer secondary side effects than ethanol. It is used in pure form and in combination with ethyl alcohol, as an additional competitive inhibitor of ADH in emergency care and further treatment of poisoning. Therefore, for example, in the invention "Derivatives of Proline as Cathepsin Inhibitors", patent RU 2535479, publ. 12/10/2014, IPC C07D 207/16, it is used to treat metabolic diseases such as diabetes, atherosclerosis, abdominal aortic aneurysm, peripheral artery disease, cancer, reduce the frequency of cardiovascular disorders in chronic kidney disease and diabetic nephropathy.

Also described is the use in a complex of emergency medical measures at a dose of 10 mg of 4-methylpyrazole per 1 kg of body weight per day intravenously, in combination with a 20-30% aqueous solution of ethanol, orally at a dose of 200 ml every 3-4 hours, or intravenously 5% solution in a 5% glucose solution, at the rate of 1.5-2 g of ethanol per 1 kg of body weight per day. (Lakusta V.N. "Methodology for the treatment of alcoholism", Chisinau, 1987, p. 87).

Also in the invention "Complex for the treatment of poisoning containing phenothiazine compound, and methods of treating poisoning", patent RU 2445954, publ. 03/27/2012, IPC A61K 31/045, the phenothiazine compound being methylene blue, the nitro ester compound being nitroglycerin, ethanol and a pharmaceutically acceptable carrier are used to treat poisoning caused by fluoroacetate, ethylene glycol and cyanide

Pyrazole derivatives are also specific inhibitors of ADH of various animal and human species, exhibiting their activity both in vitro and in vivo. Such substances are competitive inhibitors of ADH. They bind to the zinc atom of the active center of the enzyme, taking the place of the substrate, with the formation of the alcohol dehydrogenase-NAD-inhibitor complex. The role of pyrazole is to slow down or completely stop the oxidation of methanol to formaldehyde, and methanol is eliminated from the body over time.

The invention of "Synthetic peptide amides", patent RU 2500685, publ. 12/10/2013, IPC C07K 5/107, A61K 38/07, A61P 11/14, A61P 17/04, A61P 25/04, which use synthetic peptide amides, into the peptide chain of which D-amino acids are incorporated and belong to such synthetic peptide amides, which are agonists of the kappa opiate receptor, relate to methods of their use as prophylactic and therapeutic agents, and the use is carried out for the prevention and treatment of pain and inflammation associated with various diseases and conditions. However, these compositions are not antidotes to methanol, as these agents only affect the Kappa receptors, which are involved in the functions of regulating pain sensitivity, drinking and nutritional motivation; thermoregulation; modulation of cardiorespiratory processes; inhibition of dopamine release; inhibition of the release of antidiuretic hormone; activity of immunocytes. In other words, they affect the increased secretion of endorphins, which only mask the effects of alcohol, in particular methanol, due to the fact that euphoria in drinking alcohol is associated with the release of opioid neurotransmitters endorphins in the corresponding areas of the brain. According to Medical Xpress. Endorphins - short-lived neurotransmitters of peptide nature - along with enkephalins provide the activity of the analgesic system. In addition to analgesia, they cause a sense of physical and mental comfort and euphoria, due to which narcotic analgesics acting on the same receptors often cause addiction and dependence.

In case of methanol poisoning, infusions of blood substitutes, glucose-salt solutions, the introduction of antiplatelet agents, glucocorticoids and nootropics are also used. For example, the invention "Biologically active agent that affects the general metabolic, excitatory and inhibitory functions of the nervous system and intellectual-mnestic functions of the brain (options)", patent RU 2461374, publ. 20.09.2012, IPC A61K 31/198 wherein the biologically active agents using amino acid glycine, vitamin B ₆ group, or a pharmaceutically acceptable salt that can be used in various fields of therapy and prevention, safe, but insufficient effect on metabolic process in poisonings methanol.

Therefore, all known methods for the treatment of methanol in acute methanol poisoning do not exclude the formation of severe long-term consequences. Also, the use of metabolic correctors as part of antidote therapy does not provide a sufficient protective preventive effect in relation to subsequent manifestations of an immunodeficiency state.

Known inventions "Granulate emethine hydrochloride, a method for its preparation and dosage form based on it", patent RU 2309731, publ. 10.11.2007, IPC A61K 9/16, A61K 31/4725, A61K 31/4375, A61P 25/32 and "A method for the treatment of alcoholism", patent RU 2153336, publ. 07/27/2000, IPC A61K 31/445, which use the granular form of emethine hydrochloride for the prevention and treatment of alcoholism containing 70-90% emethine hydrochloride, polyvinylpyrrolidone and eudragit, or emethine hydrochloride in doses that suppress enzyme activity, respectively. They mainly use medication with the use of drugs that cause a predominantly conditioned reflex reaction (aversion) to alcohol. ("Alcoholism", A manual for doctors edited by G.V. Morozov et al., M., Medicine, 1983, pp. 321-328.). In this case, the granules are obtained using polymethacrylates, which are known for pharmaceutical purposes under the trade name Eudragit EUDRAGI and Emetin (Emetinum) - the alkaloid contained in the root of Ipecac. Usually, emetine hydrochloride (Emetlni hydrochloridum;) is used in medical practice to treat amoebic dysentery. However, amethine (emethine hydrochloride) does not have protective therapeutic properties and antidote properties, inhibiting ADH in case of poisoning only with ethanol. In addition, the medicinal product emetin is not a biologically active additive (BAA) and self-administration is dangerous. Serious secondary negative consequences of using the alkaloid emetine hydrochloride as a therapeutic agent for methanol poisoning are also possible. The invention "A method of treating alcoholism patent", patent SU 1734752, publ. 05/23/1992, IPC A61K 31/00 and the invention "A method for the treatment of alcoholism", patent RU 2153336, publ. 07.27.2000, IPC A61K 31/445).

The invention is known "Synthetic peptide amides and their dimers", patent RU 2510399, publ.: 03/27/2014, IPC C07K 5/10, A61K 38/07, A61P 11/14, A61P 17/04, A61P 25/04, in which the structural formula describes a synthetic peptide amide. However, these pharmaceutical compositions, described by the structural formula and containing peptide amide compounds and their dimers, are suitable for the prevention and treatment of pain and inflammation associated with various diseases and conditions are synthetic compounds that have all of the above disadvantages in their use, but are not an antidote methanol.

The invention of "Synthetic peptide amides", patent RU 2500685, publ. 12/10/2013, IPC C07K 5/107, A61K 38/07, A61P 11/14, A61P 17/04, A61P 25/04, which use synthetic peptide amides, into the peptide chain of which D-amino acids are incorporated and belong to such synthetic peptide amides, which are agonists of the kappa opiate receptor, relate to methods of their use as prophylactic and therapeutic agents, and the use is carried out for the prevention and treatment of pain and inflammation associated with various diseases and conditions. However, these compositions are not antidotes to methanol, as these agents only affect the Kappa receptors, which are involved in the functions of regulating pain sensitivity, drinking and nutritional motivation; thermoregulation; modulation of cardiorespiratory processes; inhibition of dopamine release; inhibition of the release of antidiuretic hormone; activity of immunocytes. In other words, they affect the increased secretion of endorphins, which only mask the effects of alcohol, in particular methanol, due to the fact that euphoria in drinking alcohol is associated with the release of opioid neurotransmitters endorphins in the corresponding areas of the brain. According to Medical Xpress. Endorphins - short-lived neurotransmitters of peptide nature - along with enkephalins provide the activity of the analgesic system. In addition to analgesia, they cause a sense of physical and mental comfort and euphoria, due to which narcotic analgesics acting on the same receptors often cause addiction and dependence.

The closest invention to the proposed composition is the invention "A method for the treatment of alcoholism", patent RU 2323727, publ. 05/10/2008, IPC A61K 31/4375, A61P 25/32, provides a complete restoration of the function of ADH and homeostasis of endogenous ethanol. It uses an effect on ethanol-oxidizing enzymes with inhibitors such as emethine hydrochloride, which can be used for drug treatment of alcoholism. However, it is used only for the treatment of patients from alcohol intoxication with ethanol by inhibiting the ADH enzyme. Amethine does not have protective prophylactic and therapeutic properties and antidote properties, inhibiting ADH in case of ethanol poisoning. Amethine is not a biologically active additive (BAA) and its use can cause serious secondary negative consequences when used as a medicine for methanol poisoning.

Thus, for the treatment of methanol poisoning, none of the known drugs has protective prophylactic properties, and the treatment is accompanied by a mandatory subsequent long-term therapy of disorders of immune homeostasis.

Methanol is widely used in production, in particular in various fields of the chemical industry, as a component of a number of motor fuels, antifreeze, in the production of polymeric materials, and as solvents for fats, oils, and other organic substances. Methanol is widely used to prevent the formation of hydrates in the shafts of gas producing wells, in gas pipelines and in the maintenance of utilities, which necessitates the prevention and targeted treatment of staff during methanol poisoning in the work area and in case of technological accidents.

Methyl alcohol is a strong poison of the nervous and cardiovascular effects with a pronounced cumulative effect. The toxic effect of methanol is accompanied by a violation of immune homeostasis, the development of severe acidosis, damage and dystrophy of the optic nerve. One of the consequences of poisoning is blindness. Life threatening not only pure methanol, but also liquids containing methanol in a relatively small amount. Ingestion of 5-10 ml of methanol leads to severe poisoning, and 30 g or more - to death. Death occurs from respiratory arrest, cerebral edema.

The maximum permissible concentration (MPC) of methanol in the air of the working area is 1 mg / m ³ , the MPC on the skin of the hands is 0.002 mg / cm ² . Methanol is absorbed in the stomach and small intestine. Methanol and its metabolites are excreted by the kidneys, and part (15%) - unchanged through the lungs.

In case of methanol poisoning, methanol biotransformation occurs with the ADH enzyme with the formation of formaldehyde. Subsequently, most of the formaldehyde, under the influence of the enzyme AcDG, is converted into formic acid - the main circulating metabolite of methanol, which triggers all pathological processes in the body. ACDH inhibitors may include salicylates, testosterone, narcot, naloxone. ACDH inhibitors can be used as prophylactic drugs when exposed to methanol on the body, and the conversion of toxic aldehydes in the body will occur with a slowdown in their production rate and an increase in the time for xenobiotics to be evacuated from the body.

The shown multi-vector nature of the dangerous manifestations of methanol poisoning requires a fundamentally new approach for the effective treatment of poisoning. Such an approach should include prevention, removal of acute poisoning and treatment without the manifestation of negative long-term consequences. The drugs used for this should be non-toxic and not initiate impaired immune homeostasis.

The set requirements are fully met by the drug developed in ROSBIO LLC (St. Petersburg) in the form of a composition of non-toxic hydrophobic amino acids. The drug inhibits ADH activity in the body, preventing the formation of highly toxic products of methanol oxidation. This is the fundamental difference between the composition and the known ADH inhibitors obtained by chemical synthesis. The observed effect is unexpected and not obvious.

The composition of amino acids is recommended as a prophylactic in the form of dietary supplements for the prevention of poisoning, as well as for the treatment of methanol poisoning and as an antidote in case of acute poisoning. The composition must be taken by workers involved in the production and processing of methanol or in cases of danger of methanol poisoning. In addition, the possibility of using the drug as an antidote in the treatment of acute occupational and domestic poisoning with methanol, as well as substitutes for ethanol, has been shown. There is currently no antidote therapy for methanol poisoning. All known therapeutic measures are aimed only at removing poison from the body and eliminating acidosis.

The technical result of the proposed composition is that it:

- has protective preventive properties;

- effective in the detoxification of methanol poisoning;

- possesses antidote properties to relieve acute toxicity of methanol.

The novelty that distinguishes the composition claimed in the patent from the prototype is that the composition of natural or synthetic hydrophobic amino acids is used as active substances for methanol detoxification to inhibit the activity of alcohol dehydrogenase and prevent the formation of secondary highly toxic products.

For testing selected hydrophobic amino acids in the following ratio, mass. %:

- tyrosine 10-19,5 - valine 13-22 - leucine 14-24 - alanine 4.0-23.3 - tryptophan 1,7-36 - proline 9.2-19

Amino acids can contain both synthetic DL-amino acids and natural L-amino acids. In the particular case, use a composition of natural L-amino acids after dry grinding or use a composition of natural L-amino acids isolated from autolysates of food yeast on ion exchangers.

One of the special cases of the composition of amino acids is that it contains D and L amino acids in the following ratio of components, mass. %:

DL-tyrosine - 11.4-13.5; DL-valine 17.1-17.3; DL leucine 5.7-19.2; DL-alanine 5.8-18.6; DL-tryptophan 28.8-30.0; L-Proline 15.4-17.4.

Or the composition may contain L and D amino acids in the following ratio of components, mass. %: D-tyrosine 13.5-19.5; L-valine 14.4-17.3; L-leucine 15.9-19.2; L-alanine 4.9-5.8; L-tryptophan 24.0-28.8; L-Proline 12.7-15.4.

Examples of carrying out the invention

I. Preventive effect of the composition of amino acids

For 10 days, a suspension of the ground powder of the amino acid composition in 1% starch mucus was administered using an atraumatic probe to mice per os in doses of 50 mg / kg per person. After 10 days, methyl alcohol was introduced into the animals. The control animals were injected with similar volumes of methyl alcohol, taking 1 ml / g.

II. Therapeutic effect of the composition of amino acids

A suspension of the ground powder of the amino acid composition in 1% starch mucus was injected with an atraumatic probe once immediately after methanol poisoning to os mice in doses of 100 mg / kg.

Control animals did not receive treatment.

Example 1

The composition of synthetic hydrophobic DL-amino acids, mass. %:

DL-tyrosine 11,4 DL-Valine 17.1 DL-Tryptophan 30,0 DL-Alanya 18.6 DL Leucine 5.7 L-proline 17.4

In this case, a reliable removal of acute methanol poisoning was observed; reliable prophylactic effect, reduction of methanol toxicity by 1.5 times; reliable therapeutic effect, reduction of methanol toxicity by 1.16 times.

Example 2

The composition of natural hydrophobic L-amino acids, mass. %:

L-tyrosine 13.5 L-Valine 17.3 L-Tryptophan 28.8 L-Alanya 5.8 L-leucine 19,2 L-proline 15.4

In this case, a reliable removal of acute methanol poisoning was observed; reliable preventive effect, reduction of methanol toxicity by 1.98 times; reliable therapeutic effect, methanol toxicity reduction 1.39 times.

Example 3

The composition of natural hydrophobic L-amino acids after dry grinding at t = 50 ° C, mass. %:

D-tyrosine 19.5 L-Valine 14,4 L-Tryptophan 24.0 L-Alanya 4.9 L-leucine 15.9 L-proline 12.7

In this case, a reliable removal of acute methanol poisoning was observed; reliable prophylactic effect, reduction of methanol toxicity by 1.5 times; reliable therapeutic effect, reduction of methanol toxicity by 1.16 times.

Example 4

The composition of hydrophobic L-amino acids isolated from autolysates of food yeast on ion exchangers, mass. %:

L-tyrosine 10.0 L-Valine 19.6 L-leucine 17.8 L-Alanya 23.3 L-Tryptophan 1.7 L-proline 9.2

In this case, a reliable removal of acute methanol poisoning was observed; reliable prophylactic effect, reduction of methanol toxicity by 1.5 times; reliable therapeutic effect, reduction of methanol toxicity by 1.24 times.

The biological study of the compounds according to the invention was carried out by removing the LD ₅₀ - the generally accepted semi-lethal dose. The effect of the biological activity of the proposed compounds in acute poisoning of rats with methanol was studied.

Testing was carried out as follows.

The safety and effectiveness of a biologically active compound were determined according to MUK 2.3.2.721-98, Ministry of Health of the Russian Federation, M., 1999, 87 pp.

Hygienic requirements for safety and nutritional value of the compound according to SanPiN 2.3.2.1078-01, M., 2002, 270 pp.

The test results of the composition of the amino acids ROSAMIN in examples 1, 2, 3, 4 for the manifestation of anti-alcohol properties are shown in the table.

Discussion

The obtained results prove that the composition of hydrophobic amino acids significantly reduce the toxicity of methanol.

Since the drug did not pass the complex of current biomedical tests, but is recommended as a biological additive and therapeutic drug, the accepted trade name of the drug is ROSAMIN. A decrease in methanol toxicity after preliminary administration of ROSAMINE to the stomach of rats was shown when compared with LD ₅₀ for control animals without ROSAMINE.

Quantitative results of methanol toxicity reduction by ROSAMIN composition As the ratio of LD _{50 experience} / LD _{50 control} are shown in the table.

Therefore, an effective positive anti-alcohol effect of the amino acid components of the claimed composition is achieved.

At the same time, ROSAMIN has protective prophylactic properties, showing a pronounced inhibitory effect of ADH activity on the oxidation of methanol and, accordingly, eliminating or significantly reducing the number of highly toxic secondary metabolites.

When taking ROSAMINE inside with a single higher dose in case of methanol poisoning, the composition acts as an antidote, which completely removes acute methanol poisoning.

The research results showed that ROSAMIN, in the case of prophylactic administration as a dietary supplement and in the case of drug administration, has an effective prophylactic and therapeutic effect in the detoxification of methanol poisoning.

Claims (12)

1. A composition of amino acids having protective and therapeutic properties in case of methanol poisoning, characterized in that the composition of amino acids from the group of natural or synthetic hydrophobic compounds that are alcohol dehydrogenase (ADH) inhibitors and containing D- and L-amino acids is used as active substances for methanol detoxification in the following ratio of components, wt.%: tyrosine 10-19,5 valine 13-22 leucine 14-24 alanine 4.0-23.3 tryptophan 1,7-36 proline 9.2-19 2. The amino acid composition according to claim 1, characterized in that the amino acid composition contains synthetic DL-amino acids. 3. The amino acid composition according to claim 1, characterized in that the amino acid composition contains natural L-amino acids. 4. The amino acid composition according to claim 3, characterized in that the composition of natural L-amino acids is used after dry grinding. 5. The amino acid composition according to claim 3, characterized in that a composition of natural L-amino acids isolated from autolysates of food yeast on ion exchangers is used. 6. The amino acid composition according to claim 1, characterized in that the amino acid composition contains D- and L-amino acids in the following ratio of components, wt.%: DL tyrosine 11.4-13.5 DL-valine 17.1-17.3 DL Leucine 15.7-19.2 DL-Alanine 5.8-18.6 DL tryptophan 28.8-30.0 L-proline 15.4-17.4 7. The amino acid composition according to claim 1, characterized in that the composition contains L- and D-amino acids in the following ratio of components, wt.%: D-tyrosine 13.5-19.5 L-valine 14.4-17.3 L-Leucine 15.9-19.2 L-alanine 4.9-5.8 L-tryptophan 24.0-28.8 L-proline 12.7-15.4