RU2765414C1 - Method for stimulating mitophagy and autophagy in cells, method for stimulating autophagy or mitophagy for treating a condition associated with mitochondrial dysfunction - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely, to methods for stimulating mitophagy. A method for stimulating autophagy in cells, involving treatment of cells with a terpenoid-based inducer, wherein the "Abisil", "Abisil-1" or "Abisil-2" agent is used as such a terpenoid-based inducer in an effective concentration. A method for stimulating autophagy in a subject, with a stage of introducing an effective dose of an inducer into the body, wherein the "Abisil", "Abisil-1" or "Abisil-2" agent is used as such an inducer and stimulation of autophagy ensures treatment of a condition associated with mitochondrial dysfunction.

EFFECT: above group of inventions provides a possibility of increasing the stability and effectiveness of impact on the cells and reducing the toxicity of plant-based agents, stimulating the process of eliminating dysfunctional cellular components by autophagy, leading to alleviation and treatment of conditions associated with mitochondrial dysfunction.

1 cl, 4 dwg, 2 tbl, 4 ex

Description

The present invention relates to the field of medicine, namely to methods for stimulating mitophagy and autophagy in a cell, which involves treating cells with a terpenoid-based inducer extracted from plants of the coniferous (Pinaceae) family, such as fir, pine or cedar, to methods for stimulating autophagy or mitophagy for treatment condition associated with mitochondrial dysfunction. In addition, the present invention relates to the use of a terpenoid-based inducer to stimulate mitophagy and autophagy in a cell, and to the use of a terpenoid-based inducer to stimulate autophagy or mitophagy to treat a condition associated with mitochondrial dysfunction.

Life expectancy in economically developed countries is gradually increasing. However, the quality of life of people deteriorates with age, as older people often suffer from incurable diseases.

Lifestyle changes over the past decades (eg, overeating, sedentary lifestyle) have led to an increase in the number of people who are overweight or obese, often signs and causes of conditions such as diabetes and heart disease.

Research has shown that by focusing on the mechanisms of aging rather than specific diseases of aging, a greater prolongation of healthy lifespan can be achieved (Brian K. Kennedy et al., "Geroscience: Linking Aging to Chronic Disease," Cell, no. 159 November 6, 2014, pp. 709-713, https://www.https://pubmed.ncbi.nlm.nih.gov/25417146).

Cellular aging is increasingly recognized as a major aging-related cause of loss of health and fitness. Aging cells, while continuing to produce new mitochondria, accumulate dysfunctional and defective mitochondria. Mitophagy, i.e. utilization of defective and dysfunctional mitochondria through autophagy, in aging cells is sluggish, fades away.

Currently, there is a consensus that impaired mitophagy plays a key role in the development of degenerative diseases associated with aging. Therefore, the main focus in the fight against a number of diseases, such as Parkinson's disease, Alzheimer's disease, dementia, congestive heart failure, sarcopenia, type 2 diabetes, age-related macular degeneration (AMD), atherosclerosis, cardiovascular disease, cancer, liver disease and diseases of the pancreas, eye diseases, arthritis, cataracts, osteoporosis, hypertension and any combination thereof, is no longer given to the symptomatic treatment of a particular disease, but to the search for ways to activate mitophagy.

Compounds that can slow down aging, so-called geroprotectors, usually slow down the onset of aging and related diseases and reduce the risk of mortality. In geroprotectors that activate the process of mitophagy in cells, either chemical compounds of a certain composition, obtained chemically, or multicomponent mixtures, usually of an indefinite composition and extracted from plant materials or waste products of yeasts and microorganisms, are usually used as an active ingredient (inducer).

For example, a method is known for the prevention or treatment of a neurodegenerative disorder in a subject, which involves the introduction into his body in a therapeutically effective amount of an inducer agent that increases Nix-mediated mitophagy in the cell, and more precisely, increases the biological activity or expression of a Nix polypeptide, its fragment, variant or analogue and/or polypeptide GABARAP-L1, its fragment, variant or analogue in the cell and containing these components (US 2019/091291 A1).

The disadvantages of the known method are the technological complexity and the high cost of obtaining a substance used as an inductor, as well as the lack of polyactivity. These shortcomings are inherent in all other known methods involving the use of substances of chemical origin as an inductor, which are xenobiotics for cells.

There are also known methods to improve the health of people, especially the elderly or weak people, which consist in the activation of mitophagy in cells by oral administration for a certain time of certain doses of a pharmaceutical composition based on urolithins, which are metabolites derived from the products of the microflora of the colon of mammals (US 2018/256538 and US 2018/256539). The disadvantages of this approach are the high cost of the active substance, due to the specific features of the processing of the feedstock and the technologically complex process of isolating the active principle from it, as well as, first of all, the instability of the pharmacological action, ranging from quite effective to toxic.

Closest to what is proposed in the invention is a method for activating mitophagy in skin cells, which consists in exposing skin cells to a cosmetic composition containing an active substance (inductor) extracted from at least one plant selected from Helianthus annuus, Ceratonia siliqua, Nigella sativa, Withania somnifera, Secale Cornutum, Sesamum indicum, Ophiopogon japonicus, Alisma plantago-aquatica, Oryza sativa, Tropaeolum majus, Cichorium intybus, Prunus persica, Verbena officinalis, Panicum miliaceum, Rosa canina, Camelina sativa and Phaseolus vulgaris, or from specific algae or yeasts specific species, as well as adjuvants and excipients (WO 2013/178965).

The known method is based on the use of an active substance extracted from plants, some of which grow in tropical countries or are cultivated in small quantities as an imported plant. Since the composition and properties of the active substances contained in plants depend on the conditions of their growth, the therapeutic effect of the substances isolated from them is unstable. Many of these plants contain not one, but a whole set of biologically active substances of multidirectional action, and some of them, for example, substances contained in Secale Cornutum, Alisma plantago-aquatica, have a strong toxic effect, cause addiction (Withania somnifera) or are simply not used in official medicine in Russia {Ophiopogon japonicus). For these reasons, the known method is industrially inapplicable without additional time-consuming research.

Stimulation of autophagy, which includes mitophagy as a special case, also promotes cell health and has a positive therapeutic effect on the subject, although, unlike mitophagy, not all mechanisms of the healing action of autophagy have been fully elucidated. There are a number of methods for stimulating autophagy and pharmaceutical preparations for this purpose.

In the predominant part of the preparations, as an active substance-inducer, substances of chemical origin of a simple composition compared to substances of plant origin are used (WO 2019/204183, JP 2019218316, KR 20190114900, US 2019/160033, US 2019/336483). Accordingly, the activity of such substances is narrowly focused, and known drugs are used mainly for cosmetic purposes, when it is difficult to establish whether the therapeutic effect is due to the action of the active substance or targeted additives.

WO 2019/099531 proposes the use of a synergistic combination of compounds from at least two and preferably three classes of compounds in an autophagy-stimulating inducer. Due to this, some increase in the efficiency of the method is achieved, but not the expansion of the scope of its effective application.

To address this shortcoming, WO 2019/204183 proposes seven options for a method of treating or promoting hair growth by administering to a subject one or more autophagy-inducing agents of chemical origin. Each of the variants of this method uses its own specific combination of agents. The very abundance of the proposed options suggests that none of them is uniquely and in all cases effective.

In addition, autophagy-inducing substances of chemical origin used according to the above-mentioned prior art are xenobiotics for cells and organisms, additionally load their metabolizing and eliminating systems, and can cause side reactions.

To a much lesser extent, these disadvantages are inherent in phytopreparations containing plant components or products of their processing as an active substance.

From US 8512764, a method is known for activating autophagy of skin cells in order to detoxify or prevent aging, which consists in applying to the skin a cosmetic composition containing, as an active ingredient (inducer), a biological substance extracted from algae, yeast or plants selected from the group Lithothamnium calcareum, Yarrowia lipolytica, Melilotus officinalis, Citrus limonum, Candida saitoana, Lens culinaris, Averrhoa carambola, Momordica charantia, as well as adjuvants and excipients.

The active substances of a part of the above plants cause a number of undesirable side effects. So, in particular, coumarin, contained in the herb of sweet clover (Melilotus officinalis), has a depressant effect on the central nervous system and has a narcotic effect. The bioactive ingredient, which is derived from Candida saitoana yeast, is so toxic that it is used as a pesticide. Substances contained in carambola (Averrhoa carambola) are toxic to uremic patients. The substances contained in momordica (Momordica charantia) are also toxic. Due to the toxicity of the active substances, the scope of application of this known method is limited to cosmetic procedures related to the treatment or care of the skin.

Closest to the proposed invention is described in AU 2016101736 method of inducing autophagy in cells by contact with triterpinoid, extracted from common ivy (Hedera helix). Such a method has been proposed for the treatment of neurodegenerative diseases, namely Parkinson's disease or Huntington's disease.

The disadvantage of this method is the limited scope of its application in the treatment of the above diseases, probably due to the properties of a particular triterpenoid isolated from ivy. To date, the existence of about 10,000 terpenes and terpenoids in plants has been established, the functions of most of which are still unknown. In addition, the leaves and fruits of ivy are poisonous, and therefore, in order to reduce the toxicity of the drug, one has to resort to a technologically complex multi-stage procedure for isolating the desired triterpenoid from ivy leaves and fruits.

The present invention was based on the task of proposing methods for stimulating autophagy and mitophagy of cells by exposing cells to an inducer, the active principle of which is selected from a wide range of inexpensive, accessible and non-toxic terpenoids obtained from plants from the Pinaceae family common in the Russian wild nature: the genus Spruce (Picla ), the genus pine (Pinus), the genus cedar (Cedrus) and the genus fir (Abies). Another object of the present invention was to develop methods for stimulating autophagy or mitophagy for the treatment of a condition associated with mitochondrial dysfunction in a subject using said inducer. Another object of the present invention was to provide a terpenoid-based inducer suitable for use in stimulating mitophagy and autophagy in a cell and suitable for use in stimulating autophagy or mitophagy to treat a condition associated with mitochondrial dysfunction.

The technical result of using the solution proposed in the invention is to increase the stability and effectiveness of the effect on cells and to reduce the toxicity of herbal preparations that stimulate the process of eliminating dysfunctional cell components by autophagy and mitophagy, leading to the relief and treatment of conditions associated with mitochondrial dysfunction.

This technical result is achieved due to the fact that when implementing the method for stimulating mitophagy in cells according to the invention, which involves treating cells with a terpenoid-based inductor, an inductor extracted from plants of the Pinaceae family is used as such an inductor based on terpenoids, for example: spruce (Picla), pine genus (Pinus), cedar genus (Cedrus) and fir genus (Abies), and having the following physical constants: acid value from 70 to 90, saponification number from 100 to 130, ether value from 10 to 60, refractive index from 1.50 to 1.52.

In one preferred embodiment, the terpenoid-based inducer is used at a concentration of 0.05 to 200 μg/ml, preferably 45 to 200 μg/ml.

In another preferred embodiment, the duration of treatment of cells with a terpenoid-based inducer is from 6 to 72 hours, preferably 16 hours.

In yet another preferred embodiment, the cells are of non-tumor tissue origin, are contained in a subject, or are found ex vivo or in vitro. Processing can be carried out in ways and methods known to those skilled in the art. For example, when carrying out the in vitro method of the invention for bringing cells into contact with an inhibitor, the cells can be cultured in the presence of the inducer in the culture medium or by adding the inducer to the cells. In carrying out the ex vivo method of the invention for bringing the inducer into contact with cells or tissues, it can, for example, be introduced into the subject's body by routes known to the person skilled in the art, in particular by any of the routes that are commonly used in the field of gene therapy. Administration of the inducer may be by various routes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, transdermal, intratracheal, intramuscular, topical, intradermal, intranasal, pulmonary, inhaled, or intrabronchial, oral, or rectal.

In another preferred embodiment, Abisil, Abisil-1 or Abisil-2 can be used as a terpenoid-based inducer, as such or with a targeted additive, which is olive, sunflower, flaxseed, soybean, coconut or argan oil and vitamin E. These preparations can be administered orally or externally, for example in the form of a 3-20% by weight oil solution, for example a 3-15% by weight oil solution, in particular 3%, 4% , 5% or 15% by weight oil solution (in olive, sunflower, flaxseed, soybean, coconut or argan oil), in the form of capsules, liposomes, sprays, creams and other dosage forms.

This technical result is also achieved due to the fact that when implementing a method for stimulating autophagy in cells, involving the treatment of cells with a terpenoid-based inducer, an inductor extracted from plants of the Pinaceae family, for example: spruce genus (Picla ), genus pine (Pinus), genus cedar (Cedrus) and genus fir (Abies), and having the following physical constants: acid number from 70 to 90, saponification number from 100 to 130, ether number from 10 to 60, refractive index from 1.50 to 1.52.

In one preferred embodiment, the terpenoid-based inducer is used at a concentration of 0.05 to 200 μg/ml, preferably 45 to 200 μg/ml.

In another preferred embodiment, the duration of treatment of cells with a terpenoid-based inducer is from 6 to 72 hours, preferably 16 hours.

In yet another preferred embodiment, the cells are of non-tumor tissue origin, are contained in a subject, or are found ex vivo or in vitro. Processing can be carried out in ways and methods known to those skilled in the art. For example, when carrying out the in vitro method of the invention for bringing cells into contact with an inhibitor, the cells can be cultured in the presence of the inducer in the culture medium or by adding the inducer to the cells. In carrying out the ex vivo method of the invention for bringing the inducer into contact with cells or tissues, it can, for example, be introduced into the subject's body by routes known to the person skilled in the art, in particular by any of the routes that are commonly used in the field of gene therapy. Administration of the inducer may be by various routes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, transdermal, intratracheal, intramuscular, topical, intradermal, intranasal, pulmonary, inhaled, or intrabronchial, oral, or rectal.

In another preferred embodiment, Abisil, Abisil-1 or Abisil-2 can be used as a terpenoid-based inducer, as such or with a targeted additive, which is olive, sunflower, flaxseed, soybean, coconut or argan oil and vitamin E. These preparations can be administered orally or externally, for example in the form of a 3-20% by weight oil solution, for example a 3-15% by weight oil solution, in particular 3%, 4% , 5% or 15% by weight oil solution (in olive, sunflower, flaxseed, soybean, coconut or argan oil), in the form of capsules, liposomes, sprays, creams and other dosage forms.

This technical result is also achieved due to the fact that when implementing a method for stimulating autophagy or mitophagy for treating a condition associated with mitochondrial dysfunction in a subject, with the stage of introducing an inductor in an effective dose into the subject's body, a terpenoid-based inducer extracted from plants is used as such an inducer. of the family Pinaceae, for example: the genus spruce (Picla), the genus pine (Pinus), the genus cedar (Cedrus) and the genus fir (Abies), and having the following physical constants: acid number from 70 to 90, saponification number from 100 to 130, essential number from 10 to 60, refractive index from 1.50 to 1.52.

The subject is a person.

A condition associated with mitochondrial dysfunction is selected from a condition including mutations and/or epigenetic abnormalities of the mitochondrial or nuclear genome leading to disruption of mitophagy, defects in mitochondrial morphology, and disturbances in mitochondrial membrane potential, and a condition including lysosomal storage diseases.

In one embodiment, the terpenoid inducer is administered to the subject at a dose of 4 to 40 mg/kg body weight, preferably 4 to 24 mg/kg body weight.

Administration of the inducer may be by various routes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, transdermal, intratracheal, intramuscular, topical, intradermal, intranasal, pulmonary, inhaled, or intrabronchial, oral, or rectal.

In another preferred embodiment, Abisil, Abisil-1 or Abisil-2 can be used as a terpenoid-based inducer as such or with a targeted additive, which is olive, sunflower, linseed, soybean, coconut or argan oil. and vitamin E. These preparations can be administered orally or externally, for example in the form of a 3-20% by weight oil solution, for example a 3-15% by weight oil solution, in particular 3%, 4%, 5% or 15% by weight oil solution (in olive, sunflower, flaxseed, soybean, coconut or argan oil), in the form of capsules, liposomes, sprays, creams and other dosage forms.

This technical result is further achieved through the use of an inductor based on terpenoids to stimulate mitophagy in cells. This application involves the treatment of cells with a terpenoid-based inductor, which inductor is used in an effective concentration of an inductor extracted from plants of the Pinaceae family, for example: spruce genus (Picla), pine genus (Pinus), cedar genus (Cedrus) and fir genus (Abies ), and having the following physical constants: acid number from 70 to 90, saponification number from 100 to 130, ester number from 10 to 60, refractive index from 1.50 to 1.52.

In one preferred embodiment, the terpenoid-based inducer is used at a concentration of 0.05 to 200 μg/ml, preferably 45 to 200 μg/ml.

In another preferred embodiment, the duration of treatment of cells with a terpenoid-based inducer is from 6 to 72 hours, preferably 16 hours.

In yet another preferred embodiment, the cells are of non-tumor tissue origin, are contained in a subject, or are found ex vivo or in vitro. Processing can be carried out in ways and methods known to those skilled in the art. For example, when carrying out the in vitro method of the invention for bringing cells into contact with an inhibitor, the cells can be cultured in the presence of the inducer in the culture medium or by adding the inducer to the cells. In carrying out the ex vivo method of the invention for bringing the inducer into contact with cells or tissues, it can, for example, be introduced into the subject's body by routes known to the person skilled in the art, in particular by any of the routes that are commonly used in the field of gene therapy. Administration of the inducer may be by various routes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, transdermal, intratracheal, intramuscular, topical, intradermal, intranasal, pulmonary, inhaled, or intrabronchial, oral, or rectal.

In another preferred embodiment, Abisil, Abisil-1 or Abisil-2 can be used as a terpenoid-based inducer, as such or with a targeted additive, which is olive, sunflower, flaxseed, soybean, coconut or argan oil and vitamin E. These preparations can be administered orally or externally, for example in the form of a 3-20% by weight oil solution, for example a 3-15% by weight oil solution, in particular 3%, 4% , 5% or 15% by weight oil solution (in olive, sunflower, flaxseed, soybean, coconut or argan oil), in the form of capsules, liposomes, sprays, creams and other dosage forms.

This technical result is also achieved through the use of a terpenoid-based inducer to stimulate autophagy in cells. This application involves the treatment of cells with a terpenoid-based inductor, which inductor is used in an effective concentration of an inductor extracted from plants of the Pinaceae family, for example: spruce genus (Picla), pine genus (Pinus), cedar genus (Cedrus) and fir genus (Abies ), and having the following physical constants: acid number from 70 to 90, saponification number from 100 to 130, ester number from 10 to 60, refractive index from 1.50 to 1.52.

In one preferred embodiment, the terpenoid-based inducer is used at a concentration of 0.05 to 200 μg/ml, preferably 45 to 200 μg/ml.

In another preferred embodiment, the duration of treatment of cells with a terpenoid-based inducer is from 6 to 72 hours, preferably 16 hours.

In yet another preferred embodiment, the cells are of non-tumor tissue origin, are contained in a subject, or are found ex vivo or in vitro. Processing can be carried out in ways and methods known to those skilled in the art. For example, when carrying out the in vitro method of the invention for bringing cells into contact with an inhibitor, the cells can be cultured in the presence of the inducer in the culture medium or by adding the inducer to the cells. In carrying out the ex vivo method of the invention for bringing the inducer into contact with cells or tissues, it can, for example, be introduced into the subject's body by routes known to the person skilled in the art, in particular by any of the routes that are commonly used in the field of gene therapy. Administration of the inducer may be by various routes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, transdermal, intratracheal, intramuscular, topical, intradermal, intranasal, pulmonary, inhaled, or intrabronchial, oral, or rectal.

In another preferred embodiment, Abisil, Abisil-1 or Abisil-2 can be used as a terpenoid-based inducer, as such or with a targeted additive, which is olive, sunflower, flaxseed, soybean, coconut or argan oil and vitamin E. These preparations can be administered orally or externally, for example in the form of a 3-20% by weight oil solution, for example a 3-15% by weight oil solution, in particular 3%, 4% , 5% or 15% by weight oil solution (in olive, sunflower, flaxseed, soybean, coconut or argan oil), in the form of capsules, liposomes, sprays, creams and other dosage forms.

This technical result is further achieved through the use of a terpenoid-based inducer to stimulate autophagy or mitophagy for the treatment of a condition associated with mitochondrial dysfunction in a subject. Said use includes the step of administering the inductor at an effective dose to the subject. As such an inductor, a terpenoid-based inductor extracted from plants of the Pinaceae family, for example: spruce genus (Picla), pine genus (Pinus), cedar genus (Cedrus) and fir genus (Abies), and having the following physical constants: acid number from 70 to 90, saponification number from 100 to 130, ester number from 10 to 60, refractive index from 1.50 to 1.52.

The subject is a person.

A condition associated with mitochondrial dysfunction is selected from a condition including mutations and/or epigenetic abnormalities of the mitochondrial or nuclear genome leading to disruption of mitophagy, defects in mitochondrial morphology, and disturbances in mitochondrial membrane potential, and a condition including lysosomal storage diseases.

In one embodiment, the terpenoid inducer is administered to the subject at a dose of 4 to 40 mg/kg body weight, preferably 4 to 24 mg/kg body weight.

Administration of the inducer may be by various routes, for example, intravenous, intra-arterial, intraperitoneal, subcutaneous, transdermal, intratracheal, intramuscular, topical, intradermal, intranasal, pulmonary, inhaled, or intrabronchial, oral, or rectal.

In another preferred embodiment, Abisil, Abisil-1 or Abisil-2 can be used as a terpenoid-based inducer as such or with a targeted additive, which is olive, sunflower, linseed, soybean, coconut or argan oil. and vitamin E. These preparations can be administered orally or externally, for example in the form of a 3-20% by weight oil solution, for example a 3-15% by weight oil solution, in particular 3%, 4%, 5% or 15% by weight oil solution (in olive, sunflower, flaxseed, soybean, coconut or argan oil), in the form of capsules, liposomes, sprays, creams and other dosage forms.

Through the use of an active substance based on terpenoids extracted from plants of the Pinaceae family, for example: spruce genus (Picla), pine genus (Pinus), cedar genus (Cedrus) and fir genus (Abies), a stable and significant activation of autophagy and mitophagy in cells is achieved. with the complete exclusion of the possibility of toxic effects on the cell.

Thanks to the use of an active substance based on terpenoids extracted from plants of the Pinaceae family, for example: spruce genus (Picla), pine genus (Pinus), cedar genus (Cedrus) and fir genus (Abies), stability of the process and result of activation of autophagy and mitophagy in cells with the complete exclusion of the possibility of toxic effects on the cell, which is explained by the stability of the composition and characteristics and the absence of toxicity of the initial plant material extracted from these plants.

The use of the above inducer based on terpenoids and, in particular, the drug "Abisil", "Abisil-1" or "Abisil-2" as such or with a target additive allows to achieve a significant activation of autophagy and mitophagy in cells without the need to use terpenoid-based inducers with other parameters that are obtained using a different technology and the effectiveness of which has yet to be tested and proved. Preparations "Abisil-1" and "Abisil" and methods for their preparation are described in patents RU 2054945, RU 2198653.

The drug "Abisil-2" and the method of its production are described in patent RU 2338547. The drug "Abisil-2" is obtained on the basis of a capsular extract of plants of the Pinaceae family subjected to short-term stress, and contains the following components:

sequiterpenoids 3-6 wt. % neutral diterpenoids 11-15 wt. % diterpenic acids 23-24 wt. % triterpene acids 8-16 wt. % unsaturated and saturated fatty acids 0.1-0.3 wt. % phenolic compounds 0.1-0.2 wt. % monoterpenoids rest

while the content of monoterpenoid bornylacetate should be at least 10.0 wt. % of the total composition of terpenoids.

The terpenoid composition is a thick liquid with a color from transparent yellow to milky white with a specific odor and has the following physical constants:

acid number 70-90 saponification number 100-130 essential number 10-60 refractive index 1,500-1,520

Below, the essence of the invention is explained with reference to the accompanying drawings, which show:

in fig. 1- assessment of the metabolic activity of cells treated with "Abisil" in various concentrations,

in fig. 2 - assessment of the decrease in mitochondrial potential caused by "Abisil" in immortalized MRC5 cells and Lech-4 lung fibroblasts,

in fig. 3 - Evaluation of the effect of "Abisil" pretreatment on the accumulation of light chain 3 and phosphatidylethanolamine (LC3II) conjugate in SV-40 transformed embryonic lung fibroblasts (MRC5-SV40) according to the results of densitometric analysis relative to internal control, and

in fig. 4 - assessment of mitophagy in MRC5-SV40 cells when treated with the drug at concentrations of 25 and 50 μg / ml after 24 and 72 hours: A, B - control sample (DMSO, 0.5%), C - 24-hour treatment at a concentration of 25 µg/ml, D - 24-hour treatment at a concentration of 50 µg/ml, E - 72-hour treatment at a concentration of 25 µg/ml, F - 72-hour treatment at a concentration of 50 µg/ml.

The possibility of carrying out the invention can be confirmed by the following examples.

Assessment of mitochondrial potential

MRC5-SV40 and Lech-4 cells were seeded in 12-well plates at 40,000 cells per well. The next day, Abisil was treated in a wide range of concentrations (25-200 μg/ml). After 16 hours, cells were stained with 100 nM and 200 nM tetramethylrhodamine methyl ester (TMRM) (Thermofisher Scientific) for 30 min at 37°C. As a positive control, 1 μM rotenone and 10 μM oxidative phosphorylation uncoupler carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich) were used. Prior to measurement, cells were stained for viability with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) at a concentration of 1 μg/ml. Fluorescence was detected using a BD LSRFortessa cytometer (Beckman Dickinson). Analysis was performed using Flowing Software 2.0 (Perttu Terho, Turku Center for Biotechnology). The results are based on three independent experiments with three biological replicates.

Assessment of autophagy and mitophagy

Autophagy was assessed on a panel of cell lines stably expressing a reporter construct of 2 fusion proteins EGFP (green fluorescent protein) and LC3. Treatment with the drug "Abisil" was carried out in a wide range of concentrations (25-200 µg/ml) for 16 hours. Rapamycin (R0395, Sigma-Aldrich) was used as a positive control.

Mitophagy was assessed using the MRC5-SV40 cell line, which stably expresses the EGFP-mCherry-FIS1 reporter construct, which contains two fluorescent proteins and in which the mCherry-GFP tandem tag is attached to the outer mitochondrial membrane localization signal derived from the mitochondrial FIS1 protein (amino acids 101- 152). FCCP (Sigma-Aldrich) was used as a positive control.

Cells were treated with Abisil at concentrations of 50 μg/ml and 25 μg/ml for 24 or 72 hours.

For the detection of mitophagy in Lech-4 cells, they were treated with Abisil at concentrations of 25 μg/ml and 50 μg/ml for 24 or 72 hours. After incubation, cells were stained for 20 minutes at 37°C with 200 nM voltage-dependent mitochondrial dye MitoTracker Red CMXRos (Thermofisher Scientific), 100 nM LysoTracker Green (Thermofisher Scientific).

Axiovert 200 microscopes (Carl Zeiss) and a Leica confocal microscope (Leica Microsystems) were used for imaging. MTT analysis

MRC5-SV40 cells were seeded in a 96-well plate at 5,000 cells per well, cultured in 100 µl of DMEM (Dulbecco's modified nutrient medium) with 5% FBS (fetal bovine serum) at 37°C and incubated for 48 h. Next, during the day was treated with the drug "Abisil" (at a concentration of 0, 9, 11, 15, 25, 50, 93, 125, 147 and 186 µg/ml) at 37°C. The level of cell metabolism was assessed using the MTS test (CellTiter 96® kit for non-radioactive cell proliferation assay (MTT), Promega) according to the manufacturer's protocol. The optical density of the colored substance, proportional to the level of metabolism of viable cells, was measured at 590 nm using a tablet reader (Tecan). Then, the IC ₅₀ values were calculated by regression analysis, as well as the concentrations at which the most intensive cellular metabolism was observed.

Western blotting

Lech-4 cells were cultured on Petri dishes with a diameter of 35 mm and at a confluence of 50% were treated with the drug "Abisil" in 5% DMEM with 5% FBS at 37°C overnight. After incubation, cells were washed with cold PBS (phosphate buffered saline) and lysed in 200 μl of reporter lysis buffer (Promega) containing protease inhibitor cocktail (#P8349, Sigma-Aldrich) according to the manufacturer's instructions. The lysates were centrifuged for 15 min at 12000g and the total protein content in the protein supernatant was measured according to Bradford. Proteins were separated by polyacrylamide gel electrophoresis in the presence of 12% sodium dodecyl sulfate (SDS) according to Laemmli and electrotransferred to an Amersham Hybond P PVDF membrane, 0.45 μm (Amersham Biosciences, GE Healthcare) in Tovbin transfer buffer (25 mM Tris, 192 mM glycine, 20% (v/v) ethanol, pH 8.3). The membranes were blocked in 4% skimmed milk in PBS (PBS with 0.05% Tween 20) for 1 hour at room temperature. Primary antibody incubation was performed overnight at 4° C. with secondary antibodies (goat anti-rabbit IgG-HRP, sc-2004, Santa Cruz Biotechnology) and for 1 hour at room temperature. Immobilon Western Chemiluminescent HRP Substrate (Merck-Millipore, Sigma-Aldrich) was used for development, detecting the chemiluminescence signal with a Bio-Rad ChemiDoc MP Imaging System (Bio-Rad). Protein bands were quantified using ImageJ software (NIH, Bethesda, MD, USA). List of primary antibodies used:

- β-actin (D6A8, #8457, Cell Signaling Technology);

- LC3 (D11, #3868, Cell Signaling Technology).

Statistical analysis

Data are presented as mean ± standard error. Statistical analysis was performed using Student's t-test. It is believed that p<0.05 indicates a statistically significant difference. The analysis was performed using the Microsoft Excel program.

Cell Processing Reagents

The cells were treated with the Abisil preparation in the form of resin from OOO Initium-Pharm. To obtain a drain for processing cell cultures, "Abisil" was dissolved in DMSO.

Cells were grown in DMEM containing 10% FBS to 50-70% confluence. Immediately before treatment, Abisil stock was diluted in a serum-free DMEM medium, treatment was carried out in DMEM medium with 5% FBS.

Negative controls were treated with the appropriate amount of DMSO in DMEM with 5% FBS.

Example 1: Effect of "Abisil" on the metabolic activity of cells The effect of 48-hour treatment "Abisil" on the metabolic activity of cells was studied using the MTT test on lines of embryonic lung fibroblasts (MRC5-SV40 and Lech-4). "Abisil" showed a significant dose-dependence (see Fig. 1). The calculated IC ₅₀ values were 149.3±0.67 for MRC5-SV40 and 152.6±0.52 μg/ml for Lech-4, respectively (Table 1). In addition, both cell lines showed a significant increase in metabolic activity (p<0.001) when treated with drugs at low concentrations (9-15 µg/ml).

Example 2: Effect of "Abisil-1" on the reduction of mitochondrial potential

Immortalized MRC5 cells and Lech-4 lung fibroblasts (MRC5-SV40 (A)) were incubated at a ratio of 1:4000 with Abisilom-1 overnight, stained with TMRM, and dye fluorescence was measured in the PE channel. The black histogram in Figure 2 represents Abisil-1 treated cells; the gray histogram represents control cells in Figure 2.

Treatment with "Abisil-1" caused an approximately 2-fold decrease in mitochondrial potential (p<0.05 (p<0.01)) (see Fig. 2 and table 2).

Thus, "Abisil-1" causes a decrease in mitochondrial potential in immortalized MRC5 cells and Lech-4 lung fibroblasts.

Example 3: Influence of "Abisil" on the intensity of autophagy and mitophagy

In this example, the effect of "Abisil" on the intensity of autophagy and mitophagy was evaluated after 24 and 72 hours.

The assessment of autophagy was performed by assessing the number of LC3II complexes in MRC5-SV40 cells, determined by Western blotting. In FIG. 3 shows the results of the densitometric analysis of the LC3II complexes relative to the internal control, which are presented as the mean ± standard deviation calculated from three independent experiments. There is a significant increase in the content of LC3II complexes (more than 10 times) when treated with "Abisil" at a concentration of 50 μg/ml, which indicates an effective stimulation of aerophagia in cells.

Example 4: Effect of "Abisil" on the reduction of mitophagy

A decrease in the mitochondrial potential of "Abisilom" may mean that it induces mitophagy. To study this, a construct encoding the GFP-mCherry-FIS1 reporter construct in MRC5-SV40 cells was introduced, which allows detection of possible mitochondrial fragmentation and mitophagy activation. FIS1 is a protein anchored to the outer mitochondrial membrane and therefore colocalized GFP and mCherry will give a yellow signal. GFP fluorescence is quenched in lysosomes due to the low pH, and therefore split mitochondria in phagolysomes can be detected by red fluorescence (see Fig. 4).

Indeed, after a 24-hour treatment, "Abisil" caused the formation of multiple mitophagosomes. However, "Abisil" did not cause a general fragmentation of mitochondria even after 72 hours of treatment.

Next, the effect of Abisil treatment on Lech-4 cells was determined and dye colocalization was studied during staining with Mitotracker Red with Lysotracker Green, which stains acidic compartments (lysosomes) in living cells. An increase in the number of phagolysosomes is observed, but they do not colocalize with the Mitotracker Red dye, which indicates that Abisil promotes autophagy, but does not specifically cause mitochondrial fragmentation in embryonic fibroblasts.

Claims (11)

1. A method for stimulating autophagy in cells, involving the treatment of cells with a terpenoid-based inducer, characterized in that the drug "Abisil", "Abisil-1" or "Abisil-2" is used in an effective concentration as such an inductor based on terpenoids. 2. The method according to claim 1, characterized in that the terpenoid-based inducer is used at a concentration of 0.05 to 200 μg/ml, preferably 45 to 200 μg/ml. 3. The method according to p. 1, characterized in that the duration of treatment of cells with an inductor based on terpenoids is from 6 to 72 hours, preferably 16 hours. 4. The method according to p. 1, characterized in that the drug "Abisil", "Abisil-1" or "Abisil-2" is used with a target additive. 5. The method according to one of paragraphs. 1-4, characterized in that the cells originate from non-tumor tissues. 6. The method of claim. 5, characterized in that the cells are contained in the subject. 7. The method according to any one of paragraphs. 1-6, characterized in that the cells are ex vivo or in vitro. 8. A method for stimulating autophagy in a subject, with the stage of introducing an effective dose of an inductor into the body, characterized in that the drug "Abisil", "Abisil-1" or "Abisil-2" is used as such an inducer and stimulation of autophagy provides treatment for a condition associated with with mitochondrial dysfunction. 9. The method according to claim 8, characterized in that the condition associated with mitochondrial dysfunction is selected from a condition including mutations and / or epigenetic disorders of the mitochondrial or nuclear genome, leading to a violation of mitophagy, defects in mitochondrial morphology and disturbances in the mitochondrial membrane potential, and conditions including lysosomal storage diseases. 10. The method according to claim 8, characterized in that the terpenoid-based inducer is administered to the subject at a dose of 4 to 40 mg/kg, preferably 4 to 24 mg/kg. 11. The method according to p. 8, characterized in that the drug "Abisil", "Abisil-1" or "Abisil-2" is used with a target additive.

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