RU2463346C2 - Method for increasing proliferative mitochondrial activity in cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for increasing proliferative mitochondrial activity in cells with the use of a stimulating preparation consists in the fact that the stimulating preparation is presented by 1-hydroxy-biphosphonic acid derivatives - piperidine or morpholine, or xydiphone, or piperidine-2, or mediphone.

EFFECT: invention provides correcting therapy and increasing cell adaptation in the patients with systemic mitochondrial insufficiency.

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Description

Technical field

The invention relates to biology and medicine, in particular to the problem of adaptation of an organism to endogenous and exogenous adverse effects, to functional insufficiency of cells of body tissues. The invention can be most effectively used in in vitro studies conducted in cell culture, as well as in vivo in the complex treatment of diseases with impaired energy metabolism.

State of the art

It is known the use of various adaptogens in vivo: vitamins, antihypoxants, antioxidants, etc. (Voronina T.A. et al. 4 International Conference "Bioantioxidant", p.91-92, M., 2002, Burlakova EB and soavt. 4 International Conference "Bioantioxidant", p.70-71, M., 2002, Pankin VZ 4 International Conference "Bioantioxidant", p.341-343, M., 2002, Ignatieva S.N. et al. Clinical laboratory diagnostics 2009, No. 1, pp. 36-39, F. Meerson "Physiology of adaptive processes", pp. 573-622, M .: Nauka, 1986). The disadvantage of these methods is the lack of correction of bioenergy processes, which are significantly reduced (depleted) in extreme and adverse conditions.

The prototype of this invention is a method for correcting bioenergetic cell deficiency with the drug lovastatin, which is used for hypercholesterolemia (atherosclerosis), which increased mitochondrial proliferation by 7.6-8.3% (JSAmstrong. Mitochondria: a target for cancer therapy.// Br.J. Pharm. 2006, v.147, 239-248; H-Ch. Lee et al. Increase of mitochondria and mitochondrial DNA in response to oxidative stress in human cells. // Biochem. J., 2000, v.348, 425- 432; MA. Shibata et al. Lovastatin inhibits tumor growth and lung metastasis in mouse mammary carcinoma model.// Cancerogenesis, 2004, v.25 (10), 1887-1898).

However, the use of these drugs is limited due to severe side effects (anemia, allergies, myopathy, inhibition of hematopoiesis, leukopenia, thrombocytopenia, dyspepsia, lactic acidosis, etc. G. Bartlett. Antimicrob Agens Chemoter., 2007, v.51, 137-140) ) We found that bisphosphonates are also able to enhance the proliferation of mitochondria in cells, in addition to calcium-regulating and membrane-stabilizing effects when used in microdoses (nanoconcentrations), enhancing mitochondrial proliferation, bisphosphonates do not have side effects in therapeutic doses, are economically available.

The aim of the invention is to increase the proliferative activity of mitochondria in cells using bisphosphonates to improve the energy supply of cells and reduce the risk of side effects, increase cell adaptation to functional insufficiency of tissues of organs and systems.

This goal is achieved through the use of various compounds of 1-hydroxy-bis-phosphonic acid, which stimulate the proliferation of mitochondria.

Disclosure of invention

The method is carried out using the effects of compounds-1-hydroxy-bis-phosphonic series on the mitochondria of cultured human lymphocytes in vitro in tissue culture and on the mitochondria of human lymphocytes in vivo.

Evidence of enhanced mitochondrial proliferation using bisphosphonates is provided by the following examples: 1) 24-hour blood cell culture on lymphocytes in the presence of bisphosphonates; and 2) 24 hours after transdermal administration of xidifon preparations (Moskhimpharmpreparat LLC, registration number 001642/01) and mediphon (certificate No. РОСС RU PR73 V33974 from 03/04/2009 to 03/27/2011, manufactured by the REFARM Institute, Moscow). Control over the proliferation of mitochondria in lymphocytes is subsequently determined by the number of formazan granules formed as a result of a cytochemical reaction according to the method of R.P. Narcissov (R. Narcissov. Image analysis of cells - the next stage in the development of clinical cytochemistry in pediatrics // Pediatrics, 1998, No. 4, 101-105), revealing the activity of the bioenergetic metabolism enzyme, succinate dehydrogenase, which is tightly bound to mitochondria.

Bisphosphonates are synthetic analogues of inorganic pyrophosphates. Inorganic pyrophosphate is formed during bioenergy metabolism and is present in human biological fluids. Bisphosphonates can affect the activity of many enzymatic processes, calcium and hormone metabolism, and the formation of prostaglandin. In their presence, the processes of oxidation of fatty and amino acids are intensified, the cycle speed of tricarboxylic acids increases. In addition, they have an anti-inflammatory effect and affect the biosynthesis of collagen (Shcherbakov B.K. et al. // Bulletin of the Academy of Sciences, Chemistry series, 1998, No. 9, pp. 1780-1783; Matkovskaya T.A., Popov K.I. ., Yuryeva EA "Bisphosphonates" // M .: "Chemistry", 2001, 222 p.).

The implementation of the invention

1. For in vitro studies, whole venous blood is used as the study material. The object of study are lymphocytes. In vitro reagents - the following incubation medium is used:

- Wednesday Needle - 20 ml;

- 1% calf serum - 0.2 ml;

- 4 mmol L-glutamic acid (12 mg per 20 ml);

- 0.45% glucose (95 mg per 20 ml);

- 6 xg / ml kanamycin (0.2).

Incubation conditions (sterile)

Incubation is carried out in standard dies (0.5 ml each) for 24 hours at 37 ° C. 0.1 ml of heparinized blood (0.5 ml of heparin and 5 ml of venous blood) is added to each cell, 0.1 ml of the incubation mixture and 15 μl of a 1 mmol / l solution of the test compounds are also added (15 nmol final concentration in the cell). As the studied compounds were used domestic bisphosphonates - derivatives of 1-hydroxy-bisphosphonic acid of the general formula:

R-C (OH) (PO3H2) 2

Where

R3 = CH3-xidiphone

R6 = (CH3) 2N-CH2-CH2 - mediphon

To prepare a control preparation, 0.1 ml of heparinized blood, 0.1 ml of the incubation mixture and 15 μl of physiological saline are added to the cell.

2. Conducting a histochemical study. Thin smears of the incubated mixture are made on clean fat-free glass slides (based on 1 glass to assess the activity of LDH in each sample), marked and dried in air at room temperature for 5 minutes, and then fixed.

Preparation of fixing solution

Pour 250 mg Trilon B into a 100 ml dry flask; Bring 60 ml of acetone with distilled water to 100 ml; pour the mixture into a flask with Trilon B; mix; put for a day in a dark place at room temperature; use supernatant; Store in the dark at room temperature.

Fixation is carried out by immersing the marked slides with smears in a fixing solution for 30 seconds (do not leave a smear in the solution for more than 60 seconds). Then the glass is rinsed in two portions of distilled water, dried in air.

Cytochemical detection of LDH activity is carried out according to the method of Narcissov R.P. (1998). The preparation of the reagent is carried out in accordance with the manufacturer's instructions attached to the reagent kit (LLC MNPK Himtekhmash State Research Institute of IREA (certificate No. 27 dated 4.10.2004 for quantitative cytochemical determination of enzymes)).

Immerse the glass in a glass with a solution containing a reagent for determining the enzyme, heated to 37 ° C. Incubated for 60 minutes in a water bath or in a thermostat at a temperature of 37 ° C. At the end of the incubation, the glasses are removed from the solution, washed with tap water and dried at room temperature.

Evaluation of the results is carried out under a light-optical microscope (magnification 10-15 × 40, water immersion). Enzymatic activity during visual morphometry is expressed in arbitrary units corresponding to the average number of granules of formazan (i.e., actually the number of mitochondria), which is the product of a cytochemical reaction. To determine the activity of the enzyme in the lymphocyte population, the average number of granules in 30 cells is counted.

Example 1, in vitro. 0.1 ml of incubation medium and 0.1 ml of heparinized venous blood, as well as 15 μl of 1 mmol / l solution of the corresponding bisphosphonate (15 nmol per cell) are added to standard plates. A cytochemical analysis of the number of mitochondria in lymphocytes is performed in smears of cells from the incubation mixture 24 hours after incubation at 37 ° C and histochemical staining.

After conducting cytochemical analysis with all samples to identify the activity of LDH, the following results were obtained:

1. The number of mitochondria per 1 lymphocyte in the control sample was 7.5-8.4 units.

2. The number of mitochondria per 1 lymphocyte in the sample with piperidine was 13.6 units. (+ 70%).

3. The number of mitochondria per 1 lymphocyte in the sample with morpholine was 15.6 units. (+ 95%).

4. The number of mitochondria per 1 lymphocyte in the sample with xidiphone was 9.3 units. (+ 11.6%).

5. The number of mitochondria per 1 lymphocyte in the sample with pyridine was 6.5 units. (-).

6. The number of mitochondria per 1 lymphocyte in the sample with piperidine-2 was 10.8 (+ 35%).

7. The number of mitochondria per 1 lymphocyte in the sample with mediphon was 11.7 units. (+ 41%).

Thus, the obtained data indicate that compounds of -1-hydroxy-bis-phosphonic acid such as piperidine (stimulating effect was 170%), morpholine (190%), xidiphone (116) have a positive proliferative effect for mitochondria of lymphocytes in blood cell culture. %), piperidin-2 (135%) and mediphon (146%).

Example 2, in vivo. For in vivo studies, the proliferative effect of the two studied domestic bisphosphonates, currently used to treat calcium metabolism disorders (xidiphon and mediphon), is determined 24 hours after transdermal administration of drugs.

2% Xidifon and 0.5% mediphon in the form of creams (Xikrem, Mediphon, REFARM) in an amount of 8-12 g (on average 200 ± 20 mg and 50 ± 5 mg xidiphon and mediphon, respectively) are applied to the back skin (collar zone , chest) by rubbing until completely absorbed for 7-8 minutes. Mitochondrial proliferation is assessed in blood smears in the same manner as in Example 1. With this method of drug administration, their proliferative therapeutic dose is stored in the body for 48-72 hours, which allows us to recommend an intermittent transdermal administration of drugs in connection with their preservation as a depot at the injection site and gradually entering the bloodstream.

The indicated method for increasing mitochondrial proliferation was studied in 4 healthy women aged 25-35 years: in 2 women, 2% cream with xidiphone (Xikrem) was applied to the skin of the back and in 2 women, 0.5% cream with mediphone. Assessment of mitochondrial proliferation is carried out on peripheral blood lymphocytes before and after 24 hours (after applying the creams) after histochemical staining of blood smears and estimation of the number of mitochondria in lymphocytes (item 2).

The following results were obtained:

1. Xidiphone:

1) before and after the experiment - 21.47 (a) and 23.91 (b) (the number of mitochondria increased by 11.4%);

2) before and after the experiment - 21.35 (a) and 24.0 (b) (the number of mitochondria increased by 12.4%),

2. Mediphon:

1) before and after the experiment - 22.8 (c) and 26.05 (g) (an increase of 14.25%)

2) before and after the experiment - 21.8 (c) and 25.1 (g) (an increase of 15.1%).

Thus, under the action of bisphosphonates in the human body, the proliferation of mitochondria increases: under the influence of xidiphon by 11-12%, and under the action of mediphon by 14-15%, although the amount of mediphon in the cream was 4 times less than xidiphon.

Studies of the adaptive reactions of the cellular system, their causes, manifestations, molecular mechanisms are among the fundamental problems of biology and medicine. Adaptation reactions are a necessary link in any pathological process, and the possibility of increasing their intensity is one of the fundamental tasks of medicine.

Mitochondrial proliferation is a process that is inherently adaptive. Mitochondria are universal and obligatory for all tissues organelles of the cytoplasm. Mitochondria are the most important center for the general and energy metabolism of a cell. An increase in the absolute number of these organelles, in particular the phenomenon of “torn red fibers” (RRF) in skeletal muscle, is a manifestation of the compensatory abilities of the body that can cope with a particular functional defect (patent No. 2357247 from 05.27.2009).

To increase the proliferative activity of mitochondria, medications prepared on the basis of bisphosphonates can be used, since they are highly effective as regulators of mitochondrial metabolism (in particular, calcium metabolism), good bioavailability and low level of side effects.

Claims (1)

A method of increasing the proliferative activity of mitochondria in cells using a stimulating drug, characterized in that derivatives of 1-hydroxy-bisphosphonic acid — piperidine, or morpholine, or xidiphone, or piperidin-2, or mediphon — are used as the stimulating drug.