RU2549495C1 - Method for producing hexamethylene tetramine and nanoselenium agent having stimulant action on body cells - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: method for producing an agent for stimulating body cells involving preparing a mixture of aqueous solution of selenious acid and PEG 400; that is followed by preparing a mixture of hydrazine hydrochloride and PEG 400; the prepared mixtures are combined; the solution is put to dialyse against distilled water; surplus of water is driven off; the produced solution is added with hexamethylene tetramine; pH is reduced to 7.2-7.4; the method is implemented in certain circumstances.

EFFECT: producing high-effective, ecologically safe agent by the synergism of colloidal selenium and hexamethylene tetramine on body cell stimulation.

1 dwg, 2 tbl, 4 ex

Description

The invention relates to the field of pharmacology and medicine, including veterinary medicine, and can be used as non-specific stimulating therapy for diseases of various etiologies to stimulate body cells.

The stimulating effect on the cells of the body is carried out by acting on the mitochondrial respiration of the cells.

Currently, a large number of substances are known, in relation to which the ability to increase increase the metabolic activity of cells by stimulating mitochondrial respiration has been shown [1, 2, 3, 4].

), H₂O₂, гидроксильного радикала (HO^-) и, возможно, синглетного кислорода ( $$O_2^{-}$$

) повреждают клеточные макромолекулы (ДНК, белки, липиды) [6, 7, 8, 9]. Полагают, что активные формы кислорода вызывают повреждения мембран, коллагена, ДНК, хроматина, структурных белков, а также участвуют в эпигенетической регуляции экспрессии ядерных и митохондриальных генов, приводя к метилированию ДНК, влияют на внутриклеточный уровень кальция, запускают каскад, ведущий к апоптозу, и т.д. [10, 6, 11, 9].Most antioxidants possess these properties [5, 6, 7, 8]. According to this theory, free radicals formed as a result of various oxidative reactions in the body exert multiple damaging effects on macromolecules (nucleic acids and proteins), causing their degradation. This theory explains a wide range of pathological processes associated with it (cardiovascular diseases, age-related immunosuppression and brain dysfunction, cataracts, cancer, and some others). Superoxide molecules produced mainly in the mitochondria of cells ( $$O_2^{-}$$

), H ₂ O ₂ , hydroxyl radical (HO ^- ) and, possibly, singlet oxygen ( $$O_2^{-}$$

) damage cellular macromolecules (DNA, proteins, lipids) [6, 7, 8, 9]. It is believed that reactive oxygen species cause damage to membranes, collagen, DNA, chromatin, structural proteins, and also participate in the epigenetic regulation of the expression of nuclear and mitochondrial genes, leading to DNA methylation, affect intracellular calcium levels, trigger a cascade leading to apoptosis, and etc. [10, 6, 11, 9].

With aging, mitochondrial energy changes significantly, in particular, the oxidation of succinic acid decreases, which is also indicated by data on a decrease in the activity of succinate dehydrogenase in tissues during aging [12, 13]. Impacts that activate the system of formation and use of succinic acid, which has antioxidant properties, can especially effectively increase the body's functional capabilities [12, 13]. Feeding sodium succinic acid to rats from 20 months of age for 1.5 years (300 mg / kg in courses of 10 days with 1-month intervals) led to an increase in average (by 6.2%, p <0.05) and maximum ( by 12.3%) their life expectancy [3]. The authors, however, did not report the frequency of spontaneous tumors in rats in these experiments.

Cross-linking inhibitors are considered as one of the possible mechanisms of aging, since this process is accompanied by the formation of defective macromolecules [14, 16]. The increase in cross-linking with age has been experimentally proven so far only for extracellular collagen and elastin proteins and, possibly, for chromatin [15]. The limitation of caloric intake, which increases the life span of animals, delays the accumulation of collagen cross-links [17].

The decrease in age of the level and metabolism of catecholamines in the brain (mainly in the hypothalamus) and the violation of their relationship with other biogenic amines, in particular with serotonin, give key importance in the mechanisms that determine age-related changes in the neuroendocrine system, and ultimately, aging organism [18]. A decrease in the catecholamine content in the hypothalamus, achieved with the help of pharmacological agents or electrolytic destruction, reduced the life span of animals and increased the incidence of neoplasms [19], while the administration of pentylenetetrazole neurostimulator to rats reduced the morphological changes in the brain that occur during aging [20].

At the present stage of the development of science, the main mechanisms of action of metabolic stimulants consider their influence on bioenergetic processes in neurons and interaction with neurotransmitters. Neurometabolic stimulants promote penetration through the blood-brain barrier and utilization of glucose, improve the metabolism of purines and pyrimidines, activate protein synthesis, RNA and ATP. The effect of some neurometabolic stimulants is mediated by the neurotransmitter systems of the brain: monoaminergic (for example, piracetam contributes to an increase in the level of dopamine and norepinephrine), cholinergic (for example, meclofenoxate contributes to an increase in the density of cholinergic receptors and the level of acetylcholine in the synapses), glutamatergic.

Mitochondria are critical for the survival and proper function of almost all types of eukaryotic cells. Mitochondria of virtually any type of cell can have birth defects or acquired defects that affect their function. Thus, the clinically significant signs and symptoms of mitochondrial defects affecting the function of the respiratory chain are heterogeneous and variable depending on the distribution of defective mitochondria among the cells and the severity of their defects and on the physiological needs for the affected cells. Non-fissile tissues with high energy needs, such as nerve tissue, skeletal muscle and cardiac muscle, are particularly sensitive to dysfunction of the mitochondrial respiratory chain, but any organ system can be affected.

The diseases and symptoms listed below include the known pathophysiological consequences of dysfunction of the mitochondrial respiratory chain and, as such, are disorders in which the agent of the present invention has therapeutic utility.

Symptoms of the disease secondary to mitochondrial dysfunction are usually associated with 1) a spillover of free radicals from the respiratory chain; 2) defects in the synthesis of ATP, leading to a lack of cellular energy, or 3) apoptosis, triggered by the release of mitochondrial signals, such as cytochrome c, that initiate or mediate apoptosis cascades. This is an important refinement of existing dogmas in understanding the pathogenesis of diseases in which mitochondrial dysfunction is involved, and in understanding how to treat such disorders.

Neurometabolic stimulants also have antioxidant, membrane-stabilizing, neuroprotective and antihypoxic effects. Improving microcirculation in the brain is important due to the optimization of the passage of red blood cells through the microvasculature and the inhibition of platelet aggregation.

The result of the combined effects of neurometabolic stimulants is an improvement in the bioelectrical activity and integrative activity of the brain, which is accompanied by characteristic changes in electrophysiological patterns (an increase in the level of wakefulness, easier passage of information between the hemispheres, an increase in the dominant peak, and an increase in the absolute and relative power of the EEG spectrum of the cortex and hippocampus). Improving information exchange in the brain, increasing cortico-subcortical control, reproducing a memorable trace lead to improved perception, memory, thinking, attention, increased learning ability, activation of intellectual functions. The ability to improve cognitive function allowed us to designate drugs of neurometabolic stimulants as “cognitive stimulants”.

The following main effects can be distinguished in the spectrum of clinical activity of neurometabolic stimulants: nootropic, mnemotropic, adaptogenic, antiasthenic, psychostimulating, antidepressant, sedative, autonomic antikinetic, antiparkinsonian, antiepileptic.

Some of the listed pharmacodynamic properties are inherent in all neurometabolic stimulants, while others are more selective. In clinical conditions, each drug is able to realize the whole spectrum of its inherent properties, and it is often not possible to evaluate each of them in isolation often.

Initially, neurometabolic stimulants were used mainly in the treatment of functional brain disorders in elderly patients with organic brain syndrome. Recently, they have become more widely used in various fields of medicine, including in geriatric, pediatric and obstetric practice, psychiatry, neurology and narcology.

Recently, a high pace of research activity aimed at finding and studying the mechanisms of action of various neurometabolic stimulants has been noted. The search continues for the basic hypothesis of the action of neurometabolic stimulants that integrates various aspects of their mechanisms of action. The search for new drugs of the class of neurometabolic stimulants that would have greater pharmacological activity and have a more selective effect on the integrative functions of the brain, improving the patient's psychopathological state, his mental activity and orientation in everyday life, remains an urgent task.

The results of neurometabolic therapy. The use of neurometabolic stimulants can lead to improvement after the start of a comprehensive treatment program in 90% of patients with a variety of disorders. The effect of neurometabolic treatment is manifested in the form of: a decrease in severity up to the complete disappearance of insomnia, headaches, dizziness; improvements in the processes of memorization and assimilation of information and increase the ability to concentrate; the complete absence or significant reduction in the manifestations of other disorders and disorders in higher nervous activity.

A known method of changing the functional activity of cells of the tissue structure of the pathological focus of a living organism (options) (see patent RU, publ. 20.11.2003). The invention relates to medicine, more specifically to selective magnetotherapy, and can be used to treat various diseases by changing the functional activity of tissue structure cells when exposed to an external low-frequency electromagnetic field. The method includes influencing the mechanism of transmembrane kinetics of cell-tissue ion ions with a mass of 0.05-1.5 kg by an external alternating low-frequency electromagnetic field, the magnetic induction of which, depending on the desired functional activity of the cells, is selected according to the first embodiment in the range of 5-20 mT, which provides a stimulating effect on the functional activity of cells of the tissue structure, according to the second option - in the range of 25-60 mT, which provides a depressing effect, while the exposure time in their variants are selected in the range of 2-30 minutes. EFFECT: provision of regular unidirectionality and repeatability of biological and therapeutic effects caused by changes in the functional activity of cells of the tissue structure of the pathological focus, which guarantee effective treatment. The main disadvantages of the method are the limitations of the object by weight (0.05-1 kg) and the long-term exposure to the electromagnetic field.

There is also a method of increasing the functional activity of lymphocytes (see RF patent No. 2182597, publ. 02.20.2002). The method is carried out by exposure to helium plasma obtained at a current strength of 30 A, a voltage of 20 V and a gas flow rate of 2 l / min, on spleen cells placed in RPMI-1640 medium. The medium additionally contains 5% inactivated human serum of group IV, 2 mM 1-glutamine, 10 mM Hepes buffer, 5 · 10 ^-5 M 2-mercaptoethanol and 50 μg / ml gentamicin. The exposure is carried out for 20 s from a distance of 20 cm from the nozzle of the plasma torch. The method allows to locally affect the functional activity of lymphocytes both in cell culture and within the whole organism, while the effect is achieved in a short time. The method can be used in clinical practice as a method of stimulating effects on biological processes in cells and tissues. The main disadvantages of the method are the use of rare IV group human serum, high gas consumption and expensive equipment. In addition, the stimulation of immune processes can have both positive and negative consequences for the body.

The present invention is directed to solving the problem of creating a highly effective means for stimulating body cells.

The technical result consists in obtaining funds by the proposed method, which has high stimulation of body cells.

The technical result is achieved by the proposed method of obtaining funds, which contains as active components colloidal selenium and hexamethylenetetramine, as well as water.

Known to the authors of the sources of patent and scientific and technical information, a means for stimulating body cells, which is a colloidal solution of nanoparticles of selenium and hexamethylenetetramine, is not described.

The use of hexamethylenetetramine in medicine as an antiseptic is known (it has a dose-dependent bactericidal or bacteriostatic effect). It is also known to use hexamethylenetetramine in the production of phenol-formaldehyde resins used in the production of plastics, adhesives, varnishes, etc .; as a food additive preservative E239, used in cheese making and in the preservation of red caviar; as a "dry alcohol" for the preparation and heating of food; in analytical chemistry - as a component of buffer solutions; as a raw material in the production of explosives hexogen and hexamethylene triperoxide diamine; as a corrosion inhibitor.

The use of colloidal selenium as an antioxidant is known. The use of hexamethylenetetramine as a drug for the treatment of urogenital infections is known.

The authors first discovered a new property of colloidal selenium and hexamethylenetetramine in a tool to repeatedly enhance the mutual effect aimed at stimulating the body's cells by enhancing cellular respiration.

A method of obtaining funds for stimulating body cells is that 250 μl of a 0.5 M aqueous solution of selenic acid is mixed with 8 ml of polyethylene glycol 400 (hereinafter PEG 400), intensively mixed with a magnetic stirrer at a minimum of 750 rpm, the pH of this mixture is 7.55, to obtain a mixture of 1. Next, 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride is added to 8 ml of PEG 400, intensively stirred on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, s obtaining a mixture of 2. With vigorous stirring, 1 cm is introduced into the mixture Referring 2 dropwise. Put the resulting solution on dialysis against distilled water to remove PEG and hydrazine hydrochloride. The excess water is distilled off on a rotary evaporator.

Hexamethylenetetramine is dissolved in the resulting solution to a final concentration of 5%. The pH was adjusted to 7.2-7.4.

The resulting product contains, wt.%:

Hexamethylenetetramine 5 Selenium 0,062 Distilled water Up to 100

The advantage of the agent obtained by the proposed method is that with the help of nanoparticles based on selenium, hexamethylenetetramine is delivered to the cell. In this case, the biodynamics of hexamethylenetetramine, combined with colloidal selenium, occurs by the lymphogenous route and to a lesser extent undergoes enzymatic degradation and neutralization by the liver. The action of hexamethylenetetramine and selenium in relation to the stimulation of body cells.

Also, a certain advantage is the fact that colloidal selenium itself is part of the metabolic chain of the body and is able to be absorbed in the intracellular space. This eliminates the undesirable consequences associated with the "disposal" of the carrier itself.

Bringing the pH to 7.2-7.4 is due to the need to create a stable system, since values of acidity above or below the specified limits will lead to the destruction of the colloidal solution.

The selection of the conditions of the technological stages in the implementation of the method is due to the need to match the problem being solved. The concentrations of the components ensure uniformity and stability of the product.

Example 1

Prepare a 0.5 M solution of selenic acid in distilled water. A mixture of selenic acid with polyethylene glycol 400 (hereinafter referred to as PEG 400) is prepared, for this purpose 250 μl of a 0.5 M aqueous solution of selenic acid are added to 8 ml of PEG 400, intensively stirred on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7, 55. A mixture of hydrazine hydrochloride with PEG 400 is prepared, for this 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride is added to 8 ml of PEG 400, intensively mixed with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68. With vigorous stirring, a solution of hydrazine is added dropwise to the selenic acid solution. Put the resulting solution on dialysis against distilled water to remove PEG 400 and hydrazine hydrochloride. Excess water (in an amount equal to the volume of hexamethylenetetramine introduced further) is distilled off on a rotary evaporator. Hexamethylenetetramine is dissolved in the resulting solution to a final concentration of 5%. The pH was adjusted to 7.2-7.4.

Total in this tool we get:

Hexamethylenetetramine - 0.05 g / ml;

Colloidal selenium - 0.00062 g / ml

As a percentage of the total volume, the composition of the product will be as follows, wt.%:

Hexamethylenetetramine 5 Colloidal selenium 0,062 Distilled water Up to 100

The agent obtained by the proposed method is a colloidal solution of selenium nanoparticles with hexamethylenetetramine particles adsorbed on its surface.

Nanoparticles in the preparation were detected by electron microscopy. The particle size is from 40 to 100 nm.

To solve this problem, we used HeLa human cancer cell cultures obtained from the cryo bank of the cell culture collection of the Institute of Biochemistry and Plant Physiology of the Russian Academy of Sciences. Cell cultures were cultured in plastic bottles in complete RPMI medium (10% fetal serum, gentamicin, ampicillin, amphotericin) at 37 ° C and 0.5% CO ₂ . Dissociation of monolayer culture cells was achieved by washing the monolayer with trypsin solution for 10 min. The number of cells 3 · 10 ⁹ in 1 ml.

The number of cells was counted in the Goryaev’s chamber in 100 large squares of the Goryaev’s grid and calculated by the formula

X = a * 250/100, i.e. X = a * 2.5;

where X is the number of cells in 1 μl of suspension; a is the number of cells in 100 large squares. The number of counted cells is multiplied by 50. rev. 10 ⁶ / ml.

To determine the activity of cells used MTT test. MTT test: a pure cell culture and supplemented cells were incubated in 500 μl in eppendorf tubes at 37 ° C for 48 hours. Each tube with cell suspensions at the end of incubation was centrifuged for 10 min at 1000 g. Re-dissolve the resulting pellet in 500 μl of MTT solution and incubate for one hour. After incubation, the cells were redissolved in 500 μl of DMSO, 200 μl of suspension was taken from each tube and placed in the wells of a 96-well flat-bottom plate. The optical density readings were read on a reader at a wavelength of λ = 490 nm.

White nonlinear mice were used to determine the acute toxicity parameters of the studied drugs when introduced into the stomach.

White male mice were kept in a vivarium according to sanitary rules and on a standard diet in accordance with the order of the Ministry of Health of the USSR No. 1045-73 dated 04/06/73 [24]; the rules of laboratory practice [23] and the order of the Ministry of Health of the USSR No. 1179 dated 10/10/83 [26] using dry briquetted feed (LLC Labkormorm, Moscow). The animals were kept in a vivarium under standard lighting (12 hours light / 12 hours dark) at an air temperature of 20 ° C and 70% relative humidity. Work with animals was carried out in accordance with the order of the Ministry of Health of the USSR No. 755 of 08/12/07 [25] and the rules adopted by the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes.

Preparation for the experiment of white mice was carried out in accordance with the guidelines of the General Pharmacopoeia Monograph “Toxicity Test” GF XI [21]. Acute toxicity was determined by the Kerber method [22, 27].

Example 2

A study of the acute toxicity of the agent obtained in example 1

We took 6 white, nonlinear mice, males, weighing 15-20 g. An insulin syringe with a soft tip-catheter was used as a probe for drug administration. The following volumes of the test drug were administered to animals:

1. Mouse No. 1 - 0.2 ml

2. Mouse No. 2 - 0.4 ml

3. Mouse No. 3 - 0.6 ml

4. Mouse No. 4 - 0.8 ml

5. Mouse No. 5 - 1.0 ml

6. Mouse No. 6 - 1.2 ml

Even reaching a dosage of 1.2 ml per head of laboratory animal No. 6, we were not able to obtain a concentration of the resulting product equal to LD 50 (the dose was 2 mg per kg). Immediately after administration of the agent, mice No. 3-6 were depressed, impaired coordination of movement, refusal of feed and water for 30 minutes. After which the condition of the animals stabilized. After 48 hours from the time of administration, it was noted that the agent administered to 6 white non-linear mice did not cause the death of any animal.

Example 3

Based on the data obtained above, we conducted basic studies on acute toxicity. We took 4 groups of animals with 6 animals each. The volume of administration of the drug was 0.6; 0.8; 1 and 1.2 ml per animal in each group, respectively. Recalculation of the input volumes of the drug per 1 kg of animal weight is presented in table 1.

The data shown in table 1 convincingly show that when administered orally to mice in doses of 0.6-1.2 ml / kg, it is not toxic to mice.

After administering the drug, a similar clinical picture was observed in mice of all groups, as in the preliminary experiments, namely, the depressed state, impaired coordination of movement, and refusal of food and water for 30 minutes. After which the condition stabilized.

When observing animals for 7 days, it was noted that the tool did not cause the death of any animal.

The introduction of a larger volume of liquid (more than 1.2 ml) to animals is not recommended according to the provisions of the pharmacopeia, as this can cause damage to the digestive system or water intoxication in laboratory animals. Therefore, according to the data obtained, an oral agent can be previously considered non-toxic.

Example 4

The study of the interaction of the funds obtained in example 1, with HeLa cells

In our early studies, we found that when incubating cells on depleted and enriched media for seven days, it was determined that in both cases their number was approximately the same (5 · 10 ⁴ cells / ml), respiratory activity was approximately equal, which indicates the absence of influence of the composition of the nutrient medium on the properties of the population of HeLa cell lines at low exposure.

In the present case, when studying the respiratory activity of HeLa cells, we took the optical density of the sample with the maximum amount of cellular material as 100% when measured with a BS 3000 P Sinnova semi-automatic biochemical analyzer (China) at a wavelength of λ = 492 nm.

With a low concentration of cells, the magnitude of the change in respiratory activity is at the level of the analytical error of the method. Sufficiently accurate results can be obtained using analytical samples with a cell number of more than 15,000 in 1 ml.

A preparation containing HeLa cells was resuspended in complete RPMI medium (including nutrient serum and antibiotics). The concentration of the drug introduced into the medium was 2 μg per 1 ml of culture medium.

In all experiments, a pure culture of HeLa cells and HeLa cells incubated with a selenium-containing agent were used as control samples. Next, the optimal incubation time was determined and the acceptable dilution interval of the selenium-containing agent for subsequent studies was identified. Cells were incubated with the drug for a day. Table 2 and Figure 1 show the results of an MTT test for the respiratory activity of HeLa cells (Se + Ge-selenium + hexamethylenetetramine (the agent obtained in Example 1); Ge — hexamethylenetetramine; K — control).

Analyzing the data obtained, it should be noted (table 2; Figure 1) that the action of both colloidal selenium and hexamethylenetetramine is observed. Moreover, hexamethylenetetramine increases the activity of cellular respiration by about 1.7 times, and the agent obtained in Example 1 increases the activity of cellular respiration by about 2.4 times, which indicates the combined effect of both components on cellular respiration. The fact that the agent containing selenium and hexamethylenetetramine, even when diluted 10 times, had a more pronounced positive effect on cellular respiration compared to the action of pure hexamethylenetetramine, indicated that it was the colloidal selenium complex and the preferred effect on the stimulation of cellular respiration. hexamethylenetetramine.

Thus, the agent obtained by the proposed method is effective for stimulating the cells of the body.

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Claims (1)

A method of obtaining a means for stimulating body cells, comprising preparing a mixture of 1 by adding 250 μl of a 0.5 M aqueous solution of selenic acid in 8 ml of PEG 400, intensive stirring on a magnetic stirrer at at least 750 rpm, the pH of this mixture is 7.55 then mix 2 is prepared by adding 250 μl of a 0.5 M aqueous solution of hydrazine hydrochloride in 8 ml of PEG 400, stirring vigorously with a magnetic stirrer at at least 750 rpm, the pH of this mixture is 0.68, with vigorous stirring is added to the mixture 1 mixture 2 dropwise, the resulting solution is put on dialysis against distilled water, removing PEG 400 and hydrazine hydrochloride, the excess water is distilled off on a rotary evaporator, hexamethylenetetramine is added to the resulting solution, the pH is adjusted to 7.2-7.4, while the components are mixed in an amount that ensures their content in the mass .%:
Hexamethylenetetramine 5 Selenium 0,062 Distilled water Up to 100

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