EA010737B1 - Medicament having heteroprotective activity and method for preparing thereof - Google Patents

Abstract

A number of inventions relates to a medicament and method for preparing thereof using animals' organs, in particular, to a medicament having heroprotective activity, which is a medical preparation for parenteral administration as solution of a peptide complex, a low-molecular fraction content being from 70 to 90% with a molecular mass comprising the peptide components within 70 to 212 Da, peptide concentration being 2.5÷2.9 mg/ml and to a method for preparing thereof from calf epiphysis (pineal body) aged less than 12 month or pigs by extracting acetic acid in the presence of zinc chloride; providing repeated deposition of formed homogenized deposition by twofold volumes of acetone at least two times and subsequent washing through a nutsch filter by twofold volumes of acetone; obtaining an aqueous solution of extract, polypeptide concentrate being 2.5÷2.9 mg/ml; centrifuging, filtering thereof with subsequent ultra-filtration purification at the apparatus with counter pressure not exceeding 1.0 kgs/cmusing materials with retention 15000 Da and adding glycocol in the ultrafiltrate to the final concentration 10÷20 mg/ml at pH=5.6÷6.6 mg/ml and subsequent sterile filtration, ampoulation and autoclaving for 8 minutes at 120°C and atmospheric pressure 1.1 kgs/cm, which provides stability of the medicament and preserving therapeutic effectiveness thereof.

Description

The group of inventions relates to a medicinal product and a method for its preparation from animal organs, in particular, to isolating a biologically active substance from the pineal gland (pineal gland) and to obtain a parenteral dosage form that can be used in medicine as a drug with geroprotective activity.

It is known that the therapeutic potential of many peptide drugs is manifested in the stimulation of immune and reparative processes. Peptides cause metabolic changes, which are controlled through the mechanisms of gene regulation [Kyautkoi U.K11. Maishi U.W. Szgop1o1oshchsa1 Azres18 o £ Sepot RerPbe Kedi1a1yup. - Vase1: Kagdeg, 2005. - 104 p.]. Immunostimulants of a peptide nature are known: tactivin (French patent No. 2570278), thymosin (6o1b81esh L.E. a1., Proc. No. 11. Asab. 8c1. I8A, 1972, ν. 69, p. 1856-1863). These drugs are a set of polypeptides of various lengths, affecting various parts of the immune response, as well as other physiological functions of the body, which leads to the appearance of unwanted side effects.

Known methods for producing biologically active substances and medicines from animal raw materials (A.S. 8I No. 1218521, 1994; A.S. 8I No. 1227198, 1986; RF patent No. 1298879, 1993; RF patent No. 1448443, 1994; RF patent No. 1522486, 1993; RF patent No. 2075944, 1997; RF patent No. 2161501, 2001; AS 8I No. 1522486, 1993; I8 patent No. 4341765; CB patent No. 1161896, 1969; GB patent No. 2583982, 1987).

A known method of producing a peptide complex from the pineal gland or pineal gland of cattle, including extraction of crushed raw materials with 3-5% acetic acid with the addition of zinc chloride, centrifugation, sedimentation of the target product from the supernatant with six volumes of acetone and ether, its renaturation in 1% acetic acid, followed by clarification and preparation of the dosage form (RF patent No. 944191, 1994).

A known method of obtaining from animal raw materials a complex of biologically active polypeptides with a geroprotective effect, from the pineal gland of the animal brain (RF patent No. 2163129, 2000), which is the closest analogue to the prototype of the proposed tool.

According to the method described in RF patent No. 2163129, a complex of biologically active polypeptides is obtained by grinding the pineal gland, extraction with a 3% solution of acetic acid in the presence of zinc chloride, treating the supernatant with acetone, washing with an organic solvent, and the extraction is carried out at a temperature of 7-15 ° C in for 48 hours, pH 3.0-4.0, the extract is separated from the ballast substances by separation, the treatment of the supernatant with acetone is carried out in a ratio of 1: 5 at! = 5 ° C, sedimented until a formed precipitate is formed, the supernatant is siphoned, the precipitate obtained is washed with acetone in a ratio of 1: (15-20), settled until the supernatant is clarified and siphoned, the precipitate is filtered and washed with acetone at room temperature, dried and an aqueous solution with peptides of 15-35 mg / ml is prepared , the resulting solution is stirred, centrifuged, then the high molecular weight water-soluble fractions are separated by ultrafiltration through materials with a retention capacity of up to 15,000 Da, the obtained target product is lyophilized.

The target product obtained by the method described in RF patent No. 2163129, contains a complex of polypeptides with a molecular weight of from 1000 to 12000 Yes, is the closest analogue - the prototype for the proposed tool with geroprotective activity.

The disadvantages of this method include the extraction during the extraction from the pineal gland tissue of a large amount (more than 70%) of non-peptide substances, which, being impurities, do not determine the geroprotective activity of the isolated active substance, as well as its low yield.

The present invention posed and solved the problem of developing a method for producing a drug having geroprotective activity in the form of a parenteral dosage form isolated from the pineal gland (pineal gland) of calves no older than 12 months of age or pigs, the technical result of which is the optimal technology for the isolation of the peptide complex with the content of the low molecular weight fraction from 70 to 90% with a molecular weight of the peptide components included in it ranging from 70 to 212 Da and obtaining an aqueous solution ora extract with a concentration of polypeptides of 2.5-2.9 mg / ml, which allows not only to clean the resulting product from impurities, but also to increase its yield.

Experimental verification showed that the proposed method provides pharmaceutical stability, maximum biological value and therapeutic efficacy of the drug with geroprotective activity, made in the form of a parenteral dosage form, which is confirmed by the experimental results given in the examples illustrating the invention.

Another aspect of the invention is an agent having geroprotective activity, in the form of a parenteral dosage form, which is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of its peptide components ranging from 70 to 212 Da, with a concentration of polypeptides 2.5-2.9 mg / ml, technically

- 1 010737 the result of which is that the isolated substance differs from known substances obtained earlier from the pineal gland of animals in terms of molecular weight of its peptide components, as well as in its non-toxicity and pyrogen-free nature due to complete purification from impurities.

In the course of studies in animal experiments, it was found that the obtained aqueous solution of the agent with a concentration of polypeptides of 2.5-2.9 mg / ml is a therapeutically effective dose, which allows us to consider the use of the agent for various types of age-related pathologies. This is confirmed by the following examples.

In the process of conducting research, the importance of the following stages of the method of obtaining funds having geroprotective activity, made in the form of a dosage form for parenteral administration (hereinafter referred to as the drug), was established:

repeated precipitation of the resulting homogenized precipitate with double volumes of acetone at least two times and subsequent washing on the suction filter with double volumes of acetone until a light gray precipitate is obtained, which indicates complete purification of the precipitate from impurities such as lipid, protein, phospholipid and others;

obtaining an aqueous solution of the extract in a concentration of polypeptides of 2.5-2.9 mg / ml; its centrifugation, filtration, followed by ultrafiltration cleaning at the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Da, and glycocol added to ultrafiltrate to a final concentration of 10-20 mg / ml at pH 5.6- 6.6;

sterilizing filtration, ampouling and autoclaving for 8 min at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² , which ensures the stability of the drug while maintaining therapeutic efficacy.

The mode and time of autoclaving were established experimentally.

It should be noted that the proposed agent with geroprotective activity differs from the known biologically active substances isolated previously from the pineal gland of animals, since it is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of its constituent peptide components ranging from 70 up to 212 Yes, isolated from the pineal gland, while the preparation obtained by the method described in RF patent No. 2163129 contains mainly a complex of polypeptides with molecular th weight from 1,000 to 12,000 Da.

The specified technical result is achieved in that the agent having geroprotective activity is made in the form of a parenteral dosage form and is a peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of the peptide components included in it ranging from 70 to 212 Yes , with a concentration of polypeptides of 2.5-2.9 mg / ml and obtained from the pineal gland of calves not older than 12 months of age or pigs by extraction with acetic acid in the presence of zinc chloride.

The specified technical result is also achieved by the fact that the proposed method of obtaining funds having geroprotective activity is characterized in that the pineal gland (pineal gland) of calves no older than 12 months of age or pigs is frozen at a temperature of at least minus 40 ° C, kept at a temperature of minus ( 20-22) ° C for at least two months, then crushed, add a 3% solution of acetic acid in a volume ratio of 1: 5 at a temperature of 20 ± 5 ° C, extraction is carried out with constant stirring, after receiving one 1% solution of zinc chloride in a volume ratio of 50: 1 is added to the suspension, it is cooled with constant stirring to a temperature of 7-16 ° C, then it is stirred for 1 hour after every 4 hours of sedimentation for 48 hours, the extract is separated from the ballast substances by separation , acetone is added to the extract in a volume ratio of 1: 5, kept at a temperature of 3-5 ° C for 4 hours, the resulting homogenized precipitate is re-precipitated with acetone at least 2 times, then the precipitate containing the active substance is washed on a suction filter with double volumes and acetone cooled to a temperature of 7-16 ° C until a light gray precipitate is obtained, rubbed through a metal sieve, dried, dissolved in distilled water at room temperature and with constant stirring to a concentration of polypeptides of 2.5-2.9 mg / ml, solution centrifuged, filtered, subjected to ultrafiltration cleaning at the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Yes, glycol is added to the ultrafiltrate to a final concentration of 10-20 mg / ml at pH 5.6-6, 6, the solution under Yergali sterilizing filtration under pressure of not more than 2.0 kgf / cm ^2, filled into vials of 2 ml and autoclaved for 8 minutes at 120 ° C and atmospheric pressure of 1.1 kgf / cm ^2.

The invention is illustrated by the figure and tables.

The figure shows the effect of the drug on the development of pineal gland explants.

In the table. Figure 1 shows the effect of the drug on life expectancy and onset of tumors in female 8AMP mice.

In the table. Figure 2 shows the effect of the drug on longevity and onset of tumors in female BAME mice.

- 2 010737

In the table. Figure 3 shows the effect of the drug on the morphological and biochemical parameters of the peripheral blood of guinea pigs in the study of toxicity.

In the table. 4 presents the dynamics of subjective indicators in patients with menopausal syndrome.

In the table. 5 shows the dynamics of changes in the content of pituitary hormones in the blood serum of patients with menopausal syndrome.

The invention is illustrated by the following examples: example 1 - a method of obtaining a preparation; examples 2 and 3 confirm the geroprotective activity of the drug; example 4 - the study of the toxicity of the drug; Example 5 confirms the therapeutic efficacy of a preparation in the form of a parenteral dosage form.

Example 1. The method of obtaining the drug.

As the raw material, the pineal gland (pineal gland) of calves (not older than 12 months of age) or pigs is used, which is frozen at a temperature of at least minus 40 ° C and kept at a temperature of minus (20-22) ° C for at least two months .

400 l of a 3% solution of acetic acid are pumped into the extraction reactor and cooled to a temperature of 20 ± 5 ° C, then 80 kg of crushed material are loaded into the reactor with constant stirring until a homogeneous mass of raw material is obtained. The extraction is carried out with constant stirring, after obtaining a uniform suspension, a 1% solution of zinc chloride in a volume ratio of 50: 1 is added to it, cooled with constant stirring to a temperature of 7-16 ° C, then it is stirred for 1 hour after every 4 hours of sedimentation for 48 hours

The extract is separated from the ballast substances by separation at 5000 ± 500 rpm for 1 hour. Acetone in a volume ratio of 1: 5 is added to the extract. It is kept at a temperature of 3-5 ° C for 4 hours. The precipitate formed is homogenized and re-precipitated with acetone at least two times. Then, the precipitate containing the active substance is washed on a suction filter with twice volumes of acetone cooled to a temperature of 7-16 ° C until a light gray precipitate is obtained. The washed precipitate is wiped through a metal sieve, spread in a thin layer into enameled cuvettes, covered with a double layer of cotton cloth and dried with periodic stirring in a fume hood until the smell of acetone is completely removed.

The output of the active substance (powder of a biologically active peptide complex isolated from the pineal gland) is 50 g per 1 kg of feedstock.

The resulting powder is dissolved in distilled water with constant stirring at room temperature for 40 minutes to a concentration of peptides of 2.5-2.9 mg / ml. The resulting solution was centrifuged at 3000 ± 200 rpm for 20 ± 5 minutes. The centrifuge is filtered through an AP-15 filter or similar.

The filtrate is subjected to ultrafiltration purification in an ultrafiltration unit with a backpressure of not more than 1.0 kgf / cm ² , through materials with a retention capacity of 15,000 Da.

In the ultrafiltrate add the calculated sample of glycol, to its final concentration of 10-20 mg / ml, mix until the glycol is completely dissolved while maintaining a pH of 5.6-6.6.

The solution is subjected to sterilizing filtration, which is carried out under pressure not exceeding 2.0 kgf / cm ² .

The resulting solution is poured into 2.0 ml ampoules and autoclaved, and the ampoules with the preparation are sterilized for 8 min at a temperature of 120 ° C and atmospheric pressure of not more than 1.1 kgf / cm ² .

The preparation is a colorless, transparent solution and contains a peptide complex with a concentration of polypeptides of 2.5-2.9 mg / ml.

Testing for the absence of high molecular weight protein components is carried out by adding to the contents of 1 ampoule of the drug 1 ml of a 10% solution of trichloroacetic acid. The transparency of the solution indicates the absence of high molecular weight protein components.

To determine peptide bonds in a preparation, a biuret reagent is added to its solution. Staining the solution with violet color indicates the peptide bonds present in the preparation.

In order to determine the polypeptides and their fractions in the preparation, the methods of ultraviolet spectrophotometry, gel chromatography, mass spectrometry, high performance liquid chromatography and polyacrylamide gel electrophoresis are used.

The ultraviolet spectrum of the drug solution is recorded in the region of wavelengths from 250 to 350 nm: the absorption maximum is observed at a wavelength of 270 ± 5 nm.

The molecular weight of the polypeptides included in the preparation is determined by the following methods: gel chromatography on Sephadex 0-25 and 0-50 (Ryagshaaa, Sweden). To calibrate a 1.6x60 cm column, use the RsrCys Mo1essiag \ Uesch111 Κίΐ M8 III (8egua, Germany);

mass spectrometry method. The spectra are obtained on a Wouadeg ΌΕ Vu8res1gosh1gu time-of-flight mass spectrometer with laser desorption and ionization using a matrix (ΜΑΓΌΙ-ΤΟΡ) at a relative intensity of the nitrogen laser 2300-2400, accelerating voltage 25000 V, delay time

- 3 010737 ns and a pressure in the vacuum chamber of 2.6x10 ⁶ torr;

by electrophoresis in a 15% polyacrylamide gel in comparison with a standard set of marker proteins.

Using the methods listed above, it was found that the composition of the drug includes polypeptides with a molecular weight of from 70 to 212 Da.

Using reverse-phase high-performance liquid chromatography in an acetonitrile gradient (Ysygokog C18 sorbent, 2x62 mm column), it was found that the preparation contains mainly low molecular weight fractions from 70 to 90%, and there are no high molecular weight components in the preparation.

Pyrogenicity of the drug is determined in rabbits by the generally accepted method (GF XI, issue 2, p. 183) with a test dose of 0.25 mg per 1 kg of animal weight in 1.0 ml of an isotonic 0.9% sodium chloride solution for injection. It is shown that the drug is pyrogen-free.

Thus, in the above method, a preparation is obtained - a parenteral dosage form, which is a peptide complex with a low molecular weight fraction from 70 to 90%, with a molecular weight of its peptide components ranging from 70 to 212 Da, with a concentration of 2.5 polypeptides -2.9 mg / ml.

Example 2. The effect of the drug on the development of pineal gland explants.

The experiments were conducted on 52 fragments of the pineal gland of the XTMag rats weighing 150-200 g. The culture medium for explant cultivation consisted of 35% Eagle's solution, 25% fetal calf serum, 35% Hanks' solution, 5% chicken embryonic extract, on Wednesday glucose (0.6%), insulin (0.5 units / ml), penicillin (100 units / ml), glutamine (2 mM) were added. Epiphysis fragments were placed in this medium and cultured on a collagen substrate in Petri dishes in a thermostat at a temperature of 36.7 ° C for 2 days. The preparation was added to the experimental medium at concentrations of 2, 10, 20, 50, and 100 ng / ml. The criterion of biological activity was the area index (PI) —the ratio of the area of the entire explant together with the growth zone to the outgoing area of the pineal gland fragment. The IP values were expressed as a percentage, the control IP value was taken as 100%.

The figure shows the effect of the drug on the development of pineal gland explants.

It was found that after 1 day of cultivation, the explants spread on a collagen substrate and the proliferation of proliferating and migrating cells began on the periphery of the explant. On the 3rd day of cultivation at a drug concentration of 20 ng / ml, a significant increase in the explant PI by 35% was observed, compared with the reference PI values. When studying the pineal gland explants for longer cultivation periods (7 days), a similar stimulating effect of the drug at the same concentration was revealed.

Thus, in relation to the tissue of the pineal gland, the drug had a tissue-specific effect, manifested in stimulating the growth of explants.

Example 3. The effect of the drug on life expectancy and spontaneous carcinogenesis in mice with accelerated aging (8LM).

One of the most promising experimental models for studying the mechanisms of aging and carcinogenesis are genetically modified 8LM mice (kepexepse asseuye thu). As a result of damage to the genome, the intensity of spontaneous mutagenesis in 8LMR mice (8LM pgope), the most prone to accelerated aging, is significantly higher than in mice of other lines, including the wild 8LMK line (8LM ge8181ap1).

In the experiment, the effect of the drug on life expectancy and tumor development in 8LMR and 8LMK mice was studied. Melatonin was used as a reference drug. Two-month-old animals of each line (111 8LMR females and 95 8LMK females) were divided into 4 groups. Group 1 mice were an intact control. Animals of the 2nd group (control with solvent) were injected subcutaneously in courses (daily for one week every month) in 0.1 ml of sterile saline. Mice of the 3rd group received melatonin in the same mode at night (from 20:00 to 8:00) with drinking water at a concentration of 20 mg / L. The mice of the 4th group according to the same scheme as the mice of the 2nd group were injected subcutaneously with the studied drug in two single doses of 1.5 μg (70 μg / kg) and 7 μg (350 μg / kg) in 0.1 ml of sterile saline, for which animals of the 4th group were divided into 2 subgroups.

When comparing the dynamics of death of 8LM mice of different lines, signs of accelerated aging of 8LMR mice were revealed (Tables 1 and 2).

Moreover, in the table. 1 and 2, the following abbreviations were adopted: SPG - average life expectancy; α is a constant in the Gompertz equation; MCPT - time for doubling mortality; # - total number of tumors (lymphomas and fibrous histiocytes), the number of fibrotic histiocytes is indicated in parentheses.

In animals of this subline, a decrease in the average life expectancy of the last 10% of individuals and its maximum duration, an increase in the rate of aging of the population, and a decrease in the time for doubling mortality were noted. Significant differences in average life expectancy in intact mice 8LMR and 8LMK could not be found.

- 4 010737

The studied drug and melatonin did not have a statistically significant effect on the average life expectancy of mice in both sublines. At the same time, both the drug and melatonin showed a geroprotective effect in 8LMR mice. Thus, melatonin increased the life expectancy of the last 10% of mice in animals of the 8LMP subline. They also increased their maximum life expectancy; the value of the constant reflecting the aging rate of the population and the time for doubling mortality decreased 1.56 times. The use of the study drug was accompanied by an increase of 1.5 months (8%) in the average life expectancy of 10% of the maximum living mice, and by the same maximum life expectancy. The dosage of the drug did not affect the severity of its geroprotective effect.

The main cause of mortality in animals of both sublines was the development of lymphomas associated with the origin of 8LM mice from the high leukemic line of ACC. For malignant lymphomas in 8AM mice, a significant increase in the size of the liver, spleen, thymus and damage to the axillary, inguinal and mesenteric lymph nodes with infiltration of their tumor cells with numerous mitoses was characteristic. In the liver, foci of lymphoma were usually located around the blood vessels. In addition to malignant lymphomas, malignant fibrous histiocytomas were found in animals in the form of tumor nodes located under the skin and growing into surrounding tissues. Tumors consisted of atypical spindle-shaped cells with multiple mitoses and proliferation of fibrous tissue. Ovarian cysts, which are thin-walled formations filled with fibrin or hemorrhagic contents, were also noted in some mice.

When analyzing the effect of the test drug and melatonin on carcinogenesis, it was shown that melatonin in 8AMP mice did not affect the incidence of tumors and the duration of their detection. The studied drug did not change the frequency of neoplasms and significantly (1.7 times) increased the life expectancy of individuals in which the tumors did not develop (Table 1).

In 8AMK mice, the use of the studied drug and melatonin did not affect the incidence of malignant neoplasms, as well as the life expectancy of animals with or without tumors (Table 2). However, under the action of the studied drug, the average life expectancy of mice significantly increased.

Thus, the drug has a pronounced effect on the mechanisms of aging and carcinogenesis, slowing down the rate of aging and reducing the incidence of malignant tumors. At the same time, the doses of 70 and 350 μg / kg turned out to be equally effective, which indicates the breadth of the therapeutic effect of the drug and the validity of the choice of therapeutically effective dose, not exceeding 70 μg / kg.

Example 4. The study of the toxicity of the drug.

The general toxic effect of the drug was investigated in accordance with the requirements of the Guidelines for the experimental (preclinical) study of new pharmacological substances (2000): acute toxicity with a single administration of the drug, as well as subacute and chronic toxicity with prolonged administration of the drug.

A study of acute toxicity was conducted on 66 white outbred male mice weighing 18-21 g. Animals were randomly divided into 6 equal groups. The drug was administered to animals once intramuscularly at doses of 35, 50, 100, 150, 200 mg / kg in 0.25 ml of a sterile 0.9% solution of N101. Animals of the control group were injected with the same volume of 0.9% solution N101.

Studies on the study of subacute toxicity were performed on 70 white mongrel rats with a body weight of 150-180 g. Once daily, animals of the experimental groups were injected intramuscularly with doses of 1, 10, 100 mg / kg in 0.5 ml of sterile 0.9 % solution No. 101. The animals of the control group were injected in the same volume with a sterile 0.9% solution of N101. Before administration of the drug on the 30, 60 and 90 days after the start of drug administration, the morphological composition and properties of peripheral blood were studied in animals. At the end of the experiment, biochemical and coagulological blood parameters were examined.

Studies on the study of chronic toxicity were carried out for 6 months, based on the duration of the recommended clinical prescription of the drug in 84 male guinea pigs weighing 250-280 g. Animals of the experimental groups received intramuscularly once daily drug for 6 months in doses of 1, 10, 100 mg / kg in 0.5 ml of a sterile 0.9% solution of N101.

In the control group, animals were injected in a similar fashion with a sterile 0.9% solution of N101 in the same volume. In animals, the number of erythrocytes, hemoglobin, reticulocytes, platelets, leukocytes, leukocyte formula, erythrocyte sedimentation rate (ESR), and erythrocyte resistance were determined by the generally accepted methods in peripheral blood. Along with this, the content of total protein in blood serum was determined by the method of Lowry, potassium and sodium by plasma spectrophotometry. After the experiment, a pathomorphological study of the brain and spinal cord, spinal ganglia, thyroid gland, parathyroid glands, adrenal glands, testes, pituitary gland, heart, lungs, aorta, liver, kidney, bladder, pancreas, stomach, small intestine, colon intestines, thymus, spleen, lymph nodes, bone marrow.

In the study of acute toxicity, it was found that a single administration of the study drug

- 5 010737 animals in a dose exceeding the therapeutic recommended for clinical use, more than 5000 times, does not cause toxic reactions, indicating a large therapeutic breadth of the drug.

The study of subacute and chronic toxicity of the drug indicates the absence of side effects with prolonged use of the drug in doses exceeding the therapeutic by 3003000 times. When studying the effect of the drug on the morphological composition and biochemical parameters of the peripheral blood of guinea pigs after 3 and 6 months after the start of the drug administration, no significant change in the indicators was revealed (Table 3).

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of the cardiovascular and respiratory systems, and liver and kidney function, pathological changes in the body were not detected.

Therefore, the drug obtained by the proposed method, with long-term administration to animals does not have toxic properties that prevent its further use as a medicine, made in the form of a dosage form for parenteral administration.

Example 5. The effectiveness of the drug in patients with menopause.

The problem of the state of health of older people is of particular importance for women 45 years of age and older, since at this age they undergo a restructuring of the functioning of the endocrine system, often accompanied by pathological manifestations. It is known that an age-related decrease in the functional activity of the pineal gland causes a violation of the mechanisms of interaction of the nervous, endocrine and immune systems and contributes to the development of various diseases and pathological conditions. Against the background of the beginning general aging of the body in women, the functions of the reproductive system are dying out due to a malfunction of the ovaries, pituitary, hypothalamus and suphypothalamic brain structures. This is manifested by a significant decrease in the hormonal reserves of the ovaries and an increase in the threshold of sensitivity of the hypothalamus and pituitary to estrogen, which leads to a violation of the correct rhythm of secretion of follicle-stimulating and luteinizing hormones. These involutional changes in the reproductive system gradually lead to the extinction of the reproductive function, and then hormonal.

Clinical trials of the effectiveness of the drug were conducted in 28 patients aged 47 to 62 years with menopausal syndrome. Patients complained of hot flashes to the head and upper body, accompanied by increased sweating, headache and changes in blood pressure, pain in the heart, chills, tachycardia attacks at rest, a tendency to fainting, nausea, a feeling of "freezing" of the heart, dizziness weakness. Psycho-emotional disorders were most often manifested by irritability, tearfulness, anxiety, sleep disturbance, decreased memory and attention, rapid fatigue, and reduced working capacity. Patients noted an increase in the incidence of respiratory infections. During a laboratory examination, hormonal status disorders were revealed in patients, characterized by a decrease in the content of FSH and LH in the peripheral blood. General clinical and biochemical parameters in the blood did not go beyond the age norm.

Patients were divided into 2 groups. The main group included 15 patients who received the drug at a dose of 5 mg in 2 ml of saline intramuscularly once daily for 10 days.

The control group consisted of 13 similar patients who were prescribed only conventional treatment, including a complex of vitamins, cavinton, clonidine, tincture of valerian or motherwort. Hormone replacement therapy was not used in patients of both groups. The study of the effectiveness of the drug was carried out on the basis of generally accepted research methods. Patients' complaints were evaluated in dynamics, a general clinical and biochemical blood test was performed. The content of hormones (FSH and LH) in the blood serum was determined by the radioimmunological method.

During the use of the drug in patients with menopausal syndrome, an improvement in subjective indicators was noted, which was manifested in a significant decrease in heart attacks, dizziness, a feeling of "fading" of the heart, and improved sleep compared to indicators in patients before treatment. In addition, the number of complaints of attacks of tachycardia, sweating, hot flashes to the head and upper body, and fluctuations in blood pressure was significantly compared with indicators in patients before treatment and with indicators in patients after treatment using generally accepted means (Table 4). Patients noted a significant increase in working capacity after a course of using the drug, which they associated with the normalization of the psychoemotional state.

It is noteworthy that the effect of the drug was characterized by a stable aftereffect. So, 1 month after the end of the course of taking the drug, symptoms such as dizziness, tinnitus, general weakness, fatigue, pelvic dysfunction, sleep disturbance and cephalgia, almost leveled. A study of the hormone content in the blood serum of patients using the drug revealed a significant decrease in the initially elevated level of FSH compared with indicators both before treatment and after treatment with conventional means in the absence of

- 6 010737 significant changes in the concentration of LH (table. 5). The study indicates a corrective effect of the drug on hormonal imbalance in patients with menopausal syndrome.

Thus, the results of the study indicate a normalizing effect of the drug in a therapeutically effective dose of 5 mg (2.5 mg / ml) on hormonal metabolism, which suggests its use in the complex treatment of menopausal syndrome and the prevention of accelerated aging, manifested in the negative course of menopause period in women.

Table 1

Index Intact mice Control (0.9% NaSG) Melatonin A drug Number of mice 24 33 24 thirty Life span (day): Average 468 + 30 537 + 26 516 + 40 582 + 20 Median 513 582 525 569 Last 10% 662 + 7 693 + 6 748 + 2 *** 741 + 9 * Maximum 669 702 750 ' 759 * "(Day’) 0.0100 0.0101 0.0064 * 0.0102 ΜΡΌΤ (day) 69 68 109 * '** 68 The number of mice with tumors 16 (67%) 20 (61%) 16 (67%) 22 (73%) The number of malignant tumors # 18 (2) 22 (2) 16 (0) 23 (3) SPG (days) of carcinoma mice 473 + 31 615 + 49 ' 520 + 98 555 + 35 SG (days) of tumor-free mice 462 + 62 374 + 69 405 + 85 584 + 84 ** The number of mice with ovarian cyst 8 (33%) 15 (45%) 11 (46%) 16 (53%)

* P <0.05 compared with that in intact mice;

** P <0.05 compared with the indicator in the control.

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table 2

Index Intact mice Control (0.9% NaCl) Melatonin A drug Number of mice 14 33 nineteen 29th Life expectancy (days): average 488 + 58 521 + 29 433 + 26 546 + 32 median 498 508 397 556 last 10% 704 738 + 14 ’ 603 + 0 ” 671 + 8 maximum 704 766 603 727 a (day · ¹ ) 0 0066 0 0076 0.0102 0.0126 ΜΚϋΤ (day) 105 91 68 55 The number of mice with tumors 11 (79%) 20 (67%) 13 (68%) 22 (76%) The number of malignant tumors # 11 (0) 20 (0) 14 (1) 23 (1) SPG (days) of carcinoma mice 516 + 39 541 + 43 478 + 51 545 + 48 SG (days) of tumor-free mice 415 + 46 445 + 28 385 + 41 461 + 45 The number of mice with ovarian cyst 4 (29%) 13 (43%) 5 (26%) 19 (66%)

* P <0.05 compared with that in intact mice;

** P <0.05 compared with the indicator in the control.

Table 3

Index The introduction of the drug (10 mg / kg) 3 months 6 months Control (n = 21) The drug (n = 21) Control (n = 21) The drug <n = 21) Red blood cells, x10 ¹² / l 5.3 + 0.5 5.2 + 0.5 5.1 + 0.2 5.3 + 0.4 Hemoglobin, g / l 14.3 + 1.1 14.1 + 1.5 14.2 + 1.4 14.2 + 1.1 Reticulocytes,% 1.3 + 0.02 1.2 + 0.04 1.1 + 0.07 1.2 + 0.06 Platelets x10 ⁹ / L 143.7 + 7.6 142.7 + 6.4 145.1 + 7.4 144.3 + 6.2 White blood cells, x10 ⁹ / l 9.6 + 0.4 9.5 + 0.2 9.4 + 0.7 9.6 + 0.2 Band neutrons,% 0.31 + 0.03 0.30 + 0.05 0.33 + 0.04 0.30 + 0.03 Segmented neutrophils,% 43,612,4 44.4 + 2.1 43.5 + 1.8 45.1 + 2.5 Eosinophils,% 0.71 + 0.07 0.67 + 0.05 0.72 + 0.04 0.69 + 0.08 Basophils,% 0.67 + 0.04 0.68 + 0.03 0.72 + 0.08 0.65 + 0.06 Monocytes,% 2.4 + 0.05 2.2 + 0.07 2.3 + 0.05 2.2 + 0.06 Lymphocytes,% 49.2 + 2.5 50.3 + 1.9 53.1 + 2.0 47.7 + 2.7 ESR, mm / h 1.84 + 0.06 1.78 + 0.07 1.76 + 0.09 1.91 + 0.05 Erythrocyte resistance,% NaC! -maximum -minimum 0.40 + 0.04 0.32 + 0.03 0.41 + 0.06 0.30 + 0.03 0.42 + 0.04 0.30 + 0.05 0.43 + 0.08 0.33 + 0.02 Total protein in blood serum, g / l 72.5 + 1.9 71.6 + 2.5 73.4 + 2.3 70.5 + 3.2 Sodium in blood serum, mmol / l 153.7 ± 5.7 155.4 + 6.8 152.4 + 6.3 154.6 + 7.2 Serum potassium, mmol / l 5.2 + 1.8 5.0 + 2.8 5.3 + 2.3 5.1 + 2.0

- 8 010737

Table 4

Index The number of patients,% Before treatment After treatment using conventional means After treatment with the drug Hot flashes to the head and upper torso 77.0 51.3 23.2 ** Increased sweating 65.1 52.4 21.3 ** Headaches 61.6 46,4 38.2 * Change in blood pressure 65.0 49.3 25.8 ** Pain in the heart 55.9 35.2 * 26.9 * Tachycardia attacks 66.9 34.6 * 23.7 * Dizziness 48,2 32.6 24.9 * Weakness 60.3 41.6 35,4 The feeling of "freezing" of the heart 55.1 24.0 * 21.6 * Fast fatiguability 77.9 51.4 * 38.3 * Reduced performance 88.2 51.6 ’ 38.4 * Irritability 91.4 58.9 * 32.0 * ' Tearfulness 67.9 41.5 * 22.6 * ’ Sleep disturbance 74.1 36.4 * 25.3 * Decreased memory and attention 54,2 39.0 33.6

* P <0.05 compared with the indicator in patients before treatment;

# P <0.05 compared with the indicator in patients after treatment using conventional means.

Table 5

Index Before treatment After treatment using conventional means After treatment with the drug FSH, ng / ml 71.0 ± 4.6 65.7 ± 4.0 48.9 + 4.1 ** LH, ng / ml 10.2 ± 1.1 12.6 + 1.9 11.0 + 1.5

* P <0.05 compared with the indicator in patients before treatment;

# P <0.05 compared with the indicator in patients after treatment using conventional means.

Claims (2)

1. The tool with geroprotective activity, which is a dosage form for parenteral administration in the form of a solution of the peptide complex with a low molecular weight fraction from 70 to 90% with a molecular weight of its peptide components ranging from 70 to 212 Da, with a concentration of polypeptides 2, 5-2.9 mg / ml, and obtained from the pineal gland (pineal gland) of calves not older than 12 months of age or pigs by extraction with acetic acid in the presence of zinc chloride. 2. The method of obtaining funds according to claim 1, characterized in that the pineal gland (pineal gland) of calves not older than 12 months of age or pigs is frozen at a temperature of at least minus 40 ° C, kept at a temperature of minus (20-22) ° C in for at least two months, then crushed, add a 3% solution of acetic acid in a volume ratio of 1: 5 at a temperature of 20 ± 5 ° C, the extraction is carried out with constant stirring, after obtaining a homogeneous suspension, a 1% solution of zinc chloride in a volume ratio is added to it 50 volumes of homogeneous suspension 1 volume of a 1% solution of zinc chloride, cooled with constant stirring to a temperature of 7-16 ° C, then stirred for 1 hour after every 4 hours of settling for 48 hours, the extract is separated from the ballast substances by separation, acetone is added to the extract in a volume ratio of raw materials : acetone, equal to 1: 5, is kept at a temperature of 3-5 ° C for 4 h, the resulting homogenized precipitate is re-precipitated with acetone at least 2 times, then the precipitate containing the active substance is washed on a suction filter with twice volumes cooled to a temperature 7–16 ° С acetone to obtain a light gray precipitate, wiped through a metal sieve, dried, dissolved in distilled water at room temperature and constant stirring to a concentration of 2.5-2.9 mg / ml polypeptides, the solution is centrifuged, filtered , they are subjected to ultrafiltration cleaning at the installation with a backpressure of not more than 1.0 kgf / cm ² through materials with a retention capacity of 15,000 Yes, glycol is added to the ultrafiltrate to a final concentration of 10-20 mg / ml at pH 5.66.6, the solution is sterilized f iltration under pressure of not more than 2.0 kgf / cm ² , poured into ampoules of 2 ml and autoclaved for 8 min at a temperature of 120 ° C and atmospheric pressure of 1.1 kgf / cm ² .