RU2194502C2 - Immunomodulator metabolite detoxicant - adaptogen radioprotector - Google Patents

Abstract

FIELD: medicine, veterinary science, pharmaceutical industry. SUBSTANCE: invention relates to development of novel medicinal agents. The proposed preparation comprises at least one placenta extract, at least one derivative of nucleic acid and at least one vitamin. Nucleic acid sodium salt or ridostin can be used as nucleic acid derivative and vitamin C or vitamins of group B can be used as vitamin. Preparation can comprise additionally at least one amino acid. Medium 199 or medium RPMI 1640 or another medium used for mammalian cell culturing and comprising vitamins and amino acids can be used as source of vitamins and amino acids. EFFECT: expanded assortment of drugs showing broad spectrum effect. 6 cl, 10 tbl

Description

 The invention relates to medicine and veterinary medicine, in particular to means for the prevention and treatment of pathological conditions accompanied by toxicosis and a decrease in the general resistance of the body during viral and bacterial infections, diseases of the liver, gastrointestinal tract, genitourinary system, immune system, stress.

 Known immunomodulator - metabolic - detoxicant - adaptogen - radioprotector, which is an extract of the placenta [1,2].

 Immunomodulators - means for selective stimulation or suppression of the immune response, stimulation of natural resistance to infections.

 Metabolics - substances that regulate carbohydrate, fat, protein, water-electrolytic and other types of metabolism.

 Detoxicants - substances that ensure the neutralization and removal of harmful, toxic and other decay products, including poisons, salts of heavy metals, radionuclides and normalize their disturbed structures and functions.

 Adaptogens - substances that improve the body's adaptability to changing external conditions and the harmful effects of the environment, helping to protect against stress.

 Radioprotectors are substances that protect the body from the adverse effects of radiation damage, stimulate postradiation survival, protect biological membranes from the damaging effects of radiation, and stimulate the restoration of structures and functions damaged by radiation.

 These actions of the known agent are not sufficiently expressed, which leads to a decrease in the effectiveness of therapy for diseases accompanied by a decrease in the body's natural resistance and the development of toxicosis. Correction of conditions of this kind requires a significant increase in the dosage of the initial drug and the duration of treatment.

 The technical result achieved by the implementation of the invention is to enhance the listed properties of a therapeutic agent.

 The specified technical result is achieved by the fact that the known immunomodulator - metabolic - detoxicant - adaptogen - radioprotector, including at least one placenta extract, contains at least one nucleic acid and / or its derivative and additionally at least one vitamin.

 The specified technical result is also achieved by the fact that it contains at least one amino acid.

 The specified technical result is also achieved by the fact that the percentage of dry residue of tissue extracts is not more than 20, nucleic acids and / or their derivatives - not more than 20, vitamins - not more than 5, amino acids - not more than 99.5 weight percent.

 The specified technical result is also achieved by the fact that as a nucleic acid or its derivative, sodium nucleinate or ridostin or larifan or derinate are used.

 The specified technical result is also achieved by the fact that the vitamins used are B vitamins, and / or vitamin C, and / or vitamin E.

 The specified technical result is also achieved by the fact that it contains, as a source of vitamins and amino acids, medium 199, or Eagle’s medium, or RPMI 1640 medium, or other medium for culturing animal cells.

 Tissue extracts are stimulants of metabolism (metabolism). Nucleic acids and their derivatives are immunomodulators.

 Some of them are also adaptogens.

 Vitamins are regulators of metabolic processes.

 Amino acids are the building material for protein molecules. Amino acids are metabolites.

 When composing the components, their combination provides a synergistic effect, since all the mentioned indicators of biological activity are higher than the same indicators of the individual ingredients.

 This conclusion can be illustrated by the following examples.

 Example 1. Immunostimulating (adjuvant) properties of the drug.

The method of local hemolysis in gel was used [3]. Mice were injected intraperitoneally with sheep erythrocytes (EBs) of 5 x 10 ⁸ in a volume of 0.5 ml). On the 5th day after immunization, spleens were taken from mice and erythrocyte suspensions were obtained. The tested cells (10 ⁶ in 0.1 ml) were introduced into molten agarose (0.7%) with freshly washed 20% sheep erythrocytes, after which the resulting mixture was layered on the surface of plastic Petri dishes with a diameter of 50 mm. After the mixture solidified, the cups were incubated at 37 ^° C. After 1 h, 1 ml of guinea pig complement was poured into the cups and incubated for an additional 1 h at 37 ^° C, after which the complement was drained and hemolysis zones were counted. The calculation of the number of antibody-forming cells (ATOC) was performed per 1 million viable splenocytes.

The following groups of mice were examined (10 animals per group):
1) control - received only EB;
2) mice that were injected with placental extract (EP) at a dose of 0.16 μl / kg (4 μg / kg dry weight) at the same time as EBs;
3) mice that were simultaneously injected with sodium nucleinate (H), belonging to immunomodulators [3], at a dose of 4 μg / kg, EP and a mixture of vitamins (B) at a dose of 0.3 ml / kg (2.85 μg / kg). The ratio of H: EP: B = 36.8%: 36.8%: 26.4%;
4) mice that were simultaneously injected with sodium nucleate (H), EP and vitamin C (BC) at a dose of 0.75 mg / kg simultaneously with EB. The ratio of H: EP: BC = 0.5%: 0.5%: 99%;
5) mice that were simultaneously injected with sodium nucleate (H), EP and vitamin B1 (BB1 at a dose of 0.05 mg / kg. H: EP ratio: BB1 = 6.9%: 6.9%: 86.2 %;
6) mice that were administered simultaneously with EB the claimed drug containing H, EP, B and a mixture of amino acids (A) at a dose of 0.3 ml / kg (67.8 μg / kg). The ratio of H: EP: B: A = 5.1%: 5.1:%: 3.6%: 86.2%;
7) mice that were administered simultaneously with EB the claimed agent containing H, B and EP2 (see example 10). The ratio of H: B: EP2 = 40.6%: 28.9%: 30.5%;
8) mice that were administered simultaneously with EB the claimed agent containing H, B and EP3 (see example 10). The ratio of H: B: EP3 = 26.9%: 19.2%: 53.9%;
9) mice that were simultaneously administered with EB the claimed agent containing N, EP and medium 199 at a dose of 0.3 ml / kg (3.5 μg / kg). The ratio of H: EP: B: A = 2.1%: 2.1%: 0.16%: 95.2%;
10) mice that were administered simultaneously with EB the claimed agent containing H, EP and RPMI 1640 medium at a dose of 0.3 ml / kg (7.27 μg / kg). The ratio of H: EP: B: A = 1.6%: 1.6%: 5.5%: 91.3%;
11) mice that were administered simultaneously with EB the claimed agent containing EP, B and ridostin in a dose of 1 μg / kg. The ratio of EP: B: H = 51.0%: 36.3%: 12.7%;
12) mice that were simultaneously administered with EB the claimed agent containing H, EP, B and serine at a dose of 0.05 mg / kg. The ratio of H: EP: B: A: = 6.6%: 6.6%: 4.7%: 82.1%.

 The results presented in table. 1 show that the addition of an immunomodulator (H) and vitamins to EP, and, to an even greater extent, the addition of nucleinate, vitamins, and amino acids to EP leads to a pronounced adjuvant effect.

 Example 2. The effect of drugs on the proliferative activity of splenocytes.

Proliferative activity was evaluated by the inclusion of a radioactive label in the DNA of cells cultured with the drug in relation to the inclusion of the label in control cultures (in the medium without the drug). The aseptically obtained suspension of splenocytes was cultured in an environment of the following composition: RPMI 1640 medium with 5% inactivated calf fetal serum, 2 mM L-glutamine, 10 mM HEPES buffer solution 50 μg ml gentamicin sulfate 5 • 10 ^-5 M 2-mercaptoethanol. Cultivation was carried out in a CO ₂ incubator in an atmosphere with 5% CO ₂ at 37 ^° C for 72 hours in 96-well flat-bottomed plates. 100 μl of a cell suspension (500,000 cells) and 100 μl of a solution of preparations at a concentration of 2 μl / ml were added to each well. 100 μl of cells and 100 μl of culture medium were added to control wells. In part of the wells, the preparations were introduced together with the T-mitogen concanavalin A (Sigma, USA), which was used at a final concentration of 2 μg / ml. 16 h before the end of cultivation, 1 μCi ³ N-thymidine with a specific activity of 37 GBq / mmol was added to each well. At the end of cultivation, the cells were transferred to glass fiber filters (GF / c, Watman, England) using a harvester (Flow, England). The radioactivity was counted on a scintillation β-counter. The results were expressed in absolute terms (pulses per minute - cpm).

 The data presented in table. 2, indicate that only the claimed agent, a mixture of EP + H + B + A and its analogues, containing medium 199 or RPMI 1640 medium as a source of amino acids and vitamins, causes reliable stimulation of lymphoid cell proliferation. However, the response to a standard mitogenic preparation, stimulating T-lymphocytes (concanavalin A) was enhanced in the presence of a mixture of EP + H + B, EP + H + BC and EP + H + BB1, which indicates the presence of immunostimulating activity in these drugs. Different placenta preparations (EP, EP1 or EP2) act almost identically.

 Example 3. Activity in radiation immunodeficiency.

 It is known that ionizing radiation in significant doses has a detrimental effect on the body, causing, among other things, serious secondary immunodeficiency.

 To study the effect of drugs on radioresistance, they were introduced, as in Example 1, intramuscularly to mice-hybrids (CBA • C57B1 / 6) F1, males weighing 20 g. There were 20 animals in the groups.

 After 1 day, the mice were irradiated at a dose of 900 rad on an ECU-50 setup, source of Co-60. Radioresistance was determined by survival for 30 days after irradiation. A positive result in this test indicates that the body has acquired resistance to the lethal effect of a lethal dose of radiation.

 The results obtained (Table 3) indicate that the preparations in combinations H + EP + B, H + EP + BC, Ridostin + EP + B, N + EP + BB1, N + EP1 + BH + EP2 + B and the claimed mixture N + EP + A + B, N + EP + medium 199, N + EP + RPMI 1640 medium, N + EP + B + serine protect animals from death. The protective effect mechanism can be due to several reasons: 1) stimulation of the proliferation of CCMs, 2) the actual radioprotective effect that protects the cell membrane from damage, 3) the antioxidant effect that prevents the destructive effects of free radicals, 4) the stimulation of production of TNF, IL-1 or interferon.

 To understand the mechanism of the radioprotective effect, we studied the effect of drugs on the proliferation of hematopoietic stem cells (HSCs) and on the production of gamma interferon.

 Example 4. Proliferation of hematopoietic stem cells (HSC).

 To determine the proportion of bone marrow HCC in the S phase of the cell cycle, i.e. in the phase of DNA synthesis (proliferation), the oxyurea suicide method was used.

After 1 and 3 days after the administration of the drugs in the doses indicated in example 1, mice were taken bone marrow from the femur, cells were incubated in RPMI 1640 medium with the addition of 10% fetal calf serum in the presence of oxyurea (1 mg / ml) or without it . After 2 hours of incubation in a CO ₂ incubator, the cells were washed by centrifugation, diluted to a concentration of 2250 thousand / ml and injected into syngeneic hybrid mice irradiated at a dose of 900 rad. After 8 days, mice (10 animals per group) were sacrificed, spleens were removed, they were fixed in Buen's solution, and the number of colonies was counted. After that, the content of HCC in the S phase was determined as a percentage ratio (ab) / b, where a is the number of colonies without oxyurea, b is the number of colonies with oxyurea.

 The data obtained (table 4) indicate that all the proposed compositions stimulate the proliferation of CCM. The maximum effect is observed 1 day after administration.

 Example 5. The study of the production of interferon.

Male CBA mice are injected intraperitoneally with standard interferon inducers at the doses indicated in Example 1. The level of interferon synthesis in the blood serum of mice is determined 24 hours after drug administration. The mass of mice is 12-14 g. Interferon titration is carried out in L-929 cell culture. As a test virus, 100 TCD50 of the mouse encephalomyocarditis virus is used. For the unit of interferon, the last dilution of the sample is taken, which provides 50% cell protection with complete destruction of the monolayer in the control. The standard IFN inducers administered intraperitoneally to mice were: Newcastle disease virus (VBI, 10 ⁸ CD ₅₀ / 0.2 ml), ridostin (yeast dsRNA, 100 μg / mouse), cycloferon (250 μg / mouse) and staphylococcal enterotoxin A (SEA, 10 μg / mouse).

 It was established (Table 5) that EP is not an inducer of interferon in the serum of mice, but it slightly increases the IFN-inducing ability of IFN inducers. A mixture of H + EP + B, H + EP + BC H + EP + BB1 has a significantly greater stimulating effect and a particularly strong stimulation of the interferon-inducing activity of standard interferon inducers is manifested in the claimed drug of the mixture H + EP + B + A, H + EP + B + serine, H + EP + medium 199, H + EP + medium RPMI 1640.

 Example 6. Correction of stress-induced immunodeficiency.

 For stress induction, experimental mice were kept under conditions of movement restriction (muscle deprivation). For 10 days, the mice were placed one by one in plastic chambers 8.5x4.0x2.0 cm in size for 7 hours daily. Control mice were kept in standard cages of 10 individuals (standard cell size 42x14x11 cm). On the 10th day of stressful exposure, experimental (and control) animals were injected with a test antigen - sheep erythrocytes, after which the animals were kept under normal conditions [4]. To determine the level of stress, the method of local hemolysis in gel described in Example 3 was used [3].

The following groups of mice were examined (10 animals per group):
1) control - receiving EB (5%) intraperitoneally in 0.5 ml;
2) subjected to stress - receiving EB on the 7th day of stress exposure;
3) mice that received EP on the 1st day of stress exposure, and EB on the 7th day;
4) mice that were injected with a mixture of vitamin C, EP and H on the 1st day of stressful exposure, and on the 7th day - EB;
5) mice that were injected with a mixture of vitamin B1, EP and H on the 1st day of stress exposure, and on the 7th day - EB;
6) mice, which on the 1st day of stress exposure were injected with a mixture of vitamins, EP and H, and on the 7th day - EB;
7) mice that on the 1st day of stress exposure were injected with the claimed drug a mixture of EP + H + B + A, and on the 7th day - EB;
8) mice that were administered the claimed agent containing H, B and EP2 on the 1st day of stress exposure (see Example 10), and on the 7th day - EB;
9) mice that were administered the claimed agent containing H, B and EP3 on the 1st day of stress exposure (see Example 10), and on the 7th day - EB;
10) mice that were administered the claimed agent on the 1st day of stress exposure containing N, EP and medium 199 at a dose of 0.3 ml / kg, and on the 7th day - EB;
11) mice that were administered the claimed agent on the 1st day of stress exposure containing H, EP and RPMI 1640 medium, and on the 7th day - EB;
12) mice that were administered the claimed agent containing EP, B and ridostin on the 1st day of stress exposure, and on the 7th day - EB;
13) mice that were administered the claimed agent containing H, EP, B and serine on the 1st day of stressful exposure, and on the 7th day - EB.

 The results presented in table. 6 show that stress caused by hypokinesia induces in mice a significant suppression of the primary immune response to EB (from 204 to 70 ATOK per 1 million splenocytes). A single injection of the placenta extract contributes to a certain increase in the number of ATOK, while the introduction of the claimed means of a mixture of EP + H + B + A restores the response to normal, completely preventing the development of stress-induced immunodeficiency. A slightly smaller effect is achieved with the introduction of other compositions.

 Example 7. Intoxication of various etiologies.

 The claimed agent, a mixture of EP + H + B + A (gamavit preparation) was used in more than 50 animals during the acute and subacute phase of the disease. When using this drug, convincing practical data were obtained on a faster detoxification process, animals were much more likely to get out of toxic conditions.

 Example 8. Anemia.

 The claimed tool is a mixture of EP + H + B + A (the drug gamavit) was used instead of ferroglukin for the treatment of anemia in calves and piglets. When using this drug, a more rapid restoration of the number of red blood cells and the level of hemoglobin was demonstrated compared with the control group in which ferroglukin was used.

 Example 9. Evaluation of the metabolic activity of the drug (by weight gain in mice).

 Male CBA mice weighing 10 g were used (previously, mice were weighed and only 10 g mice were used in the experiment). Daily, for 7 days, the mice were weighed and then intraperitoneally injected with the solutions at the doses indicated in Example 1 on days 1, 3, and 5. On day 7 weighed mice.

The following groups of mice were examined (10 animals per group):
1) control - received intraperitoneally physiological saline;
2) mice that were injected with EP;
3) mice that were injected with EP + H + BC;
4) mice injected with EP + H + BB1;
5) mice injected with EP + H + B;
6) mice that were injected with the claimed tool a mixture of EP + H + B + A;
7) mice injected with H + EP2 + B;
8) mice injected with H + EP3 + B;
9) mice injected with H + EP + 199;
10) mice injected with H + EP + RPMI;
11) mice injected with Reed + EP + B;
12) mice injected with H + EP + B + Serine.

 The results presented in table. 7, indicate a significant increase in metabolic activity under the influence of most of the mixtures used, but a particularly strong effect was observed after administration of the claimed agent (a mixture of EP + H + A + B or its analogues with 199, RPMI 1640 media).

 The total available data are presented in table. 8. It can be seen that the effect of the claimed means of a mixture of EP + H + B + A and its analogs with 199 or RPMI 1640 media significantly exceeds the prototype drug (EP) in most of the parameters studied. The total effect caused by the poorer mixture (EP + H + BC, EP + H + BB1, EP + H + B) is slightly lower.

 Example 10. Immunostimulating (adjuvant) properties of various placenta extracts.

Used the method of local hemolysis in the gel described in example 1. The following groups of mice were examined (10 animals per group):
1) control - received only EB;
2) mice that were injected with a placenta extract simultaneously with EB, similar to an allogeneic preparation of human placenta, as described in patent RU 2137486 (EP-1) at a dose of 0.16 μl / kg (4 μg / kg);
3) mice that were simultaneously injected with EB extract of the placenta obtained in accordance with patent US 4873222 (EP-2) at a dose of 0.16 μl / kg (3 μg / kg);
4) mice that were simultaneously injected with the placenta extract for injection at the same time as pharmaceuticals (see MD Mashkovsky, Medicines, M .: Medicine, 1987, v. 2, p. 154) (EP-3) at a dose of 0 16 μl / kg (8 μg / kg);
5) mice that were injected with a suspension of the placenta for injection simultaneously with EB - a pharmaceutical preparation (see MD Mashkovsky, Medicines, M .: Medicine, 1987, v. 2, p. 154) (EP-4) at a dose of 0 16 μl / kg (4 μg / kg);
6) mice that were injected with placenta extract simultaneously with EB - PAN veterinary preparation (EP-5) at a dose of 0.16 μl / kg (4 μg / kg), patent RU 2148408 dated September 14, 1999 - Method for producing biologically active tissue the drug.

 The results presented in table. 9 show that the administration of EP obtained from various sources to mice together with EB led to the formation of approximately the same number of antibody-forming cells. The data obtained indicate the same immunostimulating properties of various placenta extracts obtained in different ways.

 Example 11. The effect on the catabolism of endogenous proteins in dogs.

 Serum bilirubin, creatinine and urea levels depend on kidney function. With renal failure and with hepatitis, serum bilirubin, urea, transaminases, creatinine accumulate.

 In urine, bilirubin is not normally detected. Urea excretion is directly proportional to the rate of catabolism of endogenous proteins. A positive balance of urea and bilirubin is observed during the growth period, during pregnancy, etc. (V. T. Morozova, I. I. Mironova, R. L. Martishevskaya. "Urinalysis", M., 1996).

 In chronic renal failure in the blood, an increase in the level of bilirubin and transaminases is detected. In the table. 9 shows data on the content of bilirubin and urea in the blood of 2 dogs with hepatitis and severe poisoning before and after treatment with hamavitis, which indicate the effect of the drug on the catabolism of endogenous proteins. In addition, the appearance of bilirubin in the urine of both dogs after gamavit therapy (urea in the urine is not quantified), which, along with the removal of bilirubin and urea from the blood, reflects catabolytic function.

LITERATURE
1. Mashkovsky M.D. Medicines Vilnius, 1993, vol. 2, p. 146.

 2. The register of medicines of Russia. Encyclopedia of Medicines, ed. 8, 2001.

 3. Jerne N.K., Nordin A.A., Henry C. The agar plague technique for recognizing antibody-producing cells. In Amos B. and Koprovsky (ed.). Cell bound antibodies. Wistar Press Inst., 1963, p. 109-122.

 4. Davis D. E., Cristian J.J. Effect of population size on the adrenal glands and productive organs of male mice in populations of fixed size. Proc. Soc. Exp. Biol. (N.Y.), 1957, V. 94, pp. 227-231.

Claims (6)

 1. Immunomodulator - metabolic - detoxicant - adaptogen - radioprotector, including at least one placenta extract, at least one nucleic acid derivative and additionally at least one vitamin.  2. Immunomodulator - metabolic - detoxicant - adaptogen - radioprotector according to claim 1, characterized in that it contains at least one amino acid.  3. Immunomodulator - metabolic - detoxicant - adaptogen - radioprotector according to any one of paragraphs. 1 and 2, characterized in that as a nucleic acid derivative, sodium nucleate or ridostin is used.  4. Immunomodulator - metabolic - detoxicant - adaptogen - radioprotector according to any one of paragraphs. 1-3, characterized in that the vitamins of group B or vitamin C are used as the vitamin.  5. Immunomodulator - metabolic - detoxicant - adaptogen - radioprotector according to any one of paragraphs. 2 and 3, characterized in that it contains as a source of vitamins and amino acids, an environment for the cultivation of animal cells, including vitamins and amino acids.  6. Immunomodulator - metabolic - detoxicant - adaptogen - radioprotector according to claim 5, characterized in that it contains medium 199 or RPMI 1640 medium as a source of vitamins and amino acids.

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