RU2168335C2 - Method of correction of damaged immune homeostasis and drug - Google Patents

Abstract

FIELD: medicine, immunology. SUBSTANCE: method involves the use of potentiated form of antigen-substance taking part in development of pathological processes. Drug is prepared from this antigen by multiple dilution and shaking or triturating by homeopathic method. Substances of polypeptide, lipopoly-saccharide or polynucleotide structure are used as parent antigens. Invention can be used in immunotherapy for correction of immune homeostasis damage associated with antigen effect. EFFECT: enhanced effectiveness of immunotherapy, use of minimal amount of drug and parent antigen. 5 cl, 13 tbl, 24 ex

Description

 The invention relates to medicine, namely to immunology, and can be used in immunotherapy for the correction of immune homeostasis, a violation of which is associated with exposure to antigen.

 The immunological methods of treating various diseases by using drugs based on the antigen that causes these diseases are widely known (see, for example, Clinical Immunology, ed. L. Yeager, M .: Medicine, 1990, v. 1, p. 204-208; auth. St. USSR 1131508 cl. A 61 K 39/00, 1984; auth. St. USSR 1169659, cl. A 61 K 39/00, 1985; aut. St. USSR 1178450, class A 61 K 39/00, 1985; RF patent 2098186, class A 61 K 39/02, 1997). However, these methods of treatment are quite long and ineffective, especially for the treatment of immunodeficiency diseases with an unknown etiology.

 Closest to the invention is a method for correcting impaired immune homeostasis, comprising administering an antigen associated with the aforementioned disorder (see RF patent 2092184, class A 61 K 39/00, 1997).

 This method involves the use as antigens - protective antigens in successively increasing doses, mainly in combination with immunoadjuvants, which complicates the process of immunotherapy and not in all cases provides an effective therapeutic result.

 Known drugs for the correction of impaired homeostasis based on a treated antigenic carrier (see ed. St. USSR 1123704, class A 61 K 39/00, 1984; RF patent 2092184, class A 61 K 39/00, 1997 .).

 However, known drugs based on an antigenic carrier provide the required intensity of the immune response only at a significant dose, while many of them are effective only in combination with adjuvants.

 The invention is aimed at improving the effectiveness of immunotherapy by normalizing the regulation of impaired psychosomatic functions and / or biological reactions with minimal amounts of the drug and the original antigen.

 The solution of this problem is ensured by the fact that in the method of correcting impaired immune homeostasis, accompanied by a change in psychosomatic functions and / or biological reactions, including the introduction of a drug based on a substance having antigenic properties, according to the invention, a potentiated form of a substance, an antigen, is used as a drug in the regulation of the mentioned functions and / or reactions obtained by repeated dilution and shaking or grinding according homeopathic method.

 In addition, the solution of the problem is ensured by the fact that in the drug for the correction of impaired immune homeostasis based on a treated antigenic carrier, according to the invention, a potentiated form of the original antigen associated with impaired immune homeostasis obtained by repeated dilution and shaking or rubbing according to the homeopathic method is used.

Moreover, as the initial antigen for the potentiated form, a substance of a polypeptide structure is used;
while the substances of the lipopolysaccharide structure are used as the initial antigen;
in this case, as the initial antigen, substances of a polynucleopeptide structure are used.

 To prepare a potentiated form from a starting material with antigenic properties, which is soluble in a neutral solvent (distilled water, physiological saline, alcohol, etc.), a substance (for example, lyophilized protein, dry lipopolysaccharide, etc.) produces a uniform decrease concentration by successive dilution of 1 part of the substance in 9 parts (for tenfold dilution) or in 99 parts (for hundredth dilution) or in 999 parts (for thousandth dilution) neutral about the solvent with multiple vertical shaking ("dynamization") of each dilution obtained and using separate containers for each subsequent dilution to obtain the required potency - dilution ratio according to the homeopathic method (see, for example, W. Schwabe "Homeopathic medicines", M., 1967 g., pp. 14-29) in the dosage forms and dilutions accepted in homeopathic practice, mainly hundred (C1 - C10000).

 To prepare a potentiated form from the initial antigen-associated with the cells, a homogenate is prepared from the above-mentioned cells or cell nuclei, from which a supernatant is obtained by centrifugation, which, by sequential dissolution and repeated intermediate shaking according to the above homeopathic method in a neutral carrier, is brought to the required dilution, mainly hundred .

 Example 1

 The potentized drug is obtained from the original antigen - the main myelin protein (MBP) (bovine) by repeatedly sequentially dissolving and shaking from an industrially produced preparation in hundredth dilution, mainly in physiological saline.

 The method of administration is inside, 1-3 ml of a potentiated solution in a dilution of C50-C200.

 Indications for use: multiple sclerosis.

The main pharmacological effect is associated with:
with a decrease in the proliferative response of sensitized T lymphocytes to MBP.

 The proliferative response was evaluated in a blast transformation reaction in a suspension of mononuclear cells of a patient’s peripheral blood in the presence of a basic myelin protein. Evaluation was carried out before administration and 7 days after administration of potentiated antigen. The proliferative response after administration of the potentiated antigen was 53% of the original; with a decrease in the level of antibodies to MBP in serum.

 The level of antibodies to MBP in serum was assessed using enzyme-linked immunosorbent assay (ELISA) on standard test systems Flow Laboratories (UK). Evaluation was carried out before administration, 7, 14, 21, and 28 days after administration of the potentiated antigen. A statistically significant decrease in antibody levels was observed after 14 days. Subsequently, the level of antibodies continued to decline.

 Example 2

 The potentiated drug is obtained from the original antigen, the E.coli lipopolysaccharide, an industrially produced preparation by repeated serial dilution in phosphate-buffered saline and shaking in hundredth dilution, mainly C1-C1000.

 Indications for use: treatment of the septic process caused by E. coli.

 In the course of laboratory studies, the effect of the potentiated E. coli lipopolysaccharide on the course of the septic process in mice infected with E. coli was determined.

In the control group of mice, intravenous administration of a suspension of E. coli bacteria at a dose of 10 ¹⁰ caused sepsis.

 Five mice of CBA male mice in the experimental group 12 hours after the injection of the bacterial suspension were additionally injected with 200 μl of potentiated lipopolysaccharide (LPS) from the E. coli cell wall in phosphate-buffered saline (PBS) in C50 dilution.

 5 mice in the control group were injected with 200 μl of FSB.

 The survival time of the mice was determined.

 The research results are shown in table 1.

Conclusion:
Parenteral administration of a potentiated species-specific lipopolysaccharide eliminates the septic state caused by bacteria of this species.

 Example 3

 A potentiated nucleic acid-based drug is obtained from the original antigen, autologous DNA, by isolating the nuclei of the patient’s peripheral blood mononuclear cells, extracting the DNA from them, and then repeatedly diluting and shaking them with physiological saline in a potency of C10-C200.

 Indications for use: treatment of systemic lupus erythematosus (SLE).

 In the course of clinical trials, patient V., aged 20, II degree of activity of SLE, was administered with 2 ml of potentiated autologous DNA in a dilution of C50 1 time every week.

 The titer of antinuclear antibodies in serum was evaluated before administration, 7, 14 and 28 days after the start of the introduction of potentiated DNA using a standard technique (immunofluorescence on sections of rat liver). A statistically significant decrease in the titer of antinuclear antibodies was observed on days 14 and 28.

 Thus, the introduction of potentiated autologous DNA in SLE led to the remission of the symptoms of the disease and to a decrease in the titer of circulating antinuclear antibodies.

 Example 4

 The potentiated drug is obtained from the starting material with antigenic properties - the supernatant of the homogenate of B16 melanoma cells by repeated serial dilution and shaking in phosphate-buffered saline in a hundred-fold dilution.

Laboratory studies of the action of potentiated tumor proteins on the course of the tumor process were carried out on a model of melanoma in mice. Male C57BL mice were subcutaneously injected with a suspension of B16 melanoma cells into the hind limb (10 ⁶ ). In parallel, potentiated proteins were obtained from the supernatant of the homogenate of B16 melanoma cells.

 After the onset of clinical symptoms of melanoma (after 7 days), 5 mice of the experimental group were injected subcutaneously into the withers area with 200 μl of potentiated proteins in phosphate-buffered saline (PBS) at a dilution of C 100. In the control (5 mice), 200 μl of FSB was injected.

 The severity of tumor progression was assessed by the degree of dissemination of the primary tumor node, by the defeat of regional lymph nodes, by the size and morphology of the foci. The survival rate of animals was also determined.

 The research results are shown in table 2.

Conclusion:
Parenteral administration of potentiated tumor proteins slows the progression of the malignant tumor and increases the survival time.

Example 5 (in vivo)
The effect of potentiated antigen on the induction of HRT reaction.

 Principle of the method: Sensitized T-effector HRT lymphocytes in the area of the administration of the resolving dose of antigen secrete mediators, leading to local tissue infiltration by mononuclear cells and NF, swelling of the stroma and local edema. If the antigen is injected into the foot of the hind limb, an increase in its mass and volume is observed.

CBA mice were sensitized by subcutaneous injection of methylated bovine serum albumin (MBSA) into the withers in various modifications:
1) 3 mice were injected with 100 μl of 0.1% MBSA and 100 μl of 0.25% Evans blue solution (adjuvant) in phosphate-buffered saline (PBS).

 2) 100 μl of 0.1% MBSA and 100 μl of FSB were injected into 3 mice.

 3) 200 μl of potentiated MBSA in PBS in C50 dilution was injected into 3 mice.

 4) 3 mice were injected with 200 μl of FSB (control).

 On the sixth day, under the corn tubercle of the fourth finger of one of the hind limbs, a resolution dose of 0.125% MBSA 20 μl was administered. 20 μl of PBS was injected into the control paw.

 Evaluation of the results.

24 hours after the resolving injection of antigen, the severity of the local reaction was assessed by the difference in weight of the experimental (P _o ) and control paws (P _k ). Both legs were cut off immediately after slaughtering the animals along the protrusion of the bone below the junction of the tibia and tibia and above the heel joint. The reaction index (IR) was calculated for each mouse according to the formula: IR = (P _about -P _to ) • 100 / P _to . With IR above 5%, the difference from the control is considered significant.

 The research results are presented in table 3.

Conclusion:
The introduction of antigen in potentiated form causes the activation of the cellular immunity, comparable with the introduction of antigen with adjuvant.

 Example 6 (in vivo).

 The effect of potentiated antigen on the humoral immune response.

 Intravenous administration of antigen to animals leads to the development of a primary immune response in the form of the production of immunoglobulins, the content of which in the serum can be semi-quantified using enzyme-linked immunosorbent assay.

Male chinchilla rabbits of the same age and weight were immunized intravenously with ovalbumin according to the following scheme:
1) 3 rabbits - 10 mg of ovalbumin in 2 ml of FSB;
2) 3 rabbits - potentiated ovalbumin in 2 ml of FSB diluted in C50;
3) 3 rabbits - 2 ml of FSB (control).

 After 5, 7 and 12 days, blood was taken from the ear veins of the rabbits, and serum was obtained. Then, in accordance with the standard method of indirect solid-phase ELISA (with goat antibodies to rabbit IgG and IgM labeled with horseradish peroxidase, substrate - CRF), the serum content of antibodies to ovalbumin was analyzed.

 By interpolating the results with 5 serum titers (in the region of the linear dependence of the optical density on the titer), the serum titer was determined, which gave an optical density at 492 nm of ELISA equal to 1.0.

 The research results are shown in table 4.

Conclusion:
The introduction of antigen in potentiated form causes an earlier and more intense primary humoral immune response.

 Example 7 (In vitro).

 The effect of potentiated antigen on the level of sensitization of lymphocytes. Assessment using the reaction of the blast transformation of lymphocytes (RBTL).

 Sensitized peripheral blood lymphocytes are able to divide in vitro in response to contact with a specific antigen, and this process reflects the functional activity of immunocompetent cells.

CBA mice were sensitized by subcutaneous injection of methylated bovine serum albumin (MBSA) into the withers in various modifications:
1) 3 mice were injected with 100 μl of 0.1% MBSA and 100 μl of 0.25% Evans blue solution (adjuvant) in phosphate-buffered saline (PBS).

 2) 6 mice were injected with 200 μl of potentiated MBSA in FSB.

 3) 3 mice were injected with 200 μl of FSB (control).

After 8 days, the mice were decapitated, blood was sterile, from which mononuclear cells were isolated in a ficoll-verographin gradient. The cell concentration was adjusted to 5 • 10 ⁵ cells / ml with RPMI 1640 medium with 10% fetal calf serum, 12 mM HEPES, glutamine (300 μg / ml), antibiotics - penicillin and streptomycin (100 μg / ml each).

By automatic micropipettes, the suspension was poured into 200 μl into the wells of a 96-well plate for immunological reactions. 20 μl of RPMI 1640 medium was added to the wells of the control cultures of each group. Antigen was added to the test wells according to the following scheme:
group 1-1: 20 μl of a 0.05% MBSA solution on FSB;
group 2-1: 20 μl of a 0.05% MBSA solution on FSB;
group 2-2: 20 μl of potentiated MBSA on the FSB in a dilution of C50;
group 3-1: 20 μl of a 0.05% MBSA solution on FSB;
group 3-2: 20 μl of potentiated MBSA on the FSB.

 Each culture variant (without antigen and with antigen) occupied 3 wells.

Culture for 48 hours was placed in a thermostat at 37 ^o C in a humid atmosphere with 5% CO ₂ . During the last 4 hours of cultivation, 20 μl of a solution of H ³ -thymidine with a specific activity of 10 μCi / ml was added to each well.

 After deposition of the cells on glass fiber filters using an automatic cell harvester (Harvester), the filters were dried; then, each section of the filter onto which cells from each well were deposited was transferred into a vial of 2 ml of scintillation fluid (4 g PPO and 0.1 g POPOP per 1 kg of toluene). Isotope incorporation was counted in a scintillation counter.

 To determine the proliferative activity of lymphocytes, the average value of isotope incorporation in 3 control cultures (spontaneous proliferation - SP), as well as the average value of isotope incorporation in 3 cultures with antigen (induced proliferation - PI) was calculated.

To assess the proliferative activity of cells used the stimulation index (IS):
IP = log (IP / SP).

 The research results are shown in table 5.

Conclusion:
The introduction of antigen in potentiated form provides a more active antigen-specific sensitization of T lymphocytes. The potentiated antigen is also a more active inducer of the proliferation of sensitized T lymphocytes.

 Example 8 (in vitro).

 The effect of potentiated antigen on the migratory activity of leukocytes.

 The leukocytes placed in the well of the agarose medium are capable of spontaneous migration, forming an annular cell accumulation zone (migration zone) around the well. By the magnitude of this zone, one can judge the degree of migration activity of leukocytes. Under the influence of immunoregulatory factors secreted by lymphocytes, inhibition of the leukocyte migration ability may be observed.

 CBA mice were sensitized by subcutaneous injection of methylated bovine serum albumin (MBSA) into the withers region: 6 mice were injected with 100 μl of 0.1% MBSA and 100 μl of 0.25% Evans blue solution (adjuvant) in phosphate-buffered saline (FSB).

 After 8 days, the mice were decapitated, heparinized blood (1-2 units of heparin / ml) was sterile, from which a leukocyte suspension was isolated. The resulting cells were washed twice with medium 199 by centrifugation at 850 rpm./min for 8-10 minutes.

 For the reaction, tablets with 0.3 ml flat-bottomed cells were used. A substrate (1 μl of a 0.8% agarose solution) was applied to the center at the bottom of each cell with a microsyringe. After 10-15 minutes at room temperature, the plate was layered on a substrate with 2 μl of a suspension of leukocytes in 0.2% agarose solution prepared on medium 199 containing 15% inactivated fetal calf serum.

The tablets were placed for 5-8 minutes in a refrigerator at 4 ^o C, after which 0.2 ml of 199 medium was introduced into each well:
1) with 0.05% MBSA;
2) with potentiated MBSA dilution C50;
3) control - without antigen.

The tablets were placed in a thermostat at 37 ^o C for 48 hours.

 Evaluation of the results was carried out under a microscope (x20) using an ocular micrometer by determining the average linear values of cell migration in four mutually perpendicular directions, measured from the edge of the agarose substrate to the boundary of the migrating cell layer, conventionally determined at the transition point of the zone of small cell agglomerates into the zone of diffusely located isolated isolated cells.

The migration index (MI) was calculated by the formula
MI = (L / L _k ) • 100%,
where L, L _k are respectively the average (medians) values of the migration zone (in units of the micrometer scale) with the experimental and control medium obtained in 12 cells. MI was calculated according to the results of 5 independent experiments.

 The research results are presented in table 6.

Conclusion:
In the presence of a potentiated antigen, sensitized T lymphocytes more actively secrete immunoregulatory mediators, which provides a more active reaction of inhibition of leukocyte migration.

 Example 9 (in vitro).

 The effect of potentiated antigen on the activity of natural killers.

 (Principle of the method) Natural killer cells (EC) are lymphoid cells responsible for the spontaneous cytolysis of tumor and virus-modified cells independent of the products of the main histocompatibility complex. EC activity was determined in a cytotoxic test for the lysis of target cells (KM) labeled with radioisotopes, the tumor line of K-562 cells (human myelogenous leukemia). A suspension of mononuclear cells of peripheral blood of 5 healthy donors was used as test effector cells.

A suspension of mononuclear cells (effector cells — EC) obtained on a ficoll-verographin gradient was diluted to a concentration of 1 • 10 ⁷ in 1 ml.

The target cells (KM) of K-562 (3rd day of cultivation) were labeled with ³ N-uridine at a dose of 3 μCi per 1 ml of cell culture. For this, the cell suspension at a concentration of 5 • 10 ⁵ in 3 ml of culture medium was incubated with an isotope (specific activity 24 Ci / mm) for 1 hour at 37 ^o C, washed three times with Needle medium with 10% bovine serum and antibiotics, centrifuged at 400g at 20 ^o C 10 minutes. The washed target cells were kept in the culture medium for 2 hours at 37 ^° C and washed again to remove derivatives of the isotope that were not included in the RNA of the target cells. After this, a working suspension of labeled KM was prepared, which contains 1 ml of 10 ⁶ cells and 5 μg of pancreatic RNAase in 1 ml.

 The cytotoxic reaction was carried out in round bottom 96-well microplates. In each well, 100 μl of working suspension and labeled KM and 100 μl of EC suspension were placed, which was diluted so that the ratio EC: KM = 50: 1 was maintained; 25: 1; 12: 1. Each dilution of EC was placed in three cells. Control samples were labeled KM without the addition of EC. 20 μl of the potentiated tumor antigen, C50, obtained by potentiating the homogenizate of K-562 cell line in FSB, was also added to the experimental series of cells. 20 μl of PBS was added to the remaining wells.

1 series: EC + KM (+ FSB)
2 series: EC + KM + potentiated antigen KM
3 series: KM (+ FSB)
The filled plate was incubated for 16 hours in a humid atmosphere at 37 ^° C. in a CO ₂ incubator. After incubation, the contents of the cells were transferred to membrane filters with a pore diameter of 2.5 μm, washed.

The functional activity of EC was evaluated by the difference in radioactivity detected on a scintillation β-counter in cells containing and not containing EC. The cytotoxicity index was determined by the formula
IC = (IMP _t / IMP _k - 1) • 100.

IMP _t , IMP _k - the number of pulses in the test cells and control cells.

 The average IC value was found in 5 healthy donors (± standard deviation).

 The research results are shown in table 7.

Conclusion:
The potentiated antigen isolated from the tumor increases the cytotoxic activity of natural killers against tumor cells.

 Example 10 (In vivo).

 The effect of potentiated antigen on allograft rejection.

 As recipients, male CBA mice were used.

 As donors, male mice of the C57B1 line were used.

 The potentiated antigen was obtained from the graft homogenate from the skin of the tail of the donor mouse. The potentiated antigen in physiological saline (2 ml, C50) was administered per os to recipient mice once a day before transplantation (5 mice, group 1) and one day after transplantation (5 mice, group 2). In the control (5 mice), nat. solution.

 Methods of skin transplantation from tail to back. The donor mouse was euthanized and its tail cut off. The tail was rubbed with ethanol and cut the skin with scissors along the tail. Tweezers completely separated the skin. Dale skin was cut into pieces 5x10 mm in size. In recipient mice, a piece of back skin was cut out on the background of deep anesthesia. The cut surface was slightly larger than the graft surface. The graft was transferred to the wound and fixed with an anchorplast. A dressing with lanolin and a wet gypsum dressing were placed on top, which was removed after a week. After removal of the gypsum, a daily inspection of the grafts and evaluation of the rejection reaction were carried out.

 The research results are shown in table 8.

Conclusion:
Oral administration to the recipient of a potentiated antigen from the skin of the donor before or after skin transplantation reduces the activity of rejection of the allograft and prolongs its survival.

 Example 11

 Patient P., 38 years old. Diagnosis: Multiple sclerosis, group II disabled person, progressive course.

 He became ill 12 years ago. The predominant symptomatology is intermittent paresis of the lower extremities (it is not able to move independently at the height of an exacerbation, it moves with crutches in a state of remission), atactic gait, intentional tremor, diffuse microsymptomatics from the side of craniofacial nerves, rare epiprots. Every year, exacerbations lengthen, accompanied by an increasingly pronounced weakness in the legs. The ongoing hormotherapy does not inhibit the progreduation of the disease.

 For the last 1.5 years, every other day in the morning he receives 2 ml of a potentiated preparation in a dilution of C100 obtained from an antigen - the main myelin protein (MBP) (bovine). The condition has noticeably improved - increased muscle strength in the limbs, no exacerbations. It moves independently. Epiprots during the period of receiving potentiated MBP were observed twice. Of the coordination disorders, only intentional tremor is present.

Laboratory data are as follows:
Decrease in the proliferative response of sensitized T lymphocytes to MBP.

 The proliferative response was evaluated in a blast transformation reaction in a suspension of mononuclear cells of a patient’s peripheral blood in the presence of a basic myelin protein. Evaluation was carried out before administration and 7 days after administration of potentiated antigen. The proliferative response after administration of the potentiated antigen was 53% of the original.

 Decrease in the level of antibodies to MBP in serum.

 The level of antibodies to MBP in serum was assessed using enzyme-linked immunosorbent assay (ELISA) on standard test systems Flow Laboratories (UK). Evaluation was carried out before administration, 7, 14, 21, and 28 days after administration of the potentiated antigen. A statistically significant decrease in antibody levels was observed after 14 days. Subsequently, the level of antibodies continued to decline.

 Example 12

 Patient S., 43 years old. Diagnosis: Hashimoto's autoimmune thyroiditis.

 Ill 3 years ago. Periodically occurring exacerbations are manifested by symptoms of hypothyroidism. When examined by an endocrinologist in January 1997, she was in a state of unstable remission; complained of difficulty in swallowing, a feeling of lack of air, general weakness. On examination, a symmetric enlargement of the thyroid gland of the II-III degree was found. The gland on palpation is dense, knotty, painful. Appointed therapy: 5 drops of an alcoholic solution twice a day of a potentiated antigen - porcine thyroglobulin in potency C12. 1.5 months after the start of treatment, there is no increase in the thyroid gland, swallowing is free. No complaints. As a preventive measure, he continues to take potato thyroglobulin 1 ml, C12, once a month.

Laboratory data are as follows:
Decrease in the proliferative response of sensitized T lymphocytes to thyroglobulin;
The proliferative response was evaluated in a blast transformation reaction in a suspension of mononuclear cells of a patient’s peripheral blood in the presence of thyroglobulin. Evaluation was carried out before administration and 7 days after administration of potentiated antigen. The proliferative response after administration of the potentiated antigen was 57% of the original.

 Decrease in the level of circulating antibodies to thyroglobulin.

 The level of antibodies to thyroglobulin in serum was assessed using enzyme-linked immunosorbent assay (ELISA) on standard test systems Flow Laboratories (UK). Evaluation was carried out before administration, 7, 14, 21, and 28 days after administration of the potentiated antigen. A statistically significant decrease in antibody levels was observed after 14 days. In the future (on days 21 and 28), the level of antibodies continued to decline.

 Example 13

 Patient T., 40 years old. Diagnosis: Rheumatoid arthritis.

 Sick for 11 years. The clinical picture is dominated by paroxysmal damage to the joints of the feet, to a lesser extent - brushes, morning stiffness. Anti-inflammatory therapy does not restrain the progression of the process: pain and swelling of the joints, as well as the severity of erosion during x-ray examination increases every year, the duration of exacerbations increases.

The ingestion of 5 ml of an aqueous solution twice a day of a potentiated antigen - human type II collagen in the dilution of C24 significantly improved the course of the disease. After 3
weeks of therapy, swelling and soreness of the joints, as well as morning stiffness, disappeared. The patient moves independently.

Laboratory data are as follows:
Decreased proliferative response of sensitized T lymphocytes to type II collagen;
The proliferative response was evaluated in a blast transformation reaction in a suspension of patient peripheral blood mononuclear cells in the presence of type II human collagen. Evaluation was carried out before administration and 7 days after administration of potentiated antigen. The proliferative response after administration of the potentiated antigen was 45% of the original.

 Decrease in the level of circulating antibodies to type II collagen.

 The level of antibodies to collagen in serum was evaluated using enzyme-linked immunosorbent assay (ELISA) on standard test systems Flow Laboratories (UK). Evaluation was carried out before administration, 7, 14, 21, and 28 days after administration of the potentiated antigen. A statistically significant decrease in antibody levels was observed after 14 days. In the future (on days 21 and 28), the level of antibodies continued to decline.

 Example 14

 Patient S., 48 years old, a disabled person of group II, has been observed for 5 years in a neuropsychiatric dispensary with a diagnosis of Psycho-organic syndrome of traumatic origin. Asthenic variant.

 The clinical picture is dominated by severe cerebration, mental exhaustion, meteorolysis. Constantly complains of headache and fatigue, poor memory. Conducted therapy: nootropin, courses of vitamins B6 and B12, sedatives, does not cause a significant change in state.

 In January - February 1997, he received preparations according to the rules of homeopathy, a potentiated preparation of a brain species-specific tissue-specific protein 14-3-2 in a potency of C200 of 5 granules, saturated with a potentized alcohol solution, once a day.

 During the course of the therapy, the phenomena of cerebral growth disappeared, and the memory improved markedly. The patient is subjectively considered to be practically healthy, able to begin the previous work of a computer operator.

 Example 15

 Patient T., 21 years old. He entered the addiction department 4 with a diagnosis of Opium addiction, II st. Abstinence syndrome. Traditional detoxification therapy, carried out for 1 day, did not bring noticeable improvement. From 2 days the patient was prescribed potentiated brain-specific protein S-100 prepared according to the rules of homeopathy in a potency of C 1000 in the form of a 45% alcohol solution - 5 drops every hour.

 2 hours after the first dose, the patient felt much better: bone pains, lacrimation, anxiety and the patient fell asleep. By the end of their stay in the department of the third day, the phenomena of withdrawal symptoms were almost stopped.

 Example 16

 Patient D., 42 years old, has been observed for 8 years in a polyclinic at a narcologist at the place of residence with a diagnosis of Chronic alcoholism of the second degree, pseudo-dipsomanic form. Despite the ongoing therapeutic measures, he continues to abuse alcohol, the duration of binges increases, and periods of abstinence decrease.

Since March 1997, he has been receiving once a week 2 ml of an aqueous solution of C50 prepared according to the rules of homeopathy of a potentiated preparation of a brain-specific protein α ₂ -glycoprotein. The desire for alcohol gradually decreased, the patient began to take alcohol less frequently, and since May 15, 1997 he has not consumed them at all. As of March 10, 1998, he is in remission; there is no attraction to alcohol.

 Example 17

 Patient P., 25 years old, has been suffering from amyotrophic sclerosis (lumbosacral form) for several years, manifesting as spastic-atrophic paresis of the legs. Conducted therapy did not bring noticeable effect. Since December 1996, once a day, in the morning, he receives 1 tablet of potentiated brain-specific glial-fibrillar protein C30. Gradually, the patient's condition improved: sensitivity appeared in the legs and feet, muscle cramps disappeared, muscle strength in the affected limbs increased, reflexes decreased. Since January 1997, a previously bedridden patient began to move with a stick.

 Example 18

 Patient X., 16 years old. He entered intensive care with traumatic shock and heavy bleeding from damaged lower extremities. When transfusing according to vital signs of blood of the same group, the patient developed an acute pyrogenic reaction, stopped by the introduction of potentiated blood in a C 12 dilution of the same donor.

 Example 19

 Patient X., 40 years old. Suffers more than 20 years of chronic alcoholism. In February 1998, the patient was injected subcutaneously with the tabulated drug teturam, which blocks the acetaldehydroxidase enzyme. 10 days after this procedure, the patient resumed drinking alcohol, as a result of which he developed a severe somatovegetative state: there was redness and a feeling of heat in the face and upper body, a feeling of tightness in the chest, shortness of breath, palpitations, a sense of fear, finely spread tremor; blood pressure decreased.

 Oral administration of 5 drops of a potentiated preparation of acetaldehydroxidase in a dilution of C 12 once within 30 minutes normalized the patient's condition.

 Example 20

 The effect of potentiated plasmin (fibrinolisan) on fibrinolysis.

Fibrinolysin (an official preparation obtained from human blood plasma) was administered to male chinchilla rabbits of the same age as follows:
1) 1 group (5 rabbits) - 100 IU of fibrinolysin in 2 ml of isotonic sodium chloride solution, intravenously.

 2) group 2 (5 rabbits) - potentiated fibrinolysin in a dilution of C50, in 2 ml of isotonic sodium chloride solution, intravenously.

 3) group 3 (5 rabbits) (control) - 2 ml of isotonic sodium chloride solution, intravenously.

 After 2 hours, whole unstabilized blood (5 ml) was taken from the rabbits and thromboelastography was performed in accordance with the standard procedure.

 Fibrinolysis indices were evaluated by the time of elasticity reduction of the formed clot after reaching maximum values to 1/5 of the maximum value (T).

 The research results are presented in table 9.

Conclusion:
Potentiated fibrinolysin accelerates fibrinolysis in whole unstabilized blood. The effect of potentiated fibrinolysin is more pronounced compared to fibrinolysin in a therapeutic dose.

 Example 21

 The effect of potentiated cytochrome C on the activity of oxidative phosphorylation.

Cytochrome C in the form of an official preparation (from the tissue of the heart of cattle) was administered to purely linear male rats of the same age according to the following scheme:
1) 5 rats - 1 mg of cytochrome C in 2 ml of isotonic sodium chloride solution, subcutaneously.

 2) 5 rats - potentiated by cytochrome C (C50) in 2 ml of isotonic sodium chloride solution, subcutaneously.

 3) 5 rats (control) - 2 ml of isotonic sodium chloride solution, subcutaneously.

 After 4 hours, the rats were sacrificed, in accordance with the standard method, intact mitochondria were isolated from the liver.

 Intact mitochondria in an environment with oxidation substrates (potassium succinate, 10 mM) and oxygen are characterized by a low respiration rate. With the addition of acceptors of energy accumulated in the respiratory chain (ADP + inorganic phosphate), the respiratory rate increases sharply. The rate of depletion of energy acceptors characterizes the activity of oxidative phosphorylation in the respiratory chain. The time of depletion of energy acceptors was determined by the polarographic curve of registration of oxygen consumption by mitochondria (ADP / t) per unit mass of a suspension of mitochondria.

 The activity of oxidative phosphorylation was evaluated by the parameter of the polarographic curve - ADP / t (mol / min • mg protein).

 The research results are shown in table 10.

Conclusion:
Potentiated cytochrome C stimulates oxidative phosphorylation (stimulates tissue respiration) more actively than cytochrome C in a therapeutic dose.

 Example 22

 The effect of potentiated insulin on the concentration of glucose in the blood.

Healthy rabbits of the chinchilla breed were injected with insulin for injection according to the following scheme:
1) 5 rabbits - 5 PIECES (action units) in 2 ml in water, acidified with hydrochloric acid, subcutaneously, once.

 2) 5 rabbits - potentiated insulin in the dilution of C50 in 2 ml of water for injection.

 3) 5 rabbits (control) - 2 ml of water for injection.

 Prior to insulin administration, blood was taken from rabbits 20 minutes, 1 and 4 hours after the injection, and the glucose content was determined by the glucose oxidase method using phenolphthalein oxidation (in mmol / l).

 The research results are shown in table 11.

Conclusion:
Potentiated insulin causes a more rapid decrease in blood glucose levels than insulin in a therapeutic dose.

 Example 23

 The effect of potentiated thyroid-stimulating hormone on the concentration of thyroxine in the blood.

Healthy rabbits of the chinchilla breed were injected with thyrotropin for injection according to the following scheme:
1) 5 rabbits - 5 PIECES (action units) in 2 ml of isotonic sodium chloride solution.

 2) 5 rabbits - potentiated thyrotropin in 2 ml in a dilution of C50 isotonic sodium chloride solution.

 3) 5 rabbits (control) - 2 ml of isotonic sodium chloride solution.

 Prior to thyrotropin administration, blood was taken from rabbits 12, 24 and 48 hours after administration and the total thyroxine level (in μg%) was determined using standard radioimmunoassay methods.

 The research results are shown in table 12.

Conclusion:
Potentiated thyrotropin more actively stimulates an increase in the level of total thyroxin in the blood than thyrotropin in a therapeutic dose.

 Example 24

 The effect of potentiated collagen on the course of the wound process.

In rabbits of the chinchilla breed, a 3 cm ² skin flap was aseptically removed and the effect of the following preparations on the course of the wound process was studied:
1) 5 rabbits - a collagen film moistened with an isotonic solution of sodium chloride;
2) 5 rabbits - potentiated collagen (type 1), in a dilution of C50, in an isotonic solution of sodium chloride;
3) 5 rabbits (control) - isotonic sodium chloride solution.

 After treatment with one of these preparations, the wound was covered with a gauze dressing. Wounds were examined daily, the area (S) of the wound surface, and the time of scar formation were estimated. The thickness of the scar was evaluated on a slice after 1 month (after euthanasia of the rabbits).

 The research results are shown in table 13.

Conclusion:
Potentiated type 1 collagen, when applied topically, accelerates the healing of aseptic wounds, accelerates scar formation, and promotes the formation of a relatively more tender scar.

Claims (5)

 1. A method of correcting impaired immune homeostasis, accompanied by a change in psychosomatic functions and / or biological reactions, comprising administering a drug based on a substance having antigenic properties, characterized in that a potentiated form of a substance, an antigen, involved in the regulation of the above functions is used as a medicine and / or reactions obtained by repeated dilution and shaking or grinding by the homeopathic method.  2. A drug for the correction of impaired immune homeostasis based on a treated antigenic carrier, characterized in that a potentiated form obtained by repeated dilution and shaking or grinding by a homeopathic method from the original antigen associated with impaired immune homeostasis is used.  3. The drug according to claim 2, characterized in that the substances of the polypeptide structure are used as the initial antigen for the potentiated form.  4. The drug according to claim 2, characterized in that the substances of the lipopolysaccharide structure are used as the initial antigen for the potentiated form.  5. The drug according to claim 2, characterized in that the substances of the polynucleotide structure are used as the initial antigen for the potentiated form.

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