Stimulator of endogenous production of cytokines and geopoliticheskih factors and the method of its use

 

(57) Abstract:

The invention relates to medicine and can be used for the prevention and treatment of diseases in which appropriate stimulation of endogenous production of cytokines and hemopoietic factors. According to the invention stimulator of endogenous production of cytokines and hemopoietic factors is oxidized glutathione, which represents a dimer of glutathione restored, with the structure of gamma-glutamylcysteinylglycine, in which two molecules of Tripeptide with the above structure are connected to each other by covalent disulfide bond between cysteine residues. According to the invention pharmacological tool, proposed for the treatment of diseases in which appropriate stimulation of endogenous production of cytokines and hemopoietic factors, contains as active ingredient an effective amount of oxidized glutathione. It is reasonable to dosage form of this pharmacological means represented by injecting a solution of oxidized glutathione, in a pharmaceutically acceptable solvent in a concentration of 0.01 to 2.0 %. According to the invention for amplification and prolongation of therapeutic effeiciency acceptable component, able to prolong the stay of glutathione in the oxidized form, such as hydrogen peroxide in a concentration of 0.003 %. According to the invention a method of treating diseases in which appropriate stimulation of endogenous production of cytokines and hemopoietic factors, includes parenteral pharmacological tools in the form of injectable dosage forms, doses of 0.01 - 0.5 mg per kg of body weight, of one or more injections a day, a course lasting one or more days or continuously until a therapeutic effect. 2 s and 5 C. p. F.-ly, 21 tab., 4 Il.

The invention relates to medicine, in particular to pharmacology and therapy, and can be used for the prevention and treatment of cancer, infectious and other diseases in which stimulation of the endogenous production of cytokines and hemopoietic factors appropriate for reasons that are obvious to a person skilled in the relevant field of medicine.

It is known that a number of humoral factors, endogenous produced in mammals, cytokines and hemopoietic factors have high biological activity and can have a significant therapeutic effect in various diseases in ceacescu medicine. So in oncological practice widely studied application of interleukin-2 (IL-2), tumor necrosis factor a (TNFa), hematopoietic factors (erythropoietin, macrophage-granulocyte and granulocyte colony-stimulating factor, GM-CSF and G-CSF). Not less intensively used cytokines and hemopoietic factors in experimental and clinical practice of infectious diseases, such as interferon (IFNg and IFNa), kolonie-stimulating factors and other Colonie-stimulating factors and erythropoietin are widely used in Hematology.

It should be noted that therapeutic use of these substances introduced exogenously, has limitations related to the lack of acceptable dosage forms or their high cost, short life of the substances of this class in biological environments, difficulties in selection of doses, as well as a variety of toxic and allergic effects.

In this regard, from the point of view of achieving a more stable and meaningful therapeutic effect is not accompanied by side effects, it is preferable to stimulation of endogenous production of autologous cytokines and hemopoietic factors directly in the body of the patient. Treatment effective what's with exogenous introduction of cytokines and hemopoietic factors.

The number of known compounds that stimulate endogenous production of cytokines and hemopoietic factors in experimental and clinical conditions, including known about the use of microbial products for the treatment of cancer, which is associated with the stimulation of endogenous production of tumor necrosis factor. Products that can appeal simultaneous production of various cytokines and hemopoietic factors, called multi-cytokine inducers (Multi-cytokine inducer). By means of such actions include the preparation of killed Streptococcus, Nocardia (Nocardia opaca) and other bacterial products.

However, almost all substances with a similar ability, or killed microorganisms or microbial products or compounds with unknown or variable structure, which significantly limits the possibility of their medical use for medicinal purposes, and in some cases makes this application possible. Thus, until the present time, no known acceptable from the point of view of modern medicine and pharmacology stimulator of endogenous production of cytokines and hemopoietic factors.

In search of medical and pharmacologically acceptable stimulator of endogenous prove property of a known substance oxidized glutathione (glutathione oxidized, glutathione disulfide, GSSG; next GSSG), administered parenterally or acting on isolated cells, stimulating the production of several cytokines and hemopoietic factors in mammals (experimental animals and humans) in both normal and pathological conditions.

Oxidized glutathione (oxidized glutathione, glutathione disulfide, GSSG; next GSSG) is known as the dimer of Tripeptide glutathione-glutamylcysteinylglycine, in which two molecules of Tripeptide with the above structure are connected to each other by covalent disulfide bond between cysteine residues. Thus, as the Tripeptide glutathione (glutathione, glutathione restored GSH; next GSH) and its dimer, GSSG are natural metabolites and are present in tissues and biological fluids of humans and animals. In this case, as in normal and pathological conditions of the natural level of GSSG in the blood and body tissues is insufficient to stimulate endogenous production of cytokines.

Known properties of GSH, as one of the most important participants in the metabolism of amino acids and the factor of maintenance of intracellular homeostasis. Great importance to have restorative properties of GSH and its function donor wossy cysteine residue. This quality GSH is defined and its role as a fundamental element of one of the most important intracellular antioxidant systems, including specific GSH and two enzymes of its reversible conversion to GSSG: glutathione peroxidase and glutathione reductase. Continuous operation of this system is necessary for inactivation or recovery as endogenously generated oxidants and active metabolites of the substances that enter the body from outside.

It is also known involvement of GSH in providing detoxification reactions occurring with the participation of a group of enzymes that are grouped by the name of glutathione S-transferase. These enzymes are capable of kongugirovanii molecule of GSH to a variety of xenobiotics, forming a link between them and the glutathione via the thiol group of the cysteine residue of Tripeptide. Subsequent degradation of the conjugate is carried out by enzymes gamma-glutamine cycle and can have significant variations, depending on the nature of the xenobiotic.

In vivo accumulation of GSSG in quantities sufficient to stimulate production of cytokines and hemopoietic factors, is due to the permanent restoration of GSSG to GSH. Restoration of GSSG to GSH, active protekauschie product, consisting of amino acids, undergoes proteolytic degradation.

Know about using GSSG as a component of dietary supplements for enhanced nutrition patients. However, the oral application the bulk of GSSG as substances with a peptide nature, degraded in the digestive tract, and the remainder is recovered in the cells of the intestine and the liver to GSH and does not come into General circulation. Thus, the supply of GSSG into the body through the digestive tract eliminates the possibility of manifestations of his activity as a stimulator of endogenous production of cytokines and hemopoietic factors.

It is known that the increase of endogenous GSH level for medicinal purposes is proposed to stimulate the immune system in the treatment of intoxication, poisoning, diabetes, cardiovascular, infectious, and other diseases.

It is also known the use of exogenous GSH or its direct (g-glutamyl-cysteine, n-acetyl-cysteine, n-acetyl-containingin) or indirect (2-oxothiazolidine-4-carboxylate) biochemical precursors, and their salts and esters as medicines or food supplements in the treatment of various diseases. By application of any and intoxications.

It is also known about the use of GSH as chemoprotective agent in the prevention of neurotoxicity in cancer chemotherapy, and combinations of GSH with anticancer drugs to increase the effectiveness of their actions.

At the same time, according to publicly available sources of information, currently known about the use of GSSG as an independent pharmaceutical agents (in the form of-material), as a stimulator of endogenous production of cytokines and hemopoietic factors, and its therapeutic effects in diseases of humans and animals or on the application of GSSG as a drug for the treatment of diseases.

The basis of the invention is the creation of pharmaceutical products containing this active substance, which stimulates the endogenous production of cytokines and hemopoietic factors for a subject, in need thereof.

"Subject in need" means a mammal, such as people, Pets and livestock, with one or more manifestation of diseases in which stimulation of the endogenous production of cytokines and hemopoietic factors expedient from the standpoint of modern biomedical newaudio stimulation of endogenous production of cytokines and hemopoietic factors from the subject, in need of this, it is proposed to use GSSG, which parenteral stimulates the production of several cytokines and hemopoietic factors in mammals (experimental animals and humans) in both normal and pathological conditions.

According to the invention stimulator of endogenous production of cytokines and hemopoietic factors is oxidized glutathione (GSSG), which is a dimer of glutathione restored, with the structure of g-glutamyl-containerline, in which two molecules of Tripeptide with the above structure are connected to each other by covalent disulfide bond between cysteine residues.

It was first established that the direct impact of exogenous GSSG in mammalian cells (human or experimental animals), and are able to produce cytokines or hemopoietic factors, has a stimulating effect on the synthesis of the latter and their release into the blood or the environment, which leads to the increase of their concentration in the serum (in vivo) or the culture fluid (in vitro and/or ex vivo). The invention allows to achieve the effect, which consists in stimulating the production of cytokines and gemopoeticescoe pharmaceutically active compositions ensuring the continuation of glutathione in the oxidized form. The results of the research also showed that this property GSSG and pharmaceutical compositions with his participation have a curative effect in various pathological situations in experiment and clinic.

Apparently detected stimulation of endogenous production of cytokines and hemopoietic factors caused by GSSG is primary in relation to antitumor, anti-infective, hematopoietic and immunostimuliruyushhim, and other pharmacological effects, providing in turn the achievement of one degree or another therapeutic or preventive actions for various diseases.

According to the invention a remedy proposed for the treatment of cancer, infectious, hematologic, and other diseases that appropriate stimulation of endogenous production of cytokines and hemopoietic factors, contains as active ingredient an effective amount of GSSG, it is reasonable to dosage form of this pharmacological means represented by injecting a solution containing GSSG at a concentration of 0.01 to 2.0

The agreement is its participation which would have contributed to the continuation of glutathione in biological fluids and tissues in the oxidized state, or could enhance the detected biological and therapeutic properties of GSSG.

According to the invention to enhance and prolong therapeutic effect of GSSG as a remedy it is advisable that the dosage form of the tool (injection solution) additionally contain pharmaceutically acceptable component, is able to extend the presence of glutathione in the oxidized form.

As pharmaceutically acceptable component, is able to extend the presence of glutathione in the oxidized form, it is proposed to use, for example, hydrogen peroxide at a concentration of 0.003

This is because in the presence of hydrogen peroxide (H2O2), which is the donor of active forms of oxygen (oxidant), restoration of GSSG to GSH implemented glutathion reductase, proceeds at a slower rate, which creates conditions for a slower recovery introduced into the biological environment exogenous GSSG.

The use in the composition of the dosage form hydrogen peroxide (H2O2in concentration, pharmaceutically beer forms of oxygen) allows you to implement only one of the possible technical solutions ensuring the continuation of glutathione in biological fluids and tissues in the oxidized form with the achievement of results, consisting in strengthening and extension of the pharmaceutical effects of GSSG.

Have installed other pharmaceutically acceptable ingredients that promote a more slow process of recovery of exogenous GSSG to GSH in biological environments. As such, in particular, are factors that can join in a competitive relationship with the restored form of coenzyme nikotinamidadenindinukleotida or NADPH (inosine and other derivatives gipoksantina), as well as substances, reversible impeding recovery of the oxidized form of NADP+in NADPH, for example such inhibitors of glucose-6-phosphate dehydrogenase as tsistamin (2,2'-diaminodiphenyldisulfide).

Because the restored nikotinamidadenindinukleotida (NADPH) is a key cofactor enzyme system glutathione reductase, catalyzes the reaction for reduction of GSSG to GSH, any other pharmaceutically acceptable chemical compounds or biophysical impacts, reducing the effectiveness of its recovery or blocking its biological okisleniem condition, and consequently, may increase or prolong therapeutic effect of GSSG.

As a result of investigations it was discovered that the pharmaceutical and therapeutic effect of GSSG increased if it is used in combination with the substances entering into a competitive relationship with NADPH, as well as substances, any abscopal reversible enzymatic reaction catalyzed by glucose-6-phosphate-dehydrogenase, resulting in the recovery of the oxidized form of NADP+.

Thus, in addition to hydrogen peroxide, other pharmaceutically acceptable component, is able to extend the presence of glutathione in the oxidized form, is hypoxanthine (inosine, or 9-b-ribofuranosylpurine), which is proposed to be used as a 0.1 solution.

The researches have shown that hypoxanthine has a potentiating effect on the pharmacological and therapeutic effects of GSSG. It was found that this property of hypoxanthineguanine comes from its ability to engage in a competitive relationship with NADPH, which is slow reduction reaction of GSSG to GSH. Moreover, it was found that this property is in addition to the above-mentioned hydrogen peroxide and hypoxantine, other pharmaceutically acceptable component, is able to extend the presence of glutathione in the oxidized form, is tsistamin (2,2'-diaminodiphenyldisulfide), to be used as a 0.1 solution.

The researches have shown that tsistamin, has a potentiating effect with respect to biological and therapeutic effects of GSSG. It was found that this property of applied comes from its ability to allow reversible inhibition of the starting enzyme pentoses cycle, glucose-6-phosphate dehydrogenase, catalyzes the reaction for reduction of NADP+in NADPH.

Thus, the invention also proposes a method of potentiating the ability of oxidized glutathione (GSSG) to stimulate endogenous production of cytokines and hemopoietic factors, namely, that GSSG is used in the form of pharmaceutical compositions containing a component that is able to extend the presence of glutathione in the oxidized form. This is achieved through the use of pharmaceutically acceptable compositions containing dosage form GSSG and dosage forms of the substances, capable to extend the presence of glutathione in biological environments in the creation of hypoxanthineguanine (or other derivatives gipoksantina, including nucleoside derivatives of inosine), and 0.1 solution applied (or other compounds capable of reversible inhibition of the starting enzyme pentoses cycle, glucose-6-phosphate dehydrogenase).

Found that parenteral (intravenous, intraperitoneal, intramuscular, and so on) application of GSSG in 1 5 ml of 0.003 solution of hydrogen peroxide, GSSG in 1 5 ml of 0.1 solution hypoxanthineguanine and GSSG in 1 5 ml of 0.1 solution applied stimulates endogenous production of TNF-alpha, IFN-alpha and IFN-gamma, IL-1, IL-2, IL-6, IL-10, erythropoietin and GM-CSF in experimental animals to a greater extent than is observed in the case of using only the GSSG.

The studies confirm the ability of the above-mentioned compounds (hydrogen peroxide, hypoxanthineguanine and applied) to strengthen the biological and therapeutic effects of GSSG and suggest their combined use with GSSG in order to increase the effectiveness of treatment of cancer, infectious, hematologic, and other diseases in which stimulation of the endogenous production of cytokines and hemopoietic factors appropriate for reasons that are obvious to a person skilled in the relevant field of medicine.

However, data were obtained that indicate possible direct antitumor effect of GSSG or GSSG used in the composition of pharmaceutically acceptable compositions, ensuring the continuation of glutathione in biological environments in the oxidized form. But the effects of GSSG apparent differential effect with respect to the normal to the reecting the fact that the use of GSSG or GSSG used in the composition of pharmaceutically acceptable compositions, ensuring the continuation of glutathione in biological environments in the oxidized form, accompanied by the initiation of tumor cell death by apoptotic mechanism. However, in the case of normal cells, their death was not observed.

According to the invention is a method of treatment of cancer, infectious, hematologic, and other diseases that appropriate stimulation of endogenous production of cytokines and hemopoietic factors, includes injecting GSSG as pharmacological tools in the form of injectable dosage forms, doses of 0.01 to 0.5 mg GSSG per kg of body weight, of one or more injections a day, a course lasting one or more days or continuously until a therapeutic effect.

However, GSSG as a pharmacological tool, its dosage form and/or pharmaceutical compositions with his participation must be entered in the body exclusively parenterally, which eliminates or minimizes degradation of a molecule of GSSG or its recovery (up to GSH), intensively flowing in the digestive tract and the liver when administered orally in the neck would be to use the oral route of administration and/or local (in situ) applications in the area of the pathological process (the wound surface, the tumor tissue, and so on). In addition, the proposed method for the treatment of diseases with the purpose of strengthening or renewal offer pharmacological means may include simultaneous implementation of chemical exposure (prooxidative oxidizing agents; compounds that affect the activity of enzymes and cofactors involved in the metabolism of oxidized and reduced glutathione) and/or physical influences, such as UV or radiolucent, if such exposures would be able to extend the presence of glutathione in biological fluids and tissues in the oxidized state.

The following examples of implementation of the invention confirm that parenteral (intraperitoneal, intravenous, intramuscular, subcutaneous, and so on) application of GSSG is capable of stimulating endogenous production of TNF-a, IFN-a and IFN-g, IL-1, IL-2, IL-6 and IL-10, erythropoietin and GM-CSF in mammals, which allows to achieve a significant therapeutic effect in humans and animals suffering from cancer or infectious diseases, depression of hematopoiesis and immunity of different origin, as well as other diseases, in which stimulation of the endogenous production of the above-mentioned ciesta medicine.

From the data of the research (see examples of implementation of the invention), it follows that previously unknown ability of GSSG to enhance the endogenous production of cytokines and hemopoietic factors and therapeutic effect in various diseases not associated with increased levels of GSH, as the use of GSH in a wide range of doses and concentrations, would not achieve the stimulation of endogenous production of cytokines and hemopoietic factors, and therapeutic effect, which was discovered by GSSG.

Under "therapeutic effect" refers to any improvement in the condition of the patient or animal under treatment by the proposed method as well as any reduction in severity and symptoms of diseases or their complications, including those caused by other treatments (such as chemotherapy or radiotherapy), which could be fixed by using physical, laboratory and instrumental methods of examination and would be statistically significant and/or clinically significant from the point of view of a specialist in the relevant field of medicine.

By "prophylactic effect" refers to the prevention of any deterioration in the condition of subvene and symptoms of diseases or their complications, including those caused by other treatments (such as chemotherapy or radiotherapy), which could be fixed by using physical, laboratory and instrumental methods of examination and would be statistically significant and/or clinically significant from the point of view of experts in the relevant field of medicine.

Under "cancer and infectious diseases", "depression of hemopoiesis and immunity of different origin" and "other diseases" refers to any cancer or infectious disease, any condition caused or accompanied by inhibition of red or white Rostock blood or depression quantitative or functional parameters of the immune system, as well as any other diseases and pathological conditions in which stimulation of the endogenous production of the above-mentioned cytokines and hemopoietic factors (TNF-a, IFN-a and IFN-g, IL-1, IL-2, IL-6 and IL-10, erythropoietin and GM-CSF) would be appropriate for reasons that are obvious to a person skilled in the relevant field of medicine.

The following examples of the invention demonstrate the possibility of its practical applicability.

Active substance PE which may be obtained by known and conventional method of peptide synthesis. Thus obtained peptide (GSSG) to its further use in vivo in humans and animals used in the form of a base or pharmaceutically acceptable salt in injectable dosage form obtained by dissolving the dry substance in sterile water for injection or any pharmaceutically acceptable solvent in a concentration of 0.01 to 2.0 For studies in vitro GSSG can be dissolved in appropriate for conducting relevant experiments the liquid culture media, isotonic salt solutions etc.

Preparation of dosage forms for use in humans and animals should be performed under conditions of sterility and apirogennost and should exclude the possibility of chemical or bacterial contamination of the dosage form.

Injectable dosage form GSSG was studied in experiments on animals and in the course of limited clinical studies in human patients.

Through the use of the maximum achievable concentration of the injection solution of sodium salt of GSSG (10,0 100 mg/ml) in water, in 0,003 solution of hydrogen peroxide or 0.1 solutions hypoxanthineguanine or applied, and because IP is rivanna (in/in, 0.5 ml) and intramuscular (I/m, 0.05 ml) the introduction have been achieved with doses of sodium salt of GSSG, the components of 5000 mg/kg (b/W), 625 mg/kg (in/in) and 62.5 mg/kg (V/m), which is more than the maximum dose recommended for human component and 0.5 mg/kg, 500, 125 and 12.5, respectively. In none of the cases was not observed mortality, as well as any toxic manifestations that actually proves nontoxicity GSSG used in the pharmaceutical form solution for injection, and nontoxicity used pharmaceutical compositions containing GSSG.

The results of pre-clinical (experimental) studies of biological, pharmacological and therapeutic properties of GSSG and its dosage forms, whether or not containing 0,003 the hydrogen peroxide solution, a 0.1 solution of hypoxanthineguanine or 0.1 the solution applied, are given in examples 1 to 5 of the invention.

Example 1. The influence of the studied substances on the production of cytokines by mononuclear leukocytes in human peripheral blood in vitro.

Evaluated the ability of oxidized glutathione (GSSG) and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine or GSSG in a 0.1 solution of the applied effect is 2">

The production of cytokines by leukocytes were initiated by addition of mitogen, concanavalin A (ConA), which was added to the cells prior to introduction into the culture of the investigated substances. Supernatant cultures were collected 24 h after the beginning of the combined effects of ConA and the investigated substances and kept before the determination of cytokines at -70oC.

To assess the functional state of the cells and their ability to undergo the mitogen-induced activation in the presence of each of the measured concentrations of the investigated substances control culture of cells containing the analyte in identical concentrations, were incubated for 72 h after the beginning of the combined effects of ConA and the investigated substances. For 16 h before the end of the incubation in the control culture was made3H-thymidine and the degree of incorporation of label into DNA was regarded as the criterion of the functional state of cellular test systems.

Venous blood from healthy donors (males) were collected in heparinised tubes, tested for the absence of endotoxin (endotoxin tested). Mononuclear fraction of peripheral blood leukocytes were obtained by centrifugation in a density gradient ficoll-metrizoate (Histopaque, Sigma).

The concentration of cells dog/ml gentamicin and 10 fetal calf serum (all reagents for tissue culture, Sigma). Cell viability was assessed in test with Trifanova blue and the cell suspension is distributed into the wells of flat-bottomed 96-well plates for tissue culture based 200000 cells per well (100 ml suspension). Cells from each donor were serving not less than 36 holes.

Evaluated the effect of 5 final concentration (5000 mg/ml, 500 g/ml, 50 g/ml, 5 g/ml and 0.5 g/ml) oxidized glutathione (GSSG) or GSSG in combination:

a) with hydrogen peroxide at a final concentration of 0.003

(b) hyposensitisation at a final concentration of 0.1

C) applied at a final concentration of 0.1

Each of the concentrations was created not less than 6 holes, making 50L "full" culture medium containing the required number of pre-dissolved substances. 6 wells were used for control (added 50 l only "full" culture medium or complete medium containing hydrogen peroxide, hypoxanthine, tsistamin).

Immediately after the payment of the investigated substances to all wells (except 3 control) were introduced into a 50 l "full" culture medium containing this amount of ConA (Sigma, tissue cultures), so that its final concentration was 4.0 g/ml.

After 24 h and is containing a series of identical concentration of the substances under study) was aspirated by using the pipettor, was subjected to centrifugation, supernatant were frozen and kept at -70oC until ELISA tests to determine the presence of cytokines. The contents of all remaining filled holes continued to incubate under the above described conditions.

After 56 h from the beginning of incubation to all remaining wells were added at 1.0 Ci3H-thymidine and continued the incubation for another 16 h, after which the contents of the wells using a cell harvester (cell harvester) was applied onto a glass-fiber filters, which consistently processed 5 trichloroacetic acid and ethanol. The filters were dried and radioactivity was determined using liquid scintillation counter 1205 Betaplate (LKB).

The values of the radioactivity of the filters for each triplet of the wells were averaged and used to calculate the index mitogenic stimulation with respect to the averaged value of including3H-thymidine in the triplet holes, which were added ConA (spontaneous incorporation of the label). The stimulation index against average ConA-stimulated incorporation of three test holes to the averaged spontaneous judged on the functional state of the cells in these wells in the presence of the estimated concentrations of GSSG (or GSSG in the stimulation.

Subsequent analysis on the levels of the cytokines were subjected to cell supernatant 24-hour cultures of only those triplets of holes in the control triplets which at the 72-hour incubation index ConA-stimulation evaluated by the inclusion of3H-thymidine, was not lower than 10, indicating a satisfactory functional state of the cells.

The content of interleukin-lb (IL-1 b); interleukin-6 (IL-6), tumor necrosis factor (THF) and interferon-a(IFN) was determined by ELISA using commercial kits Medgenix (Belgium) and was expressed in PCG/ml of culture medium.

The results are presented in table. 1 4. According to the table. 1 4 the addition of GSSG in the culture medium is statistically significantly and dose-dependently enhances the production of cytokines by mononuclear leukocytes donors. However, the additional presence of hydrogen peroxide leads to increase of the control (in the absence of GSSG) indicators of the production of IL-6 and TNF. In addition, in the presence of hydrogen peroxide GSSG has more pronounced (1.5 to 2 fold) stimulatory effect on the production of the investigated cytokines: IL-1b concentrations of 5.0 5000 mg/ml, for IL-6 and TNF in the whole range of concentrations and for IFN concentrations the dose-dependent increase in cytokine production, especially in relation to IL-6 and TNF (see tab. 3 and 4).

Thus, the effect of GSSG on mononuclear leukocytes of peripheral blood in vitro leads to convincing stimulation of cytokine production in the culture medium than confirmed by the stimulating effect of GSSG on the natural cytokine-producing capacity of human blood cells. The use of GSSG in combination with hydrogen peroxide, hyposensitisation, also applied, leads to a more pronounced manifestation of cytomegalovirus effects GSSG.

Example 2. The influence of the studied substances on the production of cytokines and hemopoietic factors, and indicators of hematopoiesis and immunity in ciclofosfamida hemo - and immunosuppression.

Effect of oxidized (GSSG) and reduced (GSH) glutathione and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine or GSSG in a 0.1 solution applied, evaluated in the model hemo - and immunosuppression in mice induced by a single injection of cytostatic agent, cyclophosphamide (CP).

In the framework of this study was to assess the influence of the 5-day application of the above substances on the ability of spleen lymphocytes of mice, podvergnutoi and lymphocytes) and bone marrow (the number of kariolou) on the 8th day after the application fits. In some animals that received UV and then subjected to immunization with sheep red blood cells (EB), was assessed by the ability to develop a humoral immune response to this antigen.

The study was conducted on mice CBA (males) weighing 18 to 20 g, which once introduced ZF (50 mg/kg, intraperitoneally). Seven groups of animals (at least 15 mice in each group) was formed in the course of the study.

Description of groups.

Control group:

N1 intact animals received instead of injections ZF injection of saline;

N2 animals that received an injection of fits, which were further used saline solution (control);

N3 animals that received an injection of fits, which were further used GSH in physiological solution at a rate of 5 mg/kg of body weight (substance comparison).

Experimental group:

N4 animals that received an injection of fits, which were further used GSSG in physiological solution at a rate of 5 mg/kg of body weight (test substance);

N5 animals that received an injection of fits, which were further used GSSG in physiological solution containing 0.003 to hydrogen peroxide at a rate of 5 mg GSSG per kg of body weight (Varian is that in the future GSSG was used in physiological solution, containing 0.1 hypoxanthineguanine 5 mg GSSG per kg of body weight (option pharmaceutically active composition of the analyte);

N7 animals that received an injection of fits, which were further used GSSG in physiological solution containing 0.1 applied, at a rate of 5 mg GSSG per kg of body weight (option pharmaceutically active composition of the analyte).

After 24 h after application of ZF 5 animals from each group were subjected to immunization with sheep red blood cells (EB, 107red blood cells in 0.5 ml saline, intraperitoneally).

From the beginning 3 days after injection ZF (from immunized animals after 24 h after immunization, the animals were started intraperitoneal injection of control or of the investigated substances, as described above. Introduction of substances continued for 5 days (daily, once a day).

After 24 h after application of the control or of the investigated substances (on 8-th day after injection ZF) mice were aftanaziva and in sterile conditions were preparing a suspension of splenocytes to assess spontaneous production of IL-2 and GM-CSF by spleen lymphocytes in vitro.

In parallel, took blood samples and bone marrow DL is specialised animals on day 7 after immunization (day 8 after injection ZF) evaluated serum titer of hemagglutinin to EB.

In table. 5 shows the performance of the production of IL-2 and GM-CSF by splenocytes, indicators cellularity blood and bone marrow and the level of humoral immune response to sheep erythrocytes in mice treated with tested compounds against cyclophosphamide-induced hemo - and immunosuppression.

According to the data presented in table. 5, the application of GSSG and GSSG in 0,003 solution of hydrogen peroxide almost normalizes the production of IL-2 and GM-CSF cells of the spleen, whereas GSH has no such effect. While GSSG and GSSG in the hydrogen peroxide solution provide reliable regenerating effect on the cells of the blood and bone marrow and the ability of animals to form immune response to EB.

In table. 6 and 7 presents the results of the impact assessment pharmaceutically active compositions (composition of GSSG in a 0.1 solution of hypoxanthineguanine and GSSG in a 0.1 solution applied) on the dynamics of the studied indicators in the context of CP-induced hemo - and immunosuppression. These data indicate a significant enhancing effects GSSG components, such as hypoxanthine and tsistamin relatively stimulating the production of IL-2, GM-CSF, recovery cellularity blood and bone marrow. In this case, as you can see, GSH.

Thus, the application of the proposed method in animals subjected to chemical (cyclophosphamide induced) hemo - and immunosuppression leads to a pronounced stimulation of endogenous production of IL-2 and GM-CSF; has a regenerating effect on the cells of the blood and bone marrow, and also forms an effective immune response.

Example 3. The influence of the studied substances on the production of cytokines and hemopoietic factors, and indicators of hematopoiesis and immunity in radiation hemo - and immunosuppression.

Effect of oxidized (GSSG) and reduced (GSH) glutathione and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine and GSSG in a 0.1 solution applied was estimated in the model radiation hemo - and immunosuppression in mice induced by a single exposure to a total dose of 1 Gy.

In the framework of this study was to assess the impact of the 7-day daily use of the above substances (the first injection of drugs through 2 h after irradiation) on the ability of spleen lymphocytes of mice subjected to radiation to produce IL-2 and GM-CSF in vitro. In addition, it was estimated cellularity blood (Chi is kolonie-forming ability of bone marrow cells and spleen.

The study was conducted on mice CBA (males) weighing 18 to 20 g, were subjected to a single x-ray training at a total dose of 1 Gy (distance from source 70 cm, capacity of 180 kV, 15 mA, the filter of 0.5 Cu, duration of exposure 2 min 28 s).

Seven groups of animals (at least 12 mice in each group) was formed in the course of the study.

Description of groups.

Control group:

N1 intact animals that received stress exposure in the form of spurious radiation, which later was used saline (intact);

N2 animals irradiated at a dose of 1 Gy, which later was used saline solution (control);

N3 animals irradiated at a dose of 1 Gy, which was later applied GSH in physiological solution at a rate of 5 mg/kg of body weight (substance comparison).

Experimental group:

N4 animals irradiated at a dose of 1 Gy, which later was used GSSG in physiological solution at a rate of 5 mg/kg of body weight (test substance);

N5 animals irradiated at a dose of 1 Gy, which later was used GSSG in physiological solution containing 0.003 to hydrogen peroxide at a rate of 5 mg GSSG per kg mass of those who R which later was used GSSG in physiological solution containing 0.1 hypoxanthineguanine 5 mg GSSG per kg of body weight (option pharmaceutically active composition of the analyte);

N7 animals irradiated at a dose of 1 Gy, which later was used GSSG in physiological solution containing 0.1 applied at a rate of 5 mg GSSG per kg of body weight (option pharmaceutically active composition of the analyte);

After 2 h after irradiation, the animals were started intraperitoneal injection of control or of the investigated substances, as described above. Introduction of substances and was continued for 7 days (every day, once a day).

After 24 h after application of the control or of the substances under study (on day 8 after irradiation) mice were aftanaziva and in sterile conditions were preparing a suspension of splenocytes to assess spontaneous production of IL-2 and GM-CSF lymphocytes of the spleen in vitro

In parallel, took samples of the blood, spleen and bone marrow to assess cellularity (number of leukocytes and lymphocytes, kariolou spleen and bone marrow).

At the same time, determined the ability of hematopoietic cells in the bone marrow and spleen method is introduced about portions of the investigated cell suspensions of bone marrow and spleen from animals studied groups.

In table. 8, 9, 10 indicators of production of IL-2 and GM-CSF by splenocytes, indicators cellularity blood, spleen and bone marrow, as well as the performance of Colonie education (Colonie-forming units, CFU) of bone marrow and spleen trained animals on day 8 after radiation exposure.

As follows from the materials table. 8 to 10, the application of GSSG and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine and GSSG in a 0.1 solution of applied statistically restores the production of IL-2 and GM-CSF cells of the spleen, whereas GSH does not have a significant effect. In this case, as GSSG and GSSG in the composition of pharmaceutically active compositions provided reliable normalizing effect on the cells of the blood, spleen and bone marrow. In some cases the effect of GSSG in the hydrogen peroxide solution turned out to be more significant. So, in the case of application of GSSG effect of this substance had no statistical significance, when compared with the control group) for indexes such as the IL-2 production by splenocytes, peripheral blood leukocytes and cellularity of the bone marrow, as well as Colonie-forming activity of bone marrow cells, whereas in the case of GSSG in the hydrogen peroxide solution, stat is odorata had hypoxanthine and tsistamin, more significantly potentiate the effect of GSSG, with the maximum effect had a pharmaceutically active composition GSSG with hyposensitisation.

Thus, the application of the proposed method in animals subjected to radiation hemo - and immunosuppression leads to a pronounced stimulation of endogenous production of IL-2 and GM-CSF, normalization cellularity blood, lymphoid and hematopoietic organs, as well as the recovery of hematopoietic activity in bone marrow and spleen.

Example 4. The influence of the studied substances in the processes of proliferation and apoptotic death of normal and transformed cells.

Evaluated the ability of oxidized glutathione (GSSG) and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine and GSSG in a 0.1 solution applied to influence the processes of proliferation and apoptotic death of normal and transformed cells. This was carried out by incubation GSSG or GSSG in the composition of pharmaceutically active compositions for 24 h with cells of the myeloid line HL-60 and donors ' lymphocytes with subsequent evaluation of the cell cycle by the method of flow cytophotometry.

Venous blood from healthy donors (males) Sobirov blood was obtained by centrifugation in a density gradient filemetadata (Histopaque, Sigma). The cell concentration was brought to 2106cells in 1 ml complete culture medium (RPMI 1640) containing 20 mm HEPES, 2 mm glutamine, 50 μg/ml gentamicin and 10 fetal calf serum. Cell viability was assessed in test with Trifanova blue and the cell suspension is distributed into the wells of 96-well plates at the rate of 200 000 cells per well.

Cells are transplantable myeloid line HL-60 were grown in RPMI medium 1640 with the addition of 10 fetal calf serum. Cultivation was performed in a sealed bottle, the volume of medium (12 ml), the change of environment was performed every 4 days by centrifugation. The nature of the growth of cell suspension.

Assessed the effect of GSSG solution (5 mg/ml), and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine and GSSG in a 0.1 solution applied. Each of the solutions was evaluated in 6 samples of normal lymphocytes and cells HL-60.

Solutions of the test substances (GSSG or GSSG in the composition of pharmaceutically active compositions) in an amount of 50 μl was added to the culture medium and cells were cultured for 1 day. Then, they were tested using flow cytofluorometry for the content of DNA in cell nuclei. In the case of an APO number of fragments of nuclei with significantly lower DNA content. The assay procedure was as follows: the cells by centrifugation was transferred to a standard isotonic phosphate buffer pH 7,4 containing RNA-ABC A (20 μg/ml), a fluorescent dye ethidium bromide (10 µg/ml) and MgCl2(5 mm). The cells are then opened by the addition of non-ionic detergent Triton X-100 (final concentration of 0.1 ). The resulting suspension cell nuclei were analyzed on a flow cytofluorimetry with argon laser as a light source (wave length of the exciting colors 488 nm). Registered red fluorescence associated with DNA ethidium bromide is a measure of DNA content in cell nuclei.

Parallel to visually analyzed changes in the morphology of the studied cells.

The effect of GSSG (or GSSG in the composition of pharmaceutically active compositions) on the proliferative activity of lymphocytes of donors are presented in table. 11. As you can see from the data table. 11, the addition of GSSG or GSSG in the composition of pharmaceutically active compositions stimulates the proliferation of lymphocytes of a healthy person, which leads to an increase in their total number. While running cytofluorimetrically analysis did not reveal the appearance of a pool of cells with Keturah transformed cells of the myeloid line HL-60, revealed the ability of GSSG (or GSSG in the composition of pharmaceutically active compounds) to inhibit the proliferation of transformed cells used (see tab. 12). The data table. 12 show that composition with hydrogen peroxide, hyposensitisation and applied have a much greater ability to inhibit the proliferation of cells in HL-60 compared to GSSG used in mono. Conducted flowing cytofluorimetrically analysis showed that the reduction of cell growth cell line HL-60 is accompanied by the appearance of characteristic morphological features of apoptotic death of the cells of the spherical became multi with multiple constrictions; was a decrease of cell nuclei with normal DNA content; increased the number of fragments of nuclei with significantly reduced DNA content (see Fig. 1 4).

Thus, in vitro studies indicate the presence of a bifunctional activity of GSSG and GSSG in the composition of pharmaceutically active compositions, which increases the residence time of this compound in the oxidized state, in relation to tumor and normal cells. GSSG or GSSG in the composition of pharmaceutically active compositions selectively Udobny effect is absent for normal cells (lymphocytes donors) and the proliferation of the latter is clearly accelerating. The use of GSSG in combination with hyposensitisation leads to a more pronounced effect of GSSG.

Example 5. The influence of the studied substances on the growth of transplantable tumors in mice.

We investigated the antitumor activity of oxidized glutathione (GSSG) and GSSG in 0,003 solution of hydrogen peroxide, GSSG in a 0.1 solution of hypoxanthineguanine, GSSG in a 0.1 solution applied on two models of tumor process, induced by the introduction into mice cell leukemia R and leukemia L1210. In this study, we evaluated the inuence of a 7-day application of the above drugs on the dynamics of the content of cytokines (IL-1; IL-2; IL-6; IFN-alpha; TNF) in the blood of mice with inoculated tumors and control animals. Simultaneously assessed the dynamics of accumulation of ascitic fluid as an indicator of tumor progression and life expectancy in the control and experimental groups as an integral indicator of antitumor activity of the studied drugs.

The study was conducted on mice DBA/2 because these animals are syngeneic with respect to human strains of tumor cells (R and L12 is each of the lines of tumor cells in 6 animals. For the culture of cells stored in liquid nitrogen, were thawed and diluted in sterile Hanks solution to a concentration of 5106cells/ml, 0.2 ml of the resulting cell suspension of each line was intraperitoneally inoculable 6 mice.

On the 8th day of the passage in the case of cells R and on day 6 the passage in the case of L1210 cells from the abdominal cavity was removed ascitic fluid and samples of tumor cells were used for the main experiments. Date of receipt of ascitic fluid was considered the day of the end of the passage and was the day of the beginning of the main experiment. Ascitic fluid was diluted with sterile Hanks solution to a concentration of 5106cells/ml in the case of cells R and to the concentration of 5105cells/ml in the case of L1210 cells.

For studies with each of the tumor lines were formed 8 groups of animals at least 15 mice each. Volume inoculums suspension of 0.2 ml per mouse dose inoculum: 106cells/mouse in the case of cells R and 105cells/mouse in the case of L1210 cells.

At 24 h after inoculation of tumor cells, the animals were started intraperitoneal administration of the investigated substances and substances of comparison. Further, injection of the substances under study is nycli - 0.01 ml/g body weight of the animal.

For experiments with each of the tumor lines was formed on 9 groups of animals as described below.

Control group:

N1 intact animals received instead of the injection of tumor cells injection, saline solution, which was introduced to them in the future (intact);

N2 animals, receiving an injection of tumor cells, which further were injected with saline.

Experimental group:

N3 animals received injections of tumor cells, which further were introduced GSSG in physiological solution at a rate of 5 mg/kg of body weight;

N4 animals, receiving an injection of tumor cells, which further were introduced GSSG in physiological solution containing 0.003 to hydrogen peroxide at a rate of 5 mg GSSG per kg of body weight (option pharmaceutically active composition of the analyte);

N5 animals, receiving an injection of tumor cells, which further were introduced GSSG in physiological solution containing 0.1 hypoxanthineguanine 5 mg GSSG per kg of body weight (option pharmaceutically active composition of the analyte);

N6 animals, receiving an injection of tumor is GSSG per kg of body weight (option pharmaceutically active composition of the analyte);

N7 animals, receiving an injection of tumor cells, which further were introduced 0,003 a hydrogen peroxide solution (component pharmaceutically active compositions in the quality control of the investigated compositions);

N8 animals, receiving an injection of tumor cells, which further were administered 0.1 solution hypoxanthineguanine (component pharmaceutically active compositions in the quality control of the investigated compositions);

N9 animals, receiving an injection of tumor cells, which further were administered 0.1 solution applied (component pharmaceutically active compositions in the quality control of the investigated compositions).

In table. 13 and 14 show the performance impact of the studied substances on endogenous production of cytokines, as well as integrative indicators of tumor progression process. Analysis of the data table. 13 and 14 shows that GSSG and GSSG in the composition of pharmaceutically active compositions, which increases the residence time of this compound in the oxidized state, have a distinct cytomegaloviruses action significantly (relative to control groups) inhibit the accumulation of ascitic fluid and increase the average life expectancy of animals, is the blood levels of IL-1 and IFN, and GSSG in a 0.1 solution of hypoxanthineguanine and GSSG in a 0.1 solution of applied causes a large accumulation of IL-2, IL-6, TNF.

The maximum level of these indicators antitumor activity of the compounds under investigation as the inhibition of the accumulation of ascites and the lifetime obtained in both models of tumor process (leukemia R and L1210) in case of application of GSSG in a 0.1 solution applied.

Thus, the application of the proposed method for the treatment of mice with inoculated tumors leads

to the pronounced stimulation of endogenous developments of the cytokines IL-2, IL-6, IFN and TNF;

to a significant growth inhibition of transplantable tumors, and inhibition of tumor progression process and increase the life span of experimental animals.

Previously unknown properties of GSSG and its pharmaceutically active compositions containing 0,003 solution of hydrogen peroxide, 0.1 solution hypoxanthineguanine or 0.1 solution applied currently in the experimental, preclinical studies suggest that GSSG, its pharmaceutical form, and pharmaceutically active compositions with his participation possess significant pharmacological activity and therapeutic effect. This obon the active compositions for the prevention and treatment of diseases, where appropriate stimulation of endogenous production of cytokines and hemopoietic factors.

Examples of clinical application of medicinal forms GSSG below (see examples 6 to 12), confirm the possibility of using GSSG as a stimulator of endogenous cytokine production and hematopoiesis, and also factor in the reproduction of the effects of cytokines in humans, including for the implementation of the method of treatment of diseases based on the properties of GSSG in clinical conditions.

Example 6. The impact of the dosage form GSSG on endogenous production of cytokines and erythropoietin in people with cancer.

In this example, information relating to the stimulating effect of GSSG on endogenous production of cytokines and hemopoietic factors in people with cancer. The GSSG solution (5 mg/ml) was administered intravenously, slowly, once in two days, at a dose of 5 mg per injection. About endogenous production of cytokines were judged on their content in the blood of patients before the first injection (blood was collected for 24 h before injection) and after the third and seventh injections of GSSG. The levels of the cytokines were determined by ELISA with innym, presented in table. 15, it is possible to judge that after the first three injections of GSSG-marked stimulation of endogenous production of cytokines (IL-1b, IL-6, TNFa, IFNa) and erythropoietin. After 7-th injection (14 days treatment) in most cases there is a multiple increase in the content of cytokines and erythropoietin in the blood of patients.

Example 7. Stimulation of endogenous production of cytokines and erythropoietin and therapeutic effect in the patient with cancer of the sigmoid colon complicated by emodepside caused by chemotherapeutic treatment.

Patient 44 years old was operated on for tumor of the sigmoid colon with growth in the ovarian and metastatic lymph nodes of the mesentery and greater omentum (T4N3M1). In the postoperative period was held chemotherapy 5-fluorouracil (dose rate of 5.5 g), which was accompanied by a pronounced gematotoksicheskoe effect.

1 month after the course of chemotherapy the patient was re-examined with ultrasound abdomen and computed tomography of the liver, which showed a single metastasis in the left lobe of the liver, oval in shape, size 13 x 10 cm was Preregistrations different degree of leukopenia and lymphopenia, anemia and thrombocytopenia), which made it impossible to conduct new courses of chemotherapy.

Results clinical and immunological examination prior to the application of the dosage form oxidized glutathione (5 mg GSSG in 1 ml of 0.003 hydrogen peroxide) are given in table. 16. The treatment by the proposed method was started with intravenous injection of 5 mg of GSSG once daily for 7 days. After a 3-day suspension dose GSSG was increased to 15 mg, intravenously, once daily for 10 days. After a 7-day break GSSG drug was administered intramuscularly in a dose of 15 mg a day (a total of 20 injections).

50 days after the start of treatment by the proposed method the patient was repeated ultrasound examination of the abdomen and computed tomography of the liver, which revealed a significant regression of the size of a single metastasis in the left lobe of the liver (more than 50 from the source). Results clinical and immunological studies performed after treatment, are shown in table. 16.

As you can see, at the end of treatment significantly improved red and white blood cells, almost normalized the number of platelets, decreased erythrocyte sedimentation rate, significantly powyzej cytokines and erythropoietin, including tumor necrosis factor, which along with the increased number of natural killer cells, may be associated with regression of metastases in the liver. These changes were accompanied by improvement in the General condition of the patient, increasing physical activity.

Provided clinical supervision of a clear therapeutic effectiveness of the proposed method. As a result of treatment with a significant stimulation of endogenous production of cytokines and hemopoietic factors revealed a decrease in the size of the metastasis in the liver and normalization of clinical and immunological parameters, the improvement of the General condition of the patient.

Example 8. Stimulation of endogenous production of cytokines and therapeutic effect in a patient with AIDS complicated cryptococcal meningoencephalitis.

Male 28 years old with previously confirmed diagnosis of AIDS, stage 3/4C (WHO Staging System) has been in a state of moderate severity. Complaints paroxysmal headache, dizziness, vomiting. West body 47 kg, Karnofsky index 60, lethargic, fever up to 39oC, dyspnea at rest. In the neurological examination revealed neck stiffness, decreased tendon reflexes of the upper and STI detected Cryptococcus neoformans, on what basis was established the presence of cryptococcal meningoencephalitis, the stage of the underlying disease (AIDS) was specified as the 4C.

Started active infusion therapy. In addition to symptomatic means the patient has received treatment with Fungizone (Amphotericin B), which has not achieved the desired effect. The severity of neurological symptoms and the patient's condition continued to deteriorate. Remained febrile body temperature (37,5 38,5oC).

At the time of the application pharmaceutical form (5 mg/ml GSSG in 1 ml of 0.003 hydrogen peroxide) patient was determined by a rapid decline in CD4+and CD8+T-lymphocytes of peripheral blood, anemia, lymphopenia (see tab. 17).

The treatment of the patient by the proposed method were carried out within 3 months (1 ml GSSG per injection). Thus, in the first month injections were performed every other day (for the first 10 days of intravenous further/m), during the 2nd month once in three days (the first 10 days/in the future p/).

By the middle of the first month of treatment the patient's condition has improved significantly decreased the severity of neurological symptoms, body temperature did not rise above 37.5oC. the thief (cytological, culturally, the reaction of the latex agglutination in the presence of cryptococcal antigen). By the end of the first month of treatment was found a significant reduction in the presence of CSF is capable of growing organisms Cryptococcus neoformans. By the end of the second month of Cytology, culture and immunological tests showed the absence of the pathogen in the spinal fluid. During the 3rd month due to significant improvement in patient's condition injections were performed 1 time per week (in/m).

Results clinical and immunological examination of the patient after treatment is shown in table. 17. As you can see, decreased symptoms of anemia, and there has been a significant increase in the number of lymphocytes and their subpopulations, which allows to state restaurovani underlying disease (AIDS) with stage 4S stage 4B.

Draws attention convincing increase in the content of cytokines in the blood, some of which (IL-2 and 6, IFNg) play a fundamental role in providing mechanisms to protect the host from pathogenic fungi.

By the time of discharge from hospital the patient was regarded as satisfactory, the body weight is 60 kg (weight gain was 21.7 from the source), the temperature of ulacia endogenous production of cytokines and therapeutic effect in a patient with AIDS complicated by infectious diarrheas.

Male 38 years old about 2 years there has been diagnosed with AIDS, stage 3C (WHO Staging System). In the past year documented repeated episodes of candidiasis of oral mucosa and esophagus, as well as almost the chronicity of infectious diarrheas intestine, manifested by loss of appetite, nausea, frequent episodes of vomiting and loose stools mixed with mucus and blood. Periodic use of cotrimoxazole (trimethoprim in combination with sulfamethoxazole, TMP-SMX) gave unstable remission with rapid resumption of symptoms.

The next bout of infectious diarrheas in the last month. Therapy with co-trimoxazole, Imodium (loperamide) is practically without effect. The condition gradually deteriorated constant body temperature 38oC and above, loose stools mixed with blood and mucus to 6 to 7 times a day, vomiting, progressive weight loss (about 15 from its normal weight). Hospitalized due to progressive deterioration.

When admitted to hospital, the General state of moderate severity index scale Karnofsky 50, the body temperature of 38.2oC, the patient is malnourished (weight 42 kg), subcutaneous fat layer is virtually absent, Her skin a large number of oozes Isospora belli.

At the time of the patient's treatment by the proposed method, the patient was determined lymphopenia, a sharp decline in CD4+and CD8+T-lymphocytes, hypoproteinemia (see tab. 18).

The treatment of the patient with the use of the dosage form oxidized glutathione (5 mg GSSG in 1 ml of 0.003 hydrogen peroxide) was conducted during 3 months (1 ml of a composition per one injection). Thus, in the first month injections were performed every other day (for the first 10 days of intravenous further/m), during the 2nd month once in three days (the first 10 days/in the future p/).

The condition of the patient is clearly beginning to improve after the first 2 weeks of treatment. By the end of the first month of treatment the frequency of bowel movements did not exceed 1 to 2 times a day, without admixture of blood, the body temperature only occasionally exceeded 37,0oC. By the end of the second month re-examination of the feces for the presence of oocysts Isospora belli showed the absence of the pathogen. During the 3rd month due to significant improvement in the state was conducted prophylactic use of the dosage form GSSG 1 time per week (in/m). Reactivation of the disease is not checked.

Results clinical and immunological examination of the patient upon completion of treatment is to promote an increase in the number of lymphocytes and their subpopulations, resulting in blood after treatment corresponds 3B stage on the scale of the who Staging System), which allows to state restaurovani underlying disease (AIDS).

In this case also draws attention to a significant increase in the content of cytokines in the blood, it is known that such of them as IL-2 and IFNg play an important role in protecting the host from protozoal infections.

After the treatment the General condition of the patient improved significantly, decreased fatigue, improved appetite. Weight gain was about 30 from the source, index Karnofsky score of 90. During the physical examination the patient was regarded as satisfactory. In the course of observation within 1.5 months after discharge from hospital recurrent diarrhea is not marked.

Example 10. Stimulation of endogenous production of erythropoietin and therapeutic effect in a patient gipoplasticheskaya anemia and pancytopenia.

Male 37 years there has been about a year about anemia of unclear Genesis, the severity of which is gradually increasing. In the last 10 months constantly worried about weakness, fatigue, dizziness, frequent nosebleeds, unusual what about for a year lost about 10 of his normal weight. Re outpatient treatment courses of oral and intravenous iron preparations, folic acid, and drugs, including12without effect.

When admitted to hospital, a state close to moderate, severe weakness, shortness of breath on mild exertion, there are bruises on the skin, separate petechial rash. In repeated blood tests - moderate/heavy normalfree anemia (erythrocytes 1,5 - 2,51012/l) with a tendency to hypochromia (color index of 0.7 - 0.9), aniso and penalities, moderate leukopenia, thrombocytopenia within (50 80109/l).

Active infusion therapy with iron, folic acid, ciankobalamin, vitamins, prednisolone, re-transfusion aricultural mass without clear effect.

According to myelogram (marrow puncture) cellularity of the bone marrow dramatically reduced, megalocnus space filled mostly fatty tissue. As myeloid and erythroid sprouts, greatly oppressed, the ratio of erythroid and myeloid cells dramatically reduced. The megakaryocytes are practically absent. The relative increase in the content of undifferentiated and plasmata, pancytopenia.

The results of the clinical analysis of blood and erythropoietin level at start of application pharmaceutical form oxidized glutathione (5 mg GSSG in 1 ml of 0.003 hydrogen peroxide) are given in table. 19. As you can see, the laboratory data are consistent with the picture gipoplasticheskaya anemia, however, is typical for this type of anemia increased level of erythropoietin in the blood is not available. Moreover, the erythropoietin level was significantly reduced with respect to the lower limit of normal (9,2 PCG/ml at a rate of 30 170 PCG/ml, which is in international units match 3 17 mIU/ml).

Application pharmaceutical form oxidized glutathione (5 mg GSSG in 1 ml of 0.003 hydrogen peroxide) was started with intramuscular injection of 1 mg of GSSG, 2 times a day for 3 days. Further doses were increased to 5 mg 2 times a day for 7 days. In the blood was observed decrease in the severity of anemia. Further, the composition was applied at 10 mg GSSG intramuscularly 1 time a day for 10 days, then on the background of distinct positive dynamics of the picture of the red blood was started on intravenous application of the composition: 10 mg GSSG 1 time in three days, which lasted for 30 days. Concomitant therapy included Vitan blood after 50 days from the start of treatment by the proposed method are given in table. 19. As you can see a picture of red and white blood cells have improved dramatically increased the number of platelets, decreased erythrocyte sedimentation rate, erythropoietin level exceeded the upper limit of normal. Clinically the patient disappeared weakness, dizziness, shortness of breath. When viewed petechial hemorrhage and bruising is not detected, episodes of nasal bleeding was not observed. Weight gain during treatment was 5.5 kg (approximately 8 normal weight).

According to repeated studies of the bone marrow (trypanophobia after treatment), myeloid tissue takes up to 60 volume bezbalochnykh spaces. In the Islands of myeloid tissue the ratio of the number of cells of the erythroid and myeloid sprouts exceeds the norm. There are elements normoblastic hyperplasia, in clusters of normoblasts detected groups of cells with signs of megaloblastoid. Meet the fat cells. The megakaryocytes are present in the major amount. The amount of iron stores increased somewhat.

Example 11. Stimulation of endogenous production of cytokines and therapeutic effect in a patient with gastric cancer complicated by canceration peritoneal ascites, splenomegaly and cholestatic hepatitis

Male 33 years observed 1993 patient operated on for malignant gastric ulcers (planar resection). When the operation detected a dense conglomerate of nodes in the hilum of the liver, interpreted as metastases.

In January 1994 on the background of chemotherapy (5FU) developed severe obstructive disease, about which produced cutaneous-resicence, the external-internal drainage of the right and left hepatic ducts, and in another 6 months choledochojejunostomy removable transretinoic drains with brown anastomosis. In November 1995, the condition worsened. Clinically and according to observations in a patient with secondary active hepatitis. The liver is enlarged and painful, appears from under the costal arch on the 5 6 see blood Biochemical parameters of pathologically altered, weakly amenable to correction treatment: hyperbilirubinemia to 40.0 due to the indirect (up 31,0); increased transaminase activity 6 times, hypoalbuminemia 26 hypergammaglobulinemia; hypercholesterolemia to 10.2 µmol/L.

When fibrogastroduodenoscopy (November 1995) in the middle third of the body of the stomach along the greater curvature exophytic growing tumor with a length of 8 cm Tumor dense. The stomach wall rigid. Histologically, the tumor was identified as adenocarcinoma moderate degree of differentiation. In DECA rash on the peritoneum. Palpable splenomegaly is defined. Case recognized inoperable.

In the absence of adequate therapy, the decision to use drug form GSSG in a 0.1 solution of hypoxanthineguanine. The drug was administered systemically (intravenous or intramuscular) and locally (around the tumor through the endoscope at those points). The average dose for intravenous and intramuscular injection of 0.1 to 0.5 mg/kg; in the local introduction of up to 50 mg topically. Parenteral administration of medication 2 times a day (morning intravenously; in the evening intramuscular injection), every other day, for three weeks; then twice a week for 4 weeks. Chip through the endoscope once every seven days.

2 months after the start of treatment by the proposed method, the picture fibrogastroduodenoscopy: the esophagus is freely pass, slimy pink, socket cardia not fully closed. On an empty stomach in the stomach a moderate amount of foamy secretion intensely colored bile, stomach area of the body is deformed in the form of "hourglass", the mucosa in the area of the narrowing of the body infiltrated and dense. The extent of tumor 5 cm

At the same time there has been a significant improvement in the hematological, biochemical and immobolizing palpation; according to the U.S. at the place of the previously defined focal lesions of fibrous tissue. Picture fibrogastroduodenoscopy (may 1996): the esophagus is not closed; in the gastric lumen light turbid liquid with saliva. The mucosa of the stomach pink. In the bottom third on the greater curvature tumorous formation length of 3.6 cm with fibrin. The stomach wall is elastic. Duodenum freely passable.

Compared with the survey before treatment (November 1995) determined the decrease of tumor size 55 Simultaneously observed strong positive dynamics in liver samples, hematological and immunological parameters (see tab. 20).

Thus, as a result of the treatment by the proposed method against significant stimulation of endogenous production of cytokines and hemopoietic factors observed partial regression of the tumor process, significantly improving the quality of life and improvement of basic physiological functions of the body.

Example 12. Stimulation of endogenous production of cytokines and therapeutic effect in a patient with neuro-endocrine skin cancer (tumor Merkel) stage III, complicated chemoimmunotherapy caused cholevinae education in the left coloroptions region, painless, gradually increasing in size. One month after the process has spread to the left axillary region with the daily increase in size, become painful. Appeared fever (t 38,9oC). Examination (histological and immunohistochemical), held in October 1995, said the diagnosis of neuro-endocrine skin cancer (tumor Merkel) stage III.

In December 1995, the course chemotherapy under the scheme CMF (cyclophosphamide + methotrexate + fluorouracil) without appreciable therapeutic effect. At the same time marked toxic effects on haematopoiesis (leukocytes 2,4109/l); growth srednesemennyh and supraclavicular lymph nodes with hyperemia of the skin.

In January and February 1996, the regimen was changed: instead of CMF completed the course CF (cisplatin, cyclophosphamide).

Against the backdrop of ongoing chemotherapy complications: cytopenia (leukocytes 1,4109/l), cardiotoxicity in the form of acute ischemic heart disease. After the 2nd course of chemotherapy sharp progression of tumour necrosis in the left axillary region with the formation of the fistula, increase infiltration of soft tissue in the left shoulder and the left axillary region: swelling of the left hand (lymphostasis); int is m effect of cytostatic therapy decided to therapy dosage form GSSG in a 0.1 solution applied (hereinafter the product), in parallel with chemotherapy (CMF scheme).

After 10 daily injections of the drug (intravenous and intramuscular injection, at a dose of one infusion of 0.1 to 0.5 mg/kg) noted improved quality of life (good appetite, mobility), drying wounded areas, the elimination of purulent discharge, scarring of the fistula, tumor shrinkage of 30 normalization of body temperature, limiting areas of congestion, the improvement of hematological parameters.

Held the 3rd and 4th courses of chemotherapy (CMF scheme) with the simultaneous introduction of the dosage form GSSG in a 0.1 solution applied: intravenous and intramuscular injections twice a day in a dose of 0.5 mg/kg intravenously and 0.2 mg/kg intramuscularly. Parenteral administration of the drug 3 times a week for the scheme and once a week a local around the lesion in two points: 30 mg in each point. On the background of the specified combination therapy is marked regression of the tumor process, excellent tolerability of chemotherapy, including the absence of pain, constant improvement of quality of life, restoration of the immune system and the blood, increasing blood cytokines and hemopoietic factors.

After 2 months of treatment by the Directors, regression of left mid-cervical and supraclavicular lymph nodes, regression of tumor size in two dimensions 70 from the source, the positive dynamics of the immunological parameters (see tab. 21), the lack of inhibitory action of cytostatic therapy on the blood.

Clinical observation shows obvious curative effect of the proposed method, for a significant stimulation of endogenous production of cytokines and hemopoietic factors identified tumor shrinkage, improve the quality of life and indicators of basic bodily functions.

1. The use of oxidized glutathione, which represents a dimer of glutathione restored Tripeptide structure-glutamylcysteinylglycine, in which two molecules of Tripeptide with the above structure are connected to each other by covalent disulfide bond between cysteine residues as a stimulator of endogenous production of cytokines and geopoliticheskih factors.

2. For the treatment of cancer, infectious, hematologic, and other diseases that appropriate stimulation of endogenous production of cytokines and hemopoietic factors containing active the effective amount of oxidized glutathione.

3. Means under item 2, characterized in that it is an injectable solution of oxidized glutathione in a pharmaceutically acceptable solvent in a concentration of 0.01 to 2.0%

4. Tool for PP.2 and 3, characterized in that in order to enhance and prolong therapeutic effect it contains a pharmaceutically acceptable component, is able to extend the presence of glutathione in the oxidized form.

5. The method according to p. 4, characterized in that it is an injectable solution of oxidized glutathione in the composition of 0.003% hydrogen peroxide solution.

6. The method of potentiation of the effects of glutathione oxidized to stimulate endogenous production of cytokines and hemopoietic factors, namely, that of oxidized glutathione is used in the form of pharmaceutical compositions containing a component that is able to extend the presence of glutathione in the oxidized form.

7. A method of treating cancer, infectious, hematologic, and other diseases that appropriate stimulation of endogenous production of cytokines and hemopoietic factors, including parenteral dosage forms pharmacological tools for PP.3 and 4 in doses of 0.01 to 0.5 mg of glutathione oxide is wow effect.

 

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SUBSTANCE: the present innovation deals with enhancing circulation and trophicoregenerative processes. The suggested curative-prophylactic balsam includes fresh-water sulfide-free average-ash sapropel mud of Kirek Lake, turpentine oil (oleoresin turpentine), peroxide, anionic SAS, starch taken at a certain quantitative ratio. The suggested balsam enables to efficiently enhance circulation and trophicoregenerative processes.

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3 ex, 1 tbl

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