Means for treating immunodeficiencies


(57) Abstract:

The tool can be used in medicine and pharmacology to treat immunodeficiencies. As a means to ensure the proposed Hexapeptide structural formula lysyl-histidyl-glycyl-lysyl-histidyl-glycine. Additionally, the tool contains a stabilizer glycine. The tool has a high effectiveness in relation to stimulation of humoral immunity, immune response and molecules intermolecular action for the treatment of immunodeficiency States. 2 C.p. f-crystals, 3 tables.

The invention relates to medicine, namely to pharmacology, and can be used for the prevention and treatment of immune disorders of various etiologies.

In modern immunopharmacology there is a certain Arsenal of medicines containing peptides for use in the treatment of congenital and acquired immunodeficiency syndrome Louis-Bar and in patients with infectious and malignant neoplasms.

It is known to use the complex thymus peptides - Taktivina in patients with congenital immunodeficiency syndrome (Louis-Bar) and in patients with Hodgkin's disease [(Lopukhin J. M., R. V. Petrov et al., Pa is rune-muscle conduction in patients with syndrome Louis-Bar, the reduction of the phenomena of intoxication in patients with Hodgkin's disease. However, the known drug does not have a strong effect on the strength of the immune response and the production of immunoglobulins. In addition, Taktivin is an unseparated mixture of peptides and therefore not be a reliable standardization.

The closest analogue of the proposed medicinal product is buropean derived from synthetic Tripeptide Lys-His-Gly-NH2(lysyl-histidyl-glycyl-amide) [Audhya T., Kroon D. J., G. Heavner, G. Goldstein Bursopoietin. Pat. 4584284 (USA) // C. A. 1986. - Vol. 105. - 173063n.]. The drug stimulates the differentiation of b-lymphocytes and can be used for the treatment of patients with disorders of humoral immunity.

The proposed invention aims at creating drugs with higher efficacy in relation to stimulation of humoral immunity (antitelomerase), strength of the immune response and molecules intercellular interaction for the treatment of congenital and acquired immunodeficiencies.

The solution of this problem is provided by the fact that as a means for treating immunodeficiencies various etiologies used the SUB>O7N12.

The proposed product contains as active ingredient a synthetic Hexapeptide, molecular weight 662,7 D.

The active substance is a white powder, odorless, easily soluble in water and isotonic sodium chloride. The substance is not soluble in alcohol and chloroform.

The proposed remedy for treating immunodeficiencies contains 0.001 to 1% (within 0.00001 - 0.01 g) of the Hexapeptide. As a stabilizer (filler) tool contains 0.05-5% (of 0.0005-0.05 g) glycine. The most preferred form of the proposed funds in the form of a 0.005% solution of the Hexapeptide for subcutaneous or intramuscular injection in vials of 1.0 ml as a stabilizer tool contains 0.5% solution of glycine. Shelf life 2 years when 4-8oC.

The drug was developed in the research-and-production enterprise "Bionics", and its structural formula is obviously not following properties to stimulate humoral immunity, enhance the strength of the immune response (expression of HLA-DR on the surface of hematopoietic cells), and activate T-immunity with therapeutic effect.

Results doclines is 2">

Experimental and clinical data show that:

1. Hexapeptide stimulates antibody productive cells in 4 times more efficient compared to the substance of the prototype (table. 1), increases the amount of immunoglobulin in patients with primary acquired hypogammaglobulinemia (example 5).

2. The claimed remedy stimulates the immune response of lymphocytes of patients with genetically determined immunodeficiency (congenital agammaglobulinemia) according to the criterion of expression of antigens of the major histocompatibility complex (HLA-DR) and molecules intercellular communication (CD2). The substance of the prototype has no such effect (table. 3).

3. The Hexapeptide has an extremely low toxicity, and provides a wide safety margin. Single dose, thousands of times larger than the average therapeutic, does not cause the death of experimental animals.

4. Determination of the chronic toxicity of the medicinal product demonstrates its harmlessness. Identified variations in hematological and biochemical parameters in the appointment of animal medicines daily for one month, therapeutic or 10-fold excess of thorns pharmaceuticals does not cause local irritation, the drug has no allergenic and mutagenic activity.

The drug has shown good clinical results in the treatment of immunodeficiency in patients with congenital agammaglobulinemia and hypogammaglobulinemia.

Clinical use in patients with congenital immunodeficiency, with gobeproductive major classes of immunoglobulins are carried out taking into account clinical and immunological status of patients. To do this, before assigning means determines the state of cellular immunity, the expression of markers of T-lymphocytes, the strength of the immune response of b-lymphocytes and the number of immunoglobulins in the serum.

The effectiveness of therapy assessed by clinical and immunological indicators, including positive dynamics of the number of immunoglobulins and to reduce or limit the severity of the clinical course of the disease. The medicine is prescribed to patients of congenital immunodeficiency States is in complex therapy with the means of pathogenetic therapy. The drug is prescribed courses for subcutaneous or intramuscular injection at a dose of 0.5-5 µg/kg of body weight once, M repeat courses within 1-3 weeks.

Specific examples of the preparation of the active substance, descriptions of the biological and therapeutic properties of drugs.

Example 1. Hexapeptide synthesized by the method of solid-phase synthesis on an automated synthesizer Beckman-990".

Aminomethylpyrimidine (1.8 g, 1 mmol) and allowed to swell in chloroform for 30 minutes, then added Boc-Gly-OCH2C6H4CH2COOH (0.65 g, 2 mmol) with N,N-dicyclohexylcarbodiimide (DCGK) (0.4 g, 2 mmol in 15 ml of chloroform for 2 hours). After washing with dimethylformamide, the polymer is treated with 20 ml of a mixture of diisopropylethylamine-acetic anhydride, 2:1 for 30 minutes. After washing with dimethylformamide and chloroform, the polymer is treated with 30 ml of a mixture of triperoxonane acid - chloroform 1:1 to 20 minutes, washed with chloroform and neutralized 7% solution diisopropylethylamine in dimethylformamide for 10 minutes, then aminoacylation washed with dimethylformamide. Joining the BOC-amino acids is performed using symmetric anhydride of BOC-amino acids: 4 mmol of the BOC-amino acid dissolved in 5 ml of chloroform, cooled to 0oC, add a solution of 2 mmol DCGK in 5 ml of chloroform. After stirring for 10 minutes at 0oC d at 28oC.

After the accession of the next amino acid participalion washed with dimethylformamide, chloroform, then remove the protective BOC-group.

Remove dinitroaniline group: 2 g peptidylarginine stirred for 1.5 hours at room temperature in 40 ml of 20% aqueous solution of thiophenol in dimethylformamide. Then participalion washed with dimethylformamide, chloroform and dried in vacuum.

Removing the peptide from the resin: 2 r peptidylarginine placed in the reaction vessel of the device for operation with hydrogen fluoride, add 1 g of n-cresol, cooled with liquid nitrogen and after degassing vessel is distilled in 10 ml of liquid anhydrous hydrogen fluoride. Bring the temperature of the reaction vessel to -20oC and stirred polymer for 30 minutes, maintaining the temperature of the mixture CCl4-solid CO2then place the reaction vessel in an ice bath and maintained under stirring for another 30 minutes. After that, hydrogen fluoride evaporated on a water-jet pump, the polymer was washed on the filter with diethyl ether. Then the peptide is extracted with 20% acetic acid and lyophilizers.

Remove trifluoracetyl group. Made from resin peptide obrabatyvayutsya and absoluut gelfiltration on a column of Sephadex G-25 in 5% acetic acid.

Example 2. Investigation of the effect of the Hexapeptide (declared substances) and buropean (substances prototype) for products antitelomerase cells in the spleen of mice.

Use of male mice CBA/CaLacSto, which are subjected to immunization by intravenous sheep erythrocytes in a dose of 2106. After 10-15 minutes after immunization mice intraperitoneally injected stated the substance and the substance of the prototype in a volume of 0.5 ml at concentrations of 0.02; 0,1; 0,5 and 2,5 µg/ml (7-8 animals per group). The control group mice (7-8 animals per group) intraperitoneally injected with a similar volume of saline. On the fourth day after immunization determine the number of antibody productive cells in the spleen of the mouse by counting the local hemolysis of sheep red blood cells in agarose gel. The results of the study are presented in table 1.

The data obtained demonstrate that the Hexapeptide (declared substance) causes a four-fold excess education antibody productive cells in the spleen of the mouse compared with the substance of the prototype. Thus, the claimed substance has a higher activity on the basis of stimulation of antitelomerase than the substance of the prototype.

Example 3. Research VL is Askerov T-lymphocytes (CD2, CD4, CD8) and b lymphocytes (CD22) cells of healthy donors.

In use cells in human peripheral blood. To obtain the fraction of mononuclear cells from heparinized (25 U/ml) blood used single-stage gradient ficoll-urografin with a density of 1.077 g/cm3.

The level of expression of membraneassociated structures CD2, CD4, CD8, CD22, HLA-DR on mononuclear cells to determine the cellular solid immunoperoxidase method, using monoclonal antibodies to structures CD2, CD4, CD8, CD22, HLA-DR ("Seromed, UK) and artemisinin F(ab') fragments of immunoglobulins conjugated to horseradish peroxidase ("Seromed, UK). The analysis is performed after an hour incubation of the cells in the presence of 0.02; 0,1; 0,5; 2,5 µg/ml of the substance and of the substance of the prototype. In control experiments mononuclear cells incubated under similar conditions in a culture medium without added substances. The cultivation is carried out in medium RPMI - 1640 with 5% fetal calf serum in a final volume of 3 ml and a concentration of 2106/ml.

Enzyme-linked immunosorbent assay performed on flat-bottomed plates ("Nunc, Denmark), pre-treated 0,001%-H06/ml, 50 ál per well. After centrifugation, the cells fixed with 50 μl of a 0.5% aqueous solution of glutaraldehyde (Sigma, USA) in phosphate-buffered saline for 15 minutes. Tablets thrice washed with phosphate-saline buffer and then treated with 0.1 M solution of glycine (Sigma, USA), after which the wells make a 1% solution of bovine serum albumin in phosphate-buffered saline and incubated for 12-14 hours at +4oC. When setting reaction in the wells contribute 100 ál of the appropriate monoclonal antibody, diluted in phosphate-buffered saline with 0.5% solution of bovine serum albumin, incubated for 1 hour at +37oC. After cleaning with 0.01% solution of Tween-20 (Merck, Germany) in phosphate-buffered saline applied conjugate (Sigma, USA) in a volume of 100 μl per well, incubated, washed and added the substrate solution, orthophenylphenol ("Sigma", USA) in 0.1 M citrate-phosphate buffer, pH 4.5 and 0,004% hydrogen peroxide. After incubation for 30 minutes the reaction is stopped 1N sulfuric acid and evaluated spectrophotometrically by determining levels of absorption at a wavelength of 492 nm. The results are expressed as the percentage change of the values of optical density in the experience, in relation to the od value of the CSOs use the system without monoclonal antibodies, identifying the background of natural peroxidase activity and nonspecific adsorption of the conjugate to cells. Each experimental group of cells examined in the triplet.

Statistical processing of experimental data is carried out with the determined reliability of difference on the criterion of student-Fisher. Determine the significance of differences between sample means of experience and control. On the calculated value of t and the number of option compared to determine the significance of differences using the table of student-Fisher. The difference is considered significant at a significance level of 5% (p<0,05).

The data obtained demonstrate that the Hexapeptide (declared substance) causes significant excess of the expression of HLA-DR and CD2 on the surface of hematopoietic cells. On the contrary, the substance of the prototype causes a decrease in the expression of these surface structures. Thus, the claimed substance unlike the prototype has the property to increase the expression of genes of the immune response (HLA-DR) and solo agammaglobulinemia on the expression of HLA-DR and markers of lymphocyte subpopulations. The level of expression of membraneassociated structures CD2, CD4, CD8, CD22, HLA-DR on mononuclear cells to determine the cellular solid immunoperoxidase in a manner similar to that specified in example 3.

Comparative evaluation of the expression of surface structures on the cells of patients with agammaglobulinemia under the influence of the requested drug (Hexapeptide) and substances prototype (buropean) are shown in table 3.

The data obtained demonstrate that the Hexapeptide (declared substance) causes significant excess of the expression of HLA-DR and CD2 on the surface of hematopoietic cells of patients with agammaglobulinemia. On the contrary, the substance of the prototype causes a decrease in the expression of these structures on the cell surface in patients. Thus, the claimed substance unlike the prototype has the property to increase the expression of genes of the immune response (HLA-DR) and the maturation of T lymphocytes (CD2) in patients with congenital immunodeficiency.

Example 5. Patient P., 25 years. Clinical diagnosis: primary acquired gipogammaglobulinemia (late start).

Upon receipt by the inspection noted the presence of dermatomyositis and chronic progressive polyarthritis. When obledo peratively ability of T-lymphocytes in the reaction of besttransport and reduction of serum immunoglobulins lgA, lgG, lgM. The drug was administered once a day for 14 days. After completing therapy, a marked increase in the number of lgG with 95 mg% to 350 mg%; lgM with 49 mg% to 103 mg%, the amplification reaction besttransport lymphocytes with 753423003 pulses/min to 1864767954 pulses/min, respectively. Clinical effect after the appointment of the drug observed on the ninth day in terms of reducing muscle pain and symptoms of arthritis. The patient noted improvement in overall health, sleep and mood. Adverse reactions to the drug is not marked.

Thus, the claimed remedy stimulates the production of immunoglobulins and has a therapeutic effect on hypogammaglobulinemia.

1. Means for treating immunodeficiencies containing peptide, characterized in that it contains peptide Hexapeptide structural formula lysyl-histidyl-glycyl-lysyl-histidyl-glycine.

2. Means under item 1, characterized in that it additionally contains a stabilizer.

3. Means under item 2, characterized in that the stabilizer it contains glycine.


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