RU2157234C1 - Agent for treatment of patients with immunodeficiency state - Google Patents

Abstract

FIELD: medicine, pharmacology. SUBSTANCE: invention proposes hexapeptide of the structural formula: lysyl-histidyl-glycyl-lysyl-histidyl-glycine as a drug. Drug has glycine as a stabilizing agent additionally. Drug shows high effectiveness of action with respect to stimulation of humoral immunity, immune response potency and molecules of intermolecular interaction in therapy of immunodeficiency states. EFFECT: enhanced effectiveness of agent. 3 cl, 3 tbl, 5 ex

Description

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

 In modern immunopharmacology, there is a certain arsenal of drugs containing peptides that are used in the treatment of congenital and acquired immunodeficiency states with Louis-Bar syndrome and in patients with infectious and malignant neoplasms.

 It is known to use a complex of thymus peptides - Tactivin in patients with congenital immunity disorder (Louis-Bar syndrome) and in patients with lymphogranulomatosis [(Lopukhin JM, Petrov RV et al., Pat. 4377511 (USA) // CA - 1983]. Tactivin causes significant improvement of the state of T-cell immunity, improvement of neuromuscular conduction in individuals with Louis-Bar syndrome, reduction of intoxication phenomena in patients with lymphogranulomatosis, however, the known drug does not have a pronounced effect on the strength of the immune response and the production of immunoglobulins. n represents an unshared peptide mixture and therefore can not be reliable standardization.

The closest analogue of the proposed drug is bursopoietin, obtained on the basis of the synthetic tripeptide Lys-His-Gly-NH ₂ (lysyl-histidyl-glycyl amide) [Audhya T., Kroon DJ, Heavner G., Goldstein G. Bursopoietin. Pat. 4584284 (USA) // CA - 1986. - Vol. 105. - 173063n.]. The drug stimulates the differentiation of B-lymphocytes and can be used to treat patients with impaired humoral immunity.

 The proposed invention is aimed at creating a drug with higher efficacy in relation to the stimulation of humoral immunity (antibody production), the strength of the immune response and intercellular interaction molecules for the treatment of congenital and acquired immunodeficiency states.

The solution of this problem is provided by the fact that as a means for the treatment of immunodeficiency states of various etiologies, a hexapeptide of the structural formula is used: lysyl-histidyl-glycyl-lysyl-histidyl-glycine. Gross formula: C ₂₈ H ₄₆ O ₇ N ₁₂ .

 The proposed tool contains as active substance a synthetic hexapeptide, a molecular weight of 662.7 D.

 The active substance is an odorless white powder, readily soluble in water and isotonic sodium chloride solution. The substance is not soluble in alcohol and chloroform.

The proposed tool for the treatment of immunodeficiency states contains 0.001-1% (0.00001-0.01 g) of the hexapeptide. As a stabilizer (filler), the product contains 0.05-5% (0.0005-0.05 g) of glycine. The most preferred form of release of the proposed funds in the form of a 0.005% solution of hexapeptide for subcutaneous or intramuscular injection in ampoules of 1.0 ml. As a stabilizer, the product contains a 0.5% glycine solution. Shelf life 2 years at 4-8 ^o C.

 The drug was developed at the Bionox research and production enterprise, and its structural formula clearly does not follow the properties of stimulating humoral immunity, enhancing the strength of the immune response (HLA-DR expression on the surface of hematopoietic cells), and activating the T-link of immunity with achievement therapeutic effect.

 The results of preclinical and clinical studies of the proposed tool show that it does not cause side effects.

Experimental and clinical data show that:
1. Hexapeptide stimulates the formation of antibody-forming cells 4 times more effective than the substance of the prototype (table. 1), increases the number of immunoglobulins in patients with primary acquired hypogammaglobulinemia (example 5).

 2. The claimed tool stimulates the immune response of lymphocytes of patients with genetically determined immunodeficiency (congenital agammaglobulinemia) by the criterion for the expression of antigens of the main histocompatibility complex (HLA DR) and intercellular interaction molecules (CD2). The substance of the prototype does not have a similar effect (table. 3).

 3. Hexapeptide has extremely low toxicity and provides a wide safety margin. A single dose, a thousand times higher than the average therapeutic, does not cause the death of experimental animals.

 4. The determination of chronic toxicity of a drug demonstrates its harmlessness. The revealed fluctuations in hematological and biochemical parameters during administration of a drug to animals every day, for one month, at a therapeutic or 10-fold higher therapeutic dose after drug withdrawal returns to its original level.

 5. The introduction of the drug does not cause local irritant effects, the drug does not have allergenicity and mutagenic activity.

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

 The clinical use of the drug in patients suffering from congenital immunodeficiency, with hypoproduction of the main classes of immunoglobulins is carried out taking into account the clinical and immunological state of the patients. For this, before prescribing, the state of cellular immunity, the expression of T-lymphocyte markers, the strength of the B-lymphocyte immune response and the amount of immunoglobulins in blood serum are determined.

 The effectiveness of therapy is evaluated by clinical and immunological parameters, including the positive dynamics of the number of immunoglobulins and reduction or decrease in the severity of the clinical course of the disease. The prescription of the drug to patients with congenital immunodeficiency is carried out in complex therapy with pathogenetic therapy. The drug is prescribed courses for subcutaneous or intramuscular injection in a dose of 0.5-5 μg / kg of body weight once, daily or with an interval of 1-3 days for 10-30 days. If necessary, according to clinical and immunological parameters, repeated courses are carried out for 1-3 weeks.

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

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

Aminomethylpolymerase (1.8 g, 1 mmol) was allowed to swell in chloroform for 30 minutes, then Boc-Gly-OCH ₂ C ₆ H ₄ CH ₂ COOH (0.65 g, 2 mmol) was added with N, N-dicyclohexylcarbodiimide (DCCA) ) (0.4 g, 2 mmol in 15 ml of chloroform for 2 hours). After washing with dimethylformamide, the polymer was treated with 20 ml of a 2: 1 diisopropylethylamine-acetic anhydride mixture for 30 minutes. After washing with dimethylformamide and chloroform, the polymer is treated with 30 ml of a trifluoroacetic acid-chloroform 1: 1 mixture for 20 minutes, washed with chloroform and neutralized with a 7% solution of diisopropylethylamine in dimethylformamide for 10 minutes, then the aminoacyl polymer is washed with dimethylformamide. Boc amino acid additions are carried out using symmetric Boc amino acid anhydride: 4 mmol of Boc amino acid is dissolved in 5 ml of chloroform, cooled to 0 ^° C., a solution of 2 mmol of DCCA in 5 ml of chloroform is added. After stirring for 10 minutes at 0 ^° C., the mixture was added to the peptidyl polymer, 10 ml of dimethylformamide was added and mixed with the peptidyl polymer for 30 minutes at 28 ^° C.

 After the addition of the next amino acid, the peptidyl polymer is washed with dimethylformamide, chloroform, and then the protective Boc group is removed.

 Removal of the dinitrophenyl group: 2 g of the peptidyl polymer are stirred for 1.5 hours at room temperature in 40 ml of a 20% solution of thiophenol in dimethylformamide. Then the peptidyl polymer is washed with dimethyl foramide, chloroform and dried in vacuum.

Peptide removal from the resin: 2 r peptidylpolymers are placed in the reaction vessel of the hydrogen fluoride apparatus, 1 g of n-cresol is added, it is cooled with liquid nitrogen, and after evacuation of the vessel, 10 ml of liquid anhydrous hydrogen fluoride are distilled into it. The temperature of the reaction vessel was brought to -20 ^° C and the polymer was stirred for 30 minutes while maintaining the temperature of the CCl ₄ -hard CO ₂ mixture, then the reaction vessel was placed in an ice bath and kept stirring for another 30 minutes. After that, hydrogen fluoride is evaporated on a water-jet pump, the polymer is washed on the filter with diethyl ether. The peptide is then extracted with 20% acetic acid and lyophilized.

Removal of trifluoroacetyl group. The peptide removed from the resin is treated with 60 ml of a unipolar aqueous piperidine solution at 0 ^° C. for 2 hours. The reaction mixture is then lyophilized and desalted by gel filtration on a Sephadex G-25 column in 5% acetic acid.

 Example 2. The study of the effect of hexapeptide (the claimed substance) and bursopoietin (the substance of the prototype) on the production of antibody-forming cells in the spleen of mice.

Male CBA / CaLacSto mice were used, which were immunized intravenously with ram erythrocytes at a dose of 2 • 10 ⁶ . 10-15 minutes after immunization, the mice are injected intraperitoneally with the claimed substance and the prototype substance in a volume of 0.5 ml at concentrations of 0.02; 0.1; 0.5 and 2.5 μg / ml (7-8 animals in the group). The mice of the control group (7-8 animals in the group) are injected intraperitoneally with the same volume of saline. On the fourth day after immunization, the number of antibody-forming cells in the mouse spleen is determined by counting the local hemolysis zones of sheep erythrocytes in an agarose gel. The results of the study are presented in table 1.

 The data obtained demonstrate that the hexapeptide (the claimed substance) causes a four-fold increase in the formation of antibody-forming cells in the mouse spleen compared with the prototype substance. Thus, the claimed substance has a higher activity on the basis of stimulation of antibody formation than the substance of the prototype.

 Example 3. The study of the effect of hexapeptide and prototype substances (bursopoietin) on the expression of the marker of the strength of the immune response (HLA-DR), markers of T-lymphocytes (CD2, CD4, CD8) and B-lymphocytes (CD22) by cells of healthy donors.

In the work using cells of human peripheral blood. To obtain a fraction of mononuclear cells from heparinized (25 IU / ml) blood, a single-stage ficoll-verographin gradient with a density of 1.077 g / cm ^{3 is used} .

The expression level of membrane-associated structures CD2, CD4, CD8, CD22, HLA-DR on mononuclear cells is determined by the solid-state immunoperoxidase cell method using monoclonal antibodies to structures CD2, CD4, CD8, CD22, HLA-DR (Seromed, UK) and anti-mouse F (ab ') - fragments of immunoglobulins conjugated to horseradish peroxidase ("Seromed, UK). The analysis is carried out after an hourly incubation of cells in the presence of 0.02; 0.1; 0.5; 2.5 μg / ml of the studied substance and the substance of the prototype. In control experiments, mononuclear cells were incubated under similar conditions in a culture medium without the addition of substances. Cell cultivation is carried out in RPMI-1640 medium with the addition of 5% fetal calf serum, in a final volume of 3 ml and a concentration of 2 • 10 ⁶ / ml.

An enzyme-linked immunosorbent assay is carried out on flat-bottomed plates ("Nunc", Denmark), pre-treated with a 0.001% solution of poly-L-lysine ("Sigma", USA) in phosphate-buffered saline, pH 7.3. The studied cells contribute at a concentration of 4 • 10 ⁶ / ml, 50 μl per well. After centrifugation, the cells are fixed with 50 μl of a 0.5% solution of glutaraldehyde (Sigma, USA) in phosphate-buffered saline for 15 minutes. The plates are washed three times with phosphate-buffered saline and then treated with 0.1 M glycine solution (Sigma, USA), after which a 1% solution of bovine serum albumin in phosphate-buffered saline is added to the wells and incubated for 12-14 hours at + 4 ^o C. When setting up the reaction, 100 μl of the corresponding monoclonal antibodies diluted in phosphate-buffered saline with 0.5% bovine serum albumin solution are added to the wells, incubated for 1 hour at +37 ^o C. After washing 0.01% Tween 20 solution ("Merck", Germany) in phosphate-buffered saline they infuse the conjugate (Sigma, USA) in a volume of 100 μl per well, incubate, wash and add a solution of the substrate orthophenylenediamine (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 with 1N sulfuric acid and evaluated spectrophotometrically, determining the absorption levels at a wavelength of 492 nm. The results are expressed as the percentage change in optical density values in the experiment, relative to the optical density in the control (cells not treated with the substance), taking into account the control of the reaction, which is used as a system without monoclonal antibodies, which allows to detect the background of natural peroxidase activity and non-specific sorption conjugate on the cells. Each experimental group of cells is examined in a triplet.

 Statistical processing of experimental data is carried out with the determination of the reliability of the difference by the Student-Fisher criterion. The significance of differences in the sample means of experience and control is determined. The calculated value of t and the number of compared options determine the significance of differences using the Student-Fisher table. The difference is considered significant at a significance level of not more than 5% (p <0.05).

 A comparative assessment of the expression of surface structures on the cells of healthy donors under the influence of the claimed drug (hexapeptide) and the prototype substance (bursopoietin) are shown in table 2.

 The data obtained demonstrate that the hexapeptide (the claimed substance) causes a significant excess of the expression of HLA-DR and CD2 on the surface of human hematopoietic cells. On the contrary, the prototype substance causes a decrease in the expression of these surface structures. Thus, the claimed substance, in contrast to the prototype, has the property of increasing the expression of the immune response genes (HLA-DR) and the maturation of T-lymphocytes (CD2).

 Example 4. Clinical and immunological study of the effect of the drug in patients with agammaglobulinemia on the expression of HLA-DR and markers of subpopulations of lymphocytes. The expression level of membrane-associated structures CD2, CD4, CD8, CD22, HLA-DR on mononuclear cells is determined by the solid-state immunoperoxidase cell method as described in example 3.

 A comparative assessment of the expression of surface structures on the cells of patients with agammaglobulinemia under the influence of the claimed drug (hexapeptide) and the prototype substance (bursopoietin) are shown in table 3.

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

 Example 5. Patient P., 25 years old. Clinical diagnosis: primary acquired hypogammaglobulinemia (with a late appearance).

 Upon admission, examination noted the presence of dermatomyositis and chronic progressive polyarthritis. When examining the patient’s immune status, a functional disorder of the T and B immunity, a decrease in the proliferative ability of T lymphocytes in the blast transformation reaction, and a decrease in the number of serum immunoglobulins IgA, IgG, IgM were noted. The drug was administered once every other day for 14 days. After completion of therapy, an increase in the number of IgG from 95 mg% to 350 mg% was noted; lgM from 49 mg% to 103 mg%, intensification of the reaction of blasttransformation of lymphocytes from 75342 ± 3003 pulses / min to 186476 ± 7954 pulses / min, respectively. The clinical effect after prescribing the drug was noted on the tenth day in the form of a reduction in muscle pain and polyarthritis. The patient notes an improvement in overall health, sleep and mood. No adverse reactions to drug administration were noted.

 Thus, the claimed agent stimulates the production of immunoglobulins and has a therapeutic effect in hypogammaglobulinemia.

Claims (3)

 1. The tool for the treatment of immunodeficiency conditions, containing a peptide, characterized in that as a peptide it contains a hexapeptide of the structural formula lysyl-histidyl-glycyl-lysyl-histidyl-glycine.  2. The tool according to p. 1, characterized in that it further comprises a stabilizer.  3. The tool according to claim 2, characterized in that it contains glycine as a stabilizer.

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