RU2157235C1 - Agent for treatment of patients with immunodeficincy state - Google Patents

Abstract

FIELD: medicine, pharmacology. SUBSTANCE: invention proposes cyclohexapeptide for treatment as drug of the following structural formula: cyclo-lysyl-histidyl-glycyl-lysyl-histidyl-glycine. Drug has glycine as stabilizing agent (vehicle) additionally. Drug shows high effectiveness of effect with respect to stimulation of humoral immunity, immune response potency and molecules of intercellular interaction in therapy of immunodeficiency states. EFFECT: enhanced effectiveness of agent. 3 cl, 2 tbl, 4 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 and used in the treatment of immunodeficiency in patients with congenital (Louis-Barr syndrome) and acquired immune pathology caused by infection and malignant neoplasms.

 It is known to use a complex of thymus peptides - Tactivin in congenital immune disorders (Louis-Bar syndrome) and in patients with lymphogranulomatosis [(Lopukhin JM, Petrov RV et al., Pat. 4377511 (USA) // CA - 1983]. Tactivin causes a significant improvement in the condition 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 antibody production, which limits its scope eniya. Furthermore, Taktivin represents undivided peptide mixture and is not reliable standardization.

The closest analogue of the proposed tool is the drug 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 cyclohexapeptide of the structural formula: cyclo-lysyl-histidyl-glycyl-lysyl-histidyl-glycine is used. Gross formula: C ₂₈ H ₄₄ O ₆ N ₁₂ .

 The proposed tool contains as an active substance a synthetic hexapeptide, a molecular weight of 644.7 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 cyclohexapeptide. As a stabilizer (filler), the product contains 0.05-5% (0.0005-0.05 g) of glycine. The drug is released in the form of a 0.005% solution of cyclohexapeptide for subcutaneous or intramuscular injection in ampoules of 1.0 ml. As a stabilizer, the proposed tool contains a 0.5% solution of glycine. Shelf life 2 years at 4-8 ^o C.

 The drug was developed at the Bionox research and production enterprise and its structural formula does not obviously follow the properties of stimulating humoral immunity, enhancing the strength of the immune response (expression of HLA-DR on the surface of hematopoietic cells), and also activating the T-link of immunity to achieve 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. Cyclohexapeptide stimulates the formation of antibody-forming cells 2 times more effective than the prototype substance (see table. 1), prevents the occurrence of bacterial carriage and activates the production of secretory lgA (Sig A).

 2. The claimed tool stimulates the strength of the immune response according to the criterion for the expression of HLA DR antigens of human lymphocytes; activates intercellular interactions in terms of CD2 antigen expression. On the contrary, the prototype substance suppresses the expression of antigens of the immune response force (HLA DR) and intercellular interaction (CD2) (see table. 2).

 3. Cyclohexapeptide 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 daily, 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 to patients 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 infectious patients with reduced function of the humoral immunity.

 The clinical use of the drug in patients with hypoproduction of secretory immunoglobulin A is carried out taking into account the clinical and immunological status of patients. So, in the population, the presence of secretory immunoglobulin A (Sig A) is noted in 60.9 ± 10.4% of cases. In infectious patients after traditional treatment, respectively, in 69.0 ± 7.1% of cases. Disruption of Sig A production is the cause of bacteriocarrier and low effectiveness of bacterial infections therapy. The use of the drug causes the activation of local immunity and the appearance of Sig A in 100.0% of cases.

 In this regard, before prescribing a drug, the state of humoral immunity and the amount of Sig A in the saliva of patients are determined.

 The effectiveness of therapy is evaluated by the positive dynamics of the amount of secretory immunoglobulin A, the elimination of bacteriocarriers and the reduction of intoxication phenomena. The prescription of the drug to patients is carried out in complex therapy with antibiotics and pathogenetic therapy. The drug is prescribed courses for subcutaneous or intramuscular injection in a dose of 0.5 - 5 μg / kg body weight once, daily or with an interval of 1-3 days for 7-15 days. If necessary, according to clinical and immunological parameters, repeated courses are carried out for 1-3 weeks.

 Specific examples of the proposed tool, a description of the biological and therapeutic properties of the drug.

 Example 1. The cyclohexapeptide 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. The addition of the Boc amino acid is carried out using the symmetric anhydride of the Boc amino acid: 4 mmol of the 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 is added to the peptidyl polymer, 10 ml of dimethylformamide are added and mixed with the peptidyl polymer for 30 minutes at 28 ^° C.

 After the addition of another 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 peptidylpolymer 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.

Removal of the peptide from the resin: 2 g of the peptidylpolymer are placed in the reaction vessel of the device for working with hydrogen fluoride, 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.

Peptide Cyclization: Peptide removed from the resin was purified by high performance liquid chromatography (HPLC). The peptide (50 mg) is then dissolved in 25 ml of dimethylformamide and added dropwise with stirring and cooling to 0 ^° C. to a solution of dimethylphosphoryl azide (10 eq) in 25 ml of dimethylformamide in which crystalline potassium carbonate is suspended. The suspension is stirred for 2 days, after which potassium carbonate is filtered off and dimethylformamide is evaporated.

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 the proposed drug and bursopoietin (substances 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 cyclohexapeptide (the claimed tool) causes a twofold increase in the formation of antibody-forming cells in the mouse spleen compared with the action of the substance of the prototype. Thus, the claimed tool 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 cyclohexapeptide 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) 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, CD22, HLA-DR on mononuclear cells is determined by the solid-state immunoperoxidase cell method using monoclonal antibodies to structures CD2, CD4, 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 were fixed with 50 μl of a 0.5% solution of glutaraldehyde (Sigma, USA) in phosphate-buffered saline for 15 minutes. The tablets are washed three times with phosphate-buffered saline and then treated with 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 with 0.01% Tween 20 solution ("Merck", Germany) on phosphate-buffered saline, apply con the jugate ("Sigma", USA) in a volume of 100 μl per well, incubated, washed and a solution of the substrate - orthophenylenediamine ("Sigma", USA) in 0.1 M citrate-phosphate buffer, pH 4.5 and 0.004% hydrogen peroxide was added . After incubation for 30 minutes, the reaction was stopped with IN 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 (cyclohexapeptide) and the prototype substance (bursopoietin), see table 2.

 The data obtained demonstrate that cyclohexapeptide (the claimed substance) causes a significant excess of HLA-DR and CD2 expression 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. Patient G., 25 years old. Clinical diagnosis: salmonellosis, gastrointestinal form, moderate course.

He became acutely ill: severe abdominal pain of a cramping nature, loose stools with mucus, repeated vomiting, body temperature increased to 39 ^o C.

 During a laboratory examination of the patient noted: RPHA with salmonella antigen: blood serum 1:20; saliva 1: 4. Serum immunoglobulins: IgA - 2.97 mg / ml, IgG - 12.45 mg / ml, IgM - 0.43 mg / ml; saliva lgA - 0.19 mg / ml, Sig A - ex. , logG - 0.25 mg / ml, logM - traces. Bacteriological examination of feces: S. Typhimurium (column B).

 Treatment. Trisol, glucose 5% w / v., Sulgin 1.0 x 4 times a day. Cyclohexapeptide 1.0 ml. 0.005% solution once a day IM for 7 days.

 On the tenth day of the disease, a bacteriological study of feces on salmonella is negative, RPHA with salmonella antigen: blood serum 1: 1280, saliva 1:64. Serum immunoglobulins: IgA - 5.70 mg / ml, IgG - 13.40 mg / ml, IgM - 0.88 mg / ml; saliva: logA - 0.16 mg / ml, Sig A - 0.42 mg / ml, logG - 0.22 mg / ml, logM - traces. On the 12th day of illness with recovery, the patient was discharged from the hospital. No adverse reactions to drug administration were noted.

 Thus, the claimed agent has a pronounced therapeutic effect, prevents the occurrence of bacterial carriage and stimulates the production of secretory lgA.

Claims (3)

 1. The tool for the treatment of immunodeficiency conditions, containing a peptide, characterized in that as a peptide it contains a cyclohexapeptide of the structural formula cyclo-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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