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 cyclopentapeptide structural formula cyclo-lysyl-histidyl-glycyl-lysyl-histidyl-glycine. Additionally, the tool contains a stabilizer (filler) glycine. The tool has a high effectiveness in relation to stimulation of humoral immunity, immune response and molecules intercellular interaction for the treatment of immunodeficiency States. 2 C.p. f-crystals, 2 tab.

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 and used in the treatment of immunodeficiency in patients with congenital syndrome Louis - Barr) and acquired immune pathology caused by infection and malignant neoplasms.

It is known to use the complex thymus peptides - Taktivina in congenital disorders of immunity syndrome (Louis-Bar) and be able T-cell immunity, improvement of neuromuscular conduction in patients with syndrome Louis-Bar, reducing the effects of intoxication in patients with Hodgkin's disease. However, the known drug has expressed no effect on the strength of the immune response and production of antibodies, which limits its scope. In addition, Taktivin is an unseparated mixture of peptides and cannot be trusted standardization.

The closest analogue of the proposed tools is the drug 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 among the th formula: cyclo-lysyl-histidyl-glycyl-lysyl-histidyl-glycine. Brutto-formula: C28H44O6N12.

The proposed product contains as active ingredient a synthetic Hexapeptide, molecular weight 644,7 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) cyclopentapeptide. As a stabilizer (filler) tool contains 0.05-5% (of 0.0005-0.05 g) glycine. The drug is released in the form of a 0.005% solution of cyclopentapeptide for subcutaneous or intramuscular injection in vials of 1.0 ml as a stabilizer of the proposed 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.

Re is cnyh action.

Experimental and clinical data show that:

1. Cyclopentapeptide stimulates antibody productive cells 2 times more efficient compared to the substance of the prototype (see table. 1), prevents bacteria carrier and activates the production of secretory lgA (Sig A).

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

3. Cyclopentapeptide 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 therapiest means patients does not cause local irritation, the drug has no allergenic and mutagenic activity.

The drug has shown good clinical results in the treatment of infectious patients with reduced function of humoral immunity.

Clinical application of the drug in patients with gobeproductive secretory immunoglobulin a is carried out with regard to clinical and immunological status of patients. So, in the population, the presence of secretory immunoglobulin a (Sig (A) is celebrated in 60,910,4% of cases. Infectious patients after conventional treatment, respectively, in 69,07,1% of cases. Violation of the products Sig And is caused by bacteria carrier and low efficiency of treatment of bacterial infections. The use of the drug causes the activation of the local immune system and the appearance of Sig And 100.0% of cases.

In this regard, before the appointment of the drug determine the state of humoral immunity and the number of Sig And in the saliva of patients.

The effectiveness of therapy assessed by positive changes in the number of secretory immunoglobulin a, eliminate bacteria carrier and reduce the effects of intoxication. The purpose lekarstvennayaforma means prescribed courses for subcutaneous or intramuscular injection at a dose of 0.5 - 5 µg/kg of body weight once daily or at intervals of 1-3 days within 7-15 days. If necessary, the clinical and immunological indicators to repeat courses in 1-3 weeks.

Specific examples of the preparation of the proposed tools, descriptions of the biological and therapeutic properties of drugs.

Example 1. Cyclopentapeptide 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 of diisopropylethylamine in dimethylformamide for 10 minutes, then aminoacylation washed with dimethylformamide. Joining Boc-amino acids is performed using symmetrical angeri the PRS 2 mmol DCGK in 5 ml of chloroform. After stirring for 10 minutes at 0oTo the mixture was added to participalion, poured 10 ml of dimethylformamide and stirred with paticipation 30 minutes at 28oC.

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

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

Removing the peptide from the resin: 2 g 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 did: made from resin peptide purified by high performance liquid chromatography (HPLC). Then the peptide (50 mg) is dissolved in 25 ml of dimethylformamide and added dropwise with stirring and cooling to 0oC to a solution of dimethylacetanilide (10 EQ) in 25 ml of dimethylformamide, in which the suspended crystalline potassium carbonate. The suspension is stirred for 2 days, then filtered potassium carbonate and evaporated dimethylformamide.

Remove trifluoracetyl group. Made from resin, the peptide is treated with 60 ml odnopolyarnogo aqueous solution of piperidine at 0oC for 2 hours. Then the reaction mixture lyophilizer and absoluut gelfiltration on a column of Sephadex G-25 in 5% acetic acid.

Example 2. Study the impact of the proposed drug 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 the same volume of Phys is elegance 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 cyclopentapeptide (declared means) causes a two-fold excess education antitelomerase cells in the spleen of the mouse compared with the effect of substances on the prototype. Thus, the claimed product has a higher activity on the basis of stimulation of antitelomerase than the substance of the prototype.

Example 3. Investigation of the influence of cycloheximide and substance of the prototype (buropean) 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) 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, CD22, HLA-DR on mononuclear cells to determine the cellular solid immunoperoxidase method, using monoclonal antibodies to structures CD2, CD4, CD22, HLA-DR ("Seromed, UK) and artemisinin F(ab') fragments of antibodies, to 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% solution of poly-L-lysine (Sigma, USA) in phosphate-buffered saline, pH of 7.3. The investigated cells contribute at a concentration of 4106/ml, 50 ál per well. After centrifugation, the cells fixed with 50 µl of 0.5% solution of glutaraldehyde (Sigma, USA) in phosphate-buffered saline for 15 minutes. Tablets thrice washed with phosphate-saline buffer and then processed 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 +37ovolume 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 IN 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 value of the optical density in the control (cells not treated with the stuff), taking into account the control reactions, which use the system without monoclonal antibodies, allowing to reveal 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 cyclopentapeptide (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 the maturation of T lymphocytes (CD2).

Example 4. Patient, 25 years. Clinical diagnosis of salmonellosis, the gastrointestinal form, moderate current.

Ill acute: severe abdominal pain cramping in nature, loose stools with mucus, repeated vomiting, body temperature rose to 39oC.

The laboratory examination of the patient noted: TPHA with Salmonella antigen: serum 1:20; saliva 1:4. Immunoglobulins serum: lgA - of 2.97 mg/ml, lgG - 12,45 mg/ml, lgM - 0.43 mg/ml; saliva lgA - 0.19 mg/ml, Sig And OTS. , lgG - 0.25 mg/ml, lgM - traces. Bacteriological examination of faeces: S. Typhimurium (gr. B).

Treatment. Trial, glucose 5%/drops, sulgin 1.0 x 4 times per day. Cyclopentapeptide of 1.0 ml. of 0.005% solution of 1 times a day/m for 7 days.

Thus, the claimed product has a pronounced therapeutic effect, prevents bacteria carrier and stimulates the production of secretory lgA.

1. Means for treating immunodeficiencies containing peptide, characterized in that as it contains a peptide cyclopentapeptide structural formula cyclo-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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