There is a certain arsenal of drugs in the modern immunopharmacology containing peptides and used for treatment of congenital and acquired immunodeficiency states with Louis-Bar syndrome and in patients with infectious and malignant tumors.

It is known the application of a complex of thymus peptides, Tactivin, in patients with congenital disorder of immunity (Louis-Bar syndrome) and in patients with lymphogranulomatosis [ (Lopukhin J. M., Petrov R. V. et al., Pat. 4377511 (USA)//C. A.- 1983]. Tactivin causes the considerable improvement of the state of T-cells immunity and improvement of nervous-muscle conduction in individuals with Louis-Bar syndrome and reduction of the intoxication phenomena in patients with lymphogranulomatosis.

However, the known drug does not have the expressed activity on indexes of force of the immune answer and production of immunoglobulins. Besides, Tactivin is an unseparated admixture of peptides and consequently it is not the subject to reliable standardisation.

The most similar analogue of the offered drug is bursopoietin, obtained on the basis of a synthetic tripeptide Lys-His-Gly-NHs (lysil-histidil-glycil-amide) [Audhya T., Kroon D. J., Heavner G., Goldstein G. Bursopoietin. Pat. 4584284 (USA)//C. A.-1986.- Vol. 105.-173063n.]. The drug stimulates the differentiation of B-lymphocytes and can be used for treatment of patients with disorders of a humoral link of immunity.

The offered invention is designed to make a drug with higher performance of activity concerning a stimulation of humoral immunity (antibodygenesis) and force of the immune answer and molecules of intercellular interaction for therapy of congenital and acquired immunodeficiency states.

The solution of this problem is provided by that the hexapeptide of the structural formula : lysil-histidil-glycil-lysil-histidil-glycine is used as a drug for treatment of immunodeficiency states of a various etiology. The gross-formula is C28H4607N, 2.

The offered drug contains as an effective substance the synthetic hexapeptide with molecular weight of 662,7 D.

The effective substance represents a white powder inodorous, easily soluble in water and isoosmotic solution of sodium chloride. Substance is not soluble in alcohol and chloroform.

The offered drug for treatment of immunodeficiency states contains 0,001-1% (0,00001- 0, 01g) of hexapeptide. As the stabiliser (filler) the drug contains 0,05-5 % (0,0005-0,05g) of glycine. The most preferable shape of production of the offered drug is 0,005% of hexapeptide solution for hypodermic or intramuscular injections in ampoules of 1, Oml. As a stabiliser the drug contains 0,5 % solution of glycine. The storage term is 2 years at 4-8°C.

The drug was developed at the research-and-production plant"Bionox"Co., Ltd.

From structural formula of the drug there is no following with conspicuity the properties to increase humoral immunity, to strengthen the immune answer (expression of HLA-DR on a surface of hemopoietic cells), and also to activate T-system of immunity with achievement of treatment effect.

The results of pre-clinical and clinical study of the offered drug show that it does not cause side effects.

Experimental and clinical data show that: 1. Hexapeptide stimulates formation of antibodygenesis cells in 4 times more effectively as compared to with the prototype substance (tab. 1), enlarges amount of immunoglobulins in patients with primary acquired hypogammaglobulinemia (example 5).

2. The applied drug stimulates immune response of lymphocytes of the patients with genetic determined immunodeficiency (congenital agammaglobulinemia) by criterion of expression of antigenes of a head complex of histological compatibility (HLA-DR) and molecules of intercellular interaction (CD2). The prototype substance does not have a similar activity (tab. 3).

3. Hexapeptide has an extremely low toxicity and provides a wide reserve of safety.

The single dose, which in one thousand times greater than medial therapeutic dose, does not cause death of the experimental animal.

4. The definition of a chronic toxicity of the drug demonstrates its harmlessness.

The revealed variations of hematological and biochemical indexes in case of use of the drug in animals daily within one month in a therapeutic dose or a dose which is greater in 10 times than therapeutic one after cancelling of the drug is reverted to a datum level.

5. The injection of the drug does not cause to local irritating activity. The drug does not have an allergenicity and mutagenous activity.

The drug has shown good clinical results in treatment of immunodeficiency states in patients with congenital agammaglobulinaemia and hypogammaglobulinaemia.

Clinical application of the drug in patients with congenital immunodeficiency, with hypoproduction of the basic classes of immunoglobulins, is performed in view of a clinical and immunological state of the patients. For this purpose before administration of the drug it is determined a state of cell immunity, expression of markers of T-lymphocytes, force of the immune answer of B-lymphocytes and amount of immunoglobulins in blood serum.

Efficiency of therapy is estimated on clinical and immunological indexes, including on positive changes of amount of immunoglobulins and cutting or decreasing of severity of clinical current of the disease. The administration of the drug to the patients with congenital immunodeficiency states is performed in complex therapy with drugs of pathogenetic therapy. The drug is used as courses for hypodermic or intramuscular injections in a dose 0,5-51lg/kg of mass of a body single-passly, daily or with an interval 1-3 days within 10-30 days. If necessary the repeated courses within 1-3 weeks is performed with depending on clinical and immunological indexes.

Below are the particular examples of production of an effective substance, description of biological and medical properties of the drug.

Example &num 1.

Hexapeptide is synthesised by a method of solid-phase synthesis on the computerised synthesiser"Beckman-990".

Aminomethylpolymerase (1.8g, 1mmole) is swelled in chloroform within 30 minutes, then is combined with Boc-Gly-OCH2C6H4CH2COOH (0,65g, 2mmole) with the help of N, N-dicyclohexylcarbodiimid (DCHC) (0.4g, 2mmole in 15moi of chloroform within 2 hours). After washing by dimethylformamide the polymer is treated by 20ml of an admixture of diisopropylethylamine-acetic anhydride 2: 1 within 30 minutes. After washing by dimethylformamide and chloroform the polymer is treated by 30ml of an admixture of trifluoracetic acid-chloroform 1: 1 within 20 minutes, washed out by chloroform and neutralised by 7% solution of diisopropylethylamine in dimethylformamide within 10 minutes, then aminoacilpolymer is washed out by dimethylformamide. The addition of Boc-amino acid is carried out with the help of a symmetric anhydride of Boc- amino acid: 4mmole of Boc-amino acid is dissolve in 5ml of chloroform, cool up to 0°C, added a solution of 2mmole DCHA in 5ml of chloroform. After agitating within 10 minutes at 0°C the admixture is added to peptide polymer and added with 10mol of dimethylformamide and agitated with peptide polymer within 30 minutes at 28°C.

After the next amino acid is added the peptide polymer is washed out by dimethylformamide and chloroform, then the protective Boc-group is deleted.

Deletion of the dinitrophenyl group: 2g of peptide polymer are agitated within 1.5 hours at ambient temperature in 40ml of 20 % solution of thiophenol in dimethylformamide. Then the peptide polymer is washed out by dimethylformamide and chloroform and dried in vacuum.

Removal of the peptide from a resin: 2g of the peptide polymer are placed in a reaction vessel of the device for operation with fluorine hydride and added with lg of n- cresol and cooled by liquid nitrogen. After vacuum blowing of the vessel 10moi of the liquid anhydrous fluorine hydride are distille in this vessel. The temperature of the reaction vessel is leaded up to-20°C. The polymer is agitated during 30 minutes supporting the temperature of the admixture CC14-solid CO2 Then the reaction vessel is placed in ice bath and stood at agitating 30 minutes. After that the fluorine hydride is evaporated on a water-suction pump, and the polymer is washed out on the filter by diethyl ether. Then the peptide is extracted by 20 % acetic acid and lyophilised.

Deletion of the trifluoracetic group. The peptide removed from resin is treated by 60ml of one-molar aqueous solution of a piperidine at 0°C within 2 hours. Then the reaction mixture is lyophilised and demineraiisated by gel-filtration on a column Sephadex G-25 in 5% acetic acid.

Example #2.

Examination of the influence of hexapeptide (applied substance) and bursopoietine (the prototype substance) on production of the antibodyforming cells in a lien of mouse.

It is used mouse-males of a line CBA/CaLacSto, which is immunised intravenously by sheep red blood cells in a dose 2x106. In 10-15 minutes after the immunisation the applied substance and the prototype substance are injected to mouse intraperitonealy in volume 0,5m1 in concentrations 0,02; 0,1; 0,5 and 2,5 ; lg/ml (7-8 animals in group). The similar volume of a physiological solution is injected to mouse of control group (7-8 animals in group) intraperitonealy. For the fourth day after the immunisation it is determined the number of the antibodyforming cells in a lien of the mouse by calculation of areas of a local hemolysis of erythrocytes of the ram in agarous gel. The results of the examination are shown in the table #1.

Table &num 1.

Influence of the bursopoietine (the prototype substance) and hexapeptide (applied substance) on amount of the antibodygenesis cells in a lien of the mouse. Drug dose Amount of the antibodygenesis cells after the ug/ml injection of: Bursopoietine Hexapeptide Control group 100 79. 125 0. 02 99 371** 67-.-147 229603 0. 1 75 192** 30-184113-327 0. 5 121 171* 24-206 110-265 2. 5 151 228* 95-240106-497 Note. It is shown the ratios of number of the antibodygenesis cells of each mouse to medial arithmetic number of the antibodygenesis cells of the control group expressed in percentage.

*-p < 0,05 as compared to the control group.

**-p < 0,05 as compared to activity of the prototype substance.

Obtained data demonstrate that hexapeptide (the applied substance) causes a four-time exceeding of formation of the antibodygenesis cells in a lien of the mouse as compared to the prototype substance. Thus, the appled substance has the higher activity as to stimulation of antibodygenesis than the prototype substance.

Example #3.

Examination of the influence of the hexapeptide and the prototype substance (bursopoietine) on the expression of the immune answer force marker (HLA-DR), markers of T-lymphocytes (CD2, CD4, CD8) and B-lymphocytes (CD22) by cells of virtually healthy donors.

The peripheric human blood cells are used in the work. The single-stage gradient of phykoll-verografin with density 1,077 g/cm3 is used for production of a fraction of the mononuclear cells from heparinised (25 ED/ml) blood.

The level of the expression of membrane-associated structures CD2, CD4, CD8, CD22, HLA-DR on the mononuclear cells is determine by cellular solid-phase imunnoperoxidatic method with use of the monoclonal antibodies to structures CD2, CD4, CD8, CD22, HLA-DR ("Seromed", Great Britain) and antimouse F (ab') fragments of the immunoglobulins, conjugated with peroxidase of the horseradish ("Seromed", Great Britain). The analysis is performed after an hour incubation of the cells at the presence of 0,02; 0,1; 0,5; 2,5 llg/ml of the applied substance and the prototype substance. The mononuclear cells in control examinations are incubated in similar conditions in culture without addition of the substances. Growing of the cells is performed in culture RPMI- 1640 with addition of 5% embrionic veal serum in terminating volume 3ml and concentration 2x106/ml.

Immunofermental analysis is performed on flat-bottomed plotting boards ("Nunc", Denmark) previously treated by 0,001 % solution of poly-L-lysine ("Sigma", USA) on the phosphate-saline buffer, pH 7,3. The investigated cells are putted in concentration 4x106/ml, 50 ; J in the hole. After centrifugation the cell are fixed with the help of 50tl 0,5 % solution of glutaric aldehyde ("Sigma", USA) on the phosphate-saline buffer within 15 minutes. The plotting boards are washed out three times by the phosphate-saline buffer and then treated by 0,1 M solution of glycine ("Sigma", USA). Then 1% solution of the bull serum albumin on the phosphate-saline buffer is putted to the holes and incubated within 12-14 hours at +4°C. At performing of the reaction 100p1 of the relevant monoclonal antibodies diluted on the phosphate-saline buffer with 0,5 % solution of the bull serum albumin are putted in the holes and incubated within 1 hour at +37°C. After washings out by 0, 01 % solution of the Twin 20 ("Merck", Germany) on the phosphate- saline buffer it is putted the conjugate ("Sigma", USA) in the volume of 1 00tl on the hole, incubated, washed and added of a solution of the substrate--ortophenyldiamine ("Sigma", USA) in 0,1 M citrate-phosphatic buffer, pH 4,5 and 0,004 % of peroxide of hydrogen. After incubation within 30 minutes the reaction is stopped by 1 N sulphuric acid and estimated by spectrophotometry, determining levels of absorption at a wavelength 492nm. The results are expressed in percentage of change of values of the optical density in the experiment, in ratio of value of optical density in the control group (cells which are not treated by the substance). It is performed allowing for control of the reaction, as which it is used the system without the monoclonal antibodies allowing to indicate a background of the natural peroxidase activity and a nonspecific sorption of the conjugate on cells. Each experimental group of the cells is studied in a triplet.

Statistical data interpretation is performed with definition of the reliability of a difference by the Student-Fisher criterion. It is determined the reliability of the differences of selective medial of the experiment and control group. It is determined the reliability of the differences on the calculated value"t"and amount of compared variant using the Student-Fisher table. The difference is considered reliable at the sugnificance level of no more than 5 % (p<0,05).

Comparative estimation of the expression of the surface structures on the cells of the healthy donors under the influence of the applied drug (hexapeptide) and the prototype substance (bursopoietine) is shown in table #2.

The obtained data are demonstrated that the hexapeptide (the applied substance) causes the reliable increase of the expression of HLA-DR and CD2 on the surface of the human hemopoietic cells. On the contrary, the prototype substance causes the decrease of the expression of the specified surface structures. Thus, the applied substance as against the prototype has property to enlarge the expression of genes of the immune answer (HLA-DR) and maturing of T-lymphocytes (CD2).

Example #4.

Clinical and immunological examination of influence of the drug in patients with agammaglobulinemia on the expression of the HLA-DR and markers of subpopulations oflymphocytes.

The level of the expression of the membrane-associated structures CD2, CD4, CD8, CD22, HLA-DR on the mononuclear cells is determine by cellular solid-phase immuneperoxidase method similarly specified at the example #3.

Comparative estimation of the expression of the surface structures on cells of patients with agammaglobulinemia under the influence of the applied drug (hexapeptide) and the prototype substance (bursopoietine) is shown in table &num 3.

The obtained data demonstrate that the hexapeptide (the applied substance) causes the reliable increase of the expression of HLA-DR and CD2 on a surface of hemopoietic cells of patients with agammaglobulinemia. On the contrary, the prototype substance causes the decrease of the expression of the specified structures on a surface of cells in patients. Thus, the applied substance as against the prototype has property to enlarge the expression of genes of the immune answer (HLA-DR) and maturing of T-lymphocytes (CD2) in patients with congenital immunodeficiency.

Example #5.

The patient R., 25 years old. The clinical diagnosis: primary acquired hypogammaglobulinemia (with the late manifestation).

The presence of a dermatomyositis and chronic progressing polyarthritis is noted at examination at arrival of the patient. At examination of the immune status of the patient the function disorder of T-and B-link of immunity, decrease of proliferate ability of T-lymphocytes in blasttransformation reaction and decrease of amount of serum immunoglobulins IgA, IgG, IgM are noted. The drug has been injected one time a day within 14 days. After termination of the therapy the augmentation of the amount of IgG from 95mg% up to 350mg% is noted ; IgM-from 49mg% up to 103mg%. Intensifying of blasttransformation reaction of lymphocytes from 75342+3003 Impulses/min up to 186476+7954 impulses/min is noted, accordingly. The clinical effect after the administration of the drug is noted for the tenth day as cutting of the muscle pains and phenomena of the polyarthritis. The patient notes the improvement of the general state of health, sleep and mood. Side effects to injection of the drug are not noted.

Thus, the applied substance stimulates the production of the immunoglobulins and expresses the therapeutic activity at hypogammaglobulinemia.

Table &num 2 Dose HLA-DR CD2 (T-lymphocytes) CD-4 CD-8 µg/ml (T-lymphocytes) (T-lymphocytes) Hexapeptide Bursopoietine Hexapeptide Bursopoietine Hexapeptide Bursopoietine Hexapeptide Bursopoietine @ Control 100,00+4,5 100,0+5,7 100,0+3,7 100,0+2,9 100,0+2,3 100,0+6,5 100,0+3,6 100,0+5,3 @ group 0,02 102,7+9,8 # 90,0+8,0 #* 108,9+14,5 # 87,3+4,6 #* 95,1+1,5 #* 82,1+10,9 #* 89,1+11,8 79,6+7,1* 9 0,1 103,3+7,0 # 93,6+9,6 # 108,9+6,5 #* 95,6+8,5 # 89,2+7,3 * 87,8+17,9 78,7+2,6* 84,9+6,5* 9 0,5 107,7+11,1 # 92,8+7,9 #* 104,8+6,9 99,1+22,0 90,0+8,7* 88,9+14,7 89,8+3,9 #* 60,1+22,0 #* 9 2,5 110,3+8,7 #* 90,3+8,7 #* 116,1+12,0* 98,4+18,0 95,1+8,4 92,2+8,8* 94,2+5,6 94,2+5,6 @ Notes: data are shown in percentage as compared to the control group. Control group is the cells, which are not treate<BR> # - reliable differences between action of hexapeptide and bursopoietine;<BR> * - reliable differences between action of the drugs and control group data.

Table &num 3 Dose HLA-DR DC2 (T-lymphocytes) CD-4 CD-8 Cd-2@ µg/ml (T-lymphocytes) (T-lymphocytes) (B-ly@ Hexapeptide Bursopoietine Hexapeptide Bursopoietine Hexapeptide Bursopoietine Hexapeptide Bursopoietine Hexa@ Control 100,0+6,4 100,0+13,0 100,0+4,6 100,0+3,6 100,0+12,9 100,0+5,0 100,0+8,0 100,0+21,9 100,0 group 0,02 103,3+12,8 96,6+12,9 103,7+13,1 # 94,7+13,1 104,3+15,6 101,0+21,5 94,7+14,9 101,2+11,3 93,5+ 0,1 101,6+8,9 # 91,0+9,4 # 108,7+16,7 # 92,3+14,3 # 100,1+22,6 98,1+9,9 101,2+14,2 #* 116,5+33,1 95,1+ 0,5 107,0+13,2 98,8+14,3 102,9+10,8 94,9+17,0 95,2+12,5 99,1+14,4 82,9+14,2 #* 106,5+16,9 # 90,8+ 2,5 108,9+13,4* 102,0+ 35,1 108,8+13,2 # 86,6+19,6 112,5+17,1 # 90,5+12,0 #* 118,4+14,4* 110,0+9,9 106,1 Notes: data are shown in percentage as compared to the control group. Control group is the cells, which are not trea<BR> # - reliable differences between action of hexapeptide and bursopoietine;<BR> * - reliable differences between action of the drugs and control group data.