RU2528337C1 - Agent for treating autoimmune diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: developed agent contains trophoblastic β-1-glycoprotein (TBG), immunoglobulin (Ig) and additionally contains tetrapeptide H-Tyr-X-Y-Glu-OH, wherein X - Gln and/or Glu, Y - Cys (acm) and/or Cys.

EFFECT: effective treatment of autoimmune diseases by single doses containing TBG 1 mg, Ig 19 mg and tetrapeptide 20 mg.

6 tbl, 13 ex

Description

The invention relates to medicine, in particular to new biologically active substances (BAS) - funds with immunoregulatory properties and can be used in practical medicine for the treatment of autoimmune diseases, as well as in experimental biochemistry and veterinary medicine.

The trophoblastic β-1-glycoprotein (TBH) is known, which has an immunoregulatory property and is intended for the treatment of various autoimmune diseases (see RF patent No. 2056852, class A61K 35/50, 1994).

However, the known tool when used in the stage of exacerbation of an autoimmune disease can exert its effect only 4-5 days after its introduction into the patient's body.

By technical nature, the closest to the claimed drug is an agent having an immunoregulatory property containing trophoblastic β-1-glycoprotein (TBG) and immunoglobulin (Ig) (see, for example, US Pat. RF No. 2303995, class A61K 38/16, 04/28/2006).

However, a known agent for the treatment of autoimmune diseases is required to be used at least twice a year. Given the fact that at each dose of this drug is required intravenous administration of the drug, there is a need for hospitalization of the patient. In addition, when using the above means, remission occurs for a short time.

The technical result is the development of a high-quality agent with immunoregulatory property and accelerated clinical effect at the acute stage in the treatment of autoimmune diseases.

This is achieved by the fact that the agent for the treatment of autoimmune diseases containing trophoblastic β-1-glycoprotein (TBH) and immunoglobulin G (Ig) according to the invention further comprises a tetrapeptide of the type H-Tyr-XY-Glu-OH, where X is Gln and / or Glu, Y - Cys (acm) and / or Cys, in addition, the ingredients are taken in fractions whose ratio is 1:19:20, respectively, and class G (Ig-G), or class A immunoglobulin is used as immunoglobulin (Ig-A), or class M (Ig-M),

The essence of the invention lies in the fact that the claimed agent containing TBH, Ig and tetrapeptide type H-Tyr-XY-Glu-OH, where X is Gln and / or Glu, Y is Cys (acm) and / or Cys, has the ability to more actively suppress the proliferative activity of mononuclear cells, induce their suppressor activity and their secretion of TGF-B1, IL-10, IL-6 cytokines. In addition, the use of the claimed funds allows you to achieve a higher clinical effect, which occurs on the 1st day after its introduction and lasts for a year.

The preparation of the claimed agent can be carried out by mixing in a glass container at a temperature of 1 ÷ 10 ° C the initial components of TBH, Ig and a tetrapeptide of the type H-Tyr-XY-Glu-OH, where X is Gln and / or Glu, Y is Cys (acm ) and / or Cys, in addition, the ingredients can be taken in fractions, the ratio of which is 1:19:20.

The therapeutic effect of the claimed drug lies in the fact that it has the ability to induce the suppressor activity of mononuclear cells of patients with autoimmune diseases and the secretion of TGF-B1, IL-10 cytokines by mononuclear cells.

This allows us to argue that the claimed tool has an immunoregulatory property. It should be noted that the preparation was carried out of the funds from the above components - TBH, Ig-G and tetrapeptide in various above shares. Given the economic approach and research results, we can recommend a ratio of TBH, Ig-G and tetrapeptide equal to 1:19:20.

Moreover, different classes (types) of Ig-G, Ig-A, and Ig-M were used as Ig and approximately the same results were obtained. However, in practice, preference can be given to the Ig-G class, since its production is simpler.

The biological activity of the claimed funds, consisting of TBH, Ig and tetrapeptide, was determined as follows.

Example 1

The effect of an agent consisting of TBH, Ig-G and a tetrapeptide of the type H-Tyr-X-Y-Glu-OH on the proliferative activity of peripheral blood mononuclear cells isolated by the Boyum method of 1969 was investigated.

The range of doses used was 0.5 μg / ml - 480 μg / ml.

The research results are shown in table 1.

Table 1 The dose of pure TBH in μg / ml in the culture medium (both in the experimental and in the control) n 0.5 1,5 3 7.5 fifteen thirty 60 120 240 480 % Inhibition index in control medium (TBH + Ig G + physiological p-p) 10 0 5 ± 0.05 15 ± 0.1 24 ± 0.2 32 ± 0.2 46 ± 0.4 38 ± 0.3 25 ± 0.2 14 ± 0.1 0 The inhibition index in% in the tool (TBH + IgG + tetrapeptide) 10 3 ± 0.02 7 ± 0.03 21 ± 0.05 37 ± 0.04 45 ± 0.06 65 ± 0.3 54 ± 0.2 36 ± 0.07 20 ± 0.07 6 ± 0.09

As can be seen from table No. 1, the agent, consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH, inhibits the proliferative activity of peripheral blood mononuclear cells caused by phytohemagglutinin, 1.5 times stronger than TBH with Ig-G.

Example 2. The effect of the claimed agent consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH on the ability to induce the suppressor activity of peripheral blood mononuclear cells was investigated. At the first stage, a suspension of MNCs is obtained by the method of cell sedimentation in a single-stage gradient of ficoll-urotrust (Boyum method). Peripheral blood is taken from the donor by venipuncture at the same time and placed in test tubes with a heparin solution at the rate of 1 ml of blood - 20-30 units. heparin. Then the blood is diluted with Hanks solution without Ca ⁺⁺ and Mg ⁺⁺ in a ratio of 1: 2 and layered on the ficoll-urotrust gradient (density 1.078). Then centrifugation is carried out in the mode of 400 g for 30 minutes A suspension of MNCs from the interphase is transferred to a centrifuge tube, Hanks solution without Ca ⁺⁺ and Mg ^{++ is} added and 3 consecutive centrifugations of 10 minutes are performed to wash the cells from the Ficoll-Urotrast solution. After the 3rd centrifugation, the MNC pellet was resuspended in 1 ml of medium 199 and the number of mononuclear cells was counted using a Goryaev camera.

At the second stage, MNCs are divided into two equal parts, the first of which is cultivated without a suppressor activator, the second with a suppressor activator, which is used as a test agent. MNCs are cultivated in penicillin vials closed with rubber stoppers No. 14.5 at a temperature of 37 ° C. Cultivation medium RPM-1640 with the addition of 20% serum IV AB blood group and glutamine.

In each vial, 5 × 10 ⁶ cells are cultured in 2.0 ml of complete medium. An agent consisting of TBH, Ig-G and a tetrapeptide of the type H-Tyr-XY-Glu-OH, in doses of 1-960 μg / ml, is added to the culture for the induction of suppressors.

Cell cultivation is carried out for 48 hours. After this, MNCs are washed from the culture medium and proliferation is blocked by treatment with mitomycin C - 40 μg / ml for 30 min at 37 ° C. Then washed with medium 199 with 5% serum IV AB (chilled) three times. The cell pellet is resuspended, the number of nucleated cells is counted, the percentage of cell viability is determined using a 0.1% trypan blue solution and diluted to the desired concentration. At the same time, all operations are done separately with control cells and with a stimulated composition, and siliconized dishes are used to wash cells.

At the next stage, freshly isolated MNCs stimulated with phytohemagglutinin (PHA), which serve as response test cells in equal proportions (0.5 × 10 ⁶ cells / ml), are added to each part of the control and stimulated with MNCs to obtain test cultures. Their cultivation is carried out for 72 hours. After this, the proliferation of test cultures is evaluated using H ³ - thymidine and the amount of suppression is judged by the degree of decrease in proliferation in them. The suppression index (IP) is determined by the formula:

The results of studies of the effect of an agent consisting of TBH, Ig-G and tetrapeptide type H-Tyr-X-Y-Glu-OH on the ability to induce suppressor activity of MHCs are shown in table 2.

The dose range is 0.5-480 mcg / ml.

table 2 The dose of pure TBH in μg / ml in the culture medium (both in the experimental and in the control) n 0.5 1,5 3 7.5 fifteen thirty 60 120 240 480 Suppression index in% in the control medium (TBH + IgG + physiological p-p) 10 0 3 ± 0.02 8 ± 0.03 17 ± 0.1 24 ± 0.12 44 ± 0.14 27 ± 0.06 15 ± 0.02 6 ± 0.02 0 The suppression index in% in the tool (TBH + IgG + tetrapeptide) 10 2 ± 0.04 5 ± 0.06 13 ± 0.07 25 ± 0.08 35 ± 0.09 64 ± 0.1 40 ± 0.1 21 ± 0.04 10 ± 0.08 4 ± 0.04

As can be seen from table No. 2, the agent consisting of TBH, Ig-G and tetrapeptide type H-Tyr-X-Y-Glu-OH induces the suppressor activity of peripheral blood mononuclear cells 1.5 times stronger than TBG with Ig-G.

Example 3. The effect of an agent consisting of TBH, Ig-G and a tetrapeptide of the type H-Tyr-XY-Glu-OH on the ability to induce the production of cytokines transforming growth factor beta 1 (TGF-B1) and interleukin-10 (IL- 10) peripheral blood mononuclear cells (MNCs). At the first stage, an MNC suspension was obtained by the method of cell sedimentation in a single-stage gradient of ficoll utrotrust (Boyum method). MNCs were divided into 2 equal parts, the first of which was cultivated without the claimed means, the second with the means.

MNCs are cultivated in penicillin vials closed with rubber stoppers No. 14.5 at a temperature of 37 ° C. The cultivation medium RP VI-1640 with the addition of 20% serum IV AB blood groups and glutamine. In each vial, 5 × 10 ⁶ MNCs were cultured in 1 ml of complete medium. To the culture of MNCs to induce the production of cytokines TGF-B 1 and IL-10 was added the inventive tool. Cell cultivation was carried out 24 hours. After this, the culture medium was separated from the cells by centrifugation (1000 g for 10 minutes and 500 μl necessary for analysis) were taken from it. When determining the amount of TGF-B1 in the culture medium, samples were initially extracted. This stage of the analysis allows the release of TGF-B 1 from the complexes, making it available for analysis. For this, 0.25 ml (250 μl) of the culture medium and 0.05 (50 μl) of the extraction solution were added to the polypropylene tube. Vortexed the contents. Incubated for 30 minutes at 4 ° C. 250 μl of working buffer solution was added to the tube for dilution of standards. At this stage, the culture medium is diluted 2.2 times. Next, the required number of 8 hole strips required for analysis was taken from a collapsible microplate coated with antibodies to TGF-β1.

To increase the reliability of the analysis results, the test and control samples were placed in duplicates using two wells for each sample. 200 μl of each standard and extracted sample or clotron were added to the respective wells. 500 μl of biotinylated anti-TGF-β1 antibodies were added to all wells, mixed by tapping on a plate. The plate was covered with film and incubated for 30 min at room temperature. The contents of the wells were completely removed. Wash all wells 4 times with working buffer, adding 400 ml each time to all wells. At the same time, each time the solution was completely filled and removed from the holes of the strips. After washing, the remaining moisture was freed by tapping inverted strips on filter paper. 100 μl of streptavidin peroxidase conjugate stock solution was added to each well, with the exception of the chromogenic blank. The plate was covered with film and incubated for 30 min at room temperature. The contents of the wells were completely removed. The wells were washed 4 times with working buffer solution. Then, 100 μl of a chromogenic solution of tetramethylbenzidine (TMB) were added to all wells. They were incubated for 20-30 min at room temperature in the dark until a blue color appeared in the wells with a calibration sample with a maximum TGF-β1 content.

100 μl of stop solution (1N sulfuric acid solution) was added to each well to stop the enzymatic reaction. The reagents were mixed by gently tapping the strip holder. The color of the solution changed from blue to yellow. The results were recorded photometrically on a photometer for enzyme immunoassay at a wavelength of 450 nm immediately after stopping the enzymatic reaction.

To determine the concentration of TGF-β1 in the studied samples, a calibration curve was constructed in the coordinates: abscissa axis — concentration of TGF-β1 in calibration samples (rg / ml); the ordinate axis is the corresponding value of the optical density. Using the calibration curve based on the obtained optical density, the concentration of TGF-B1 in the samples was determined. If the samples were previously diluted, the obtained concentration values were multiplied by a dilution factor (10, 100, 1000, etc.). To determine the amount of IL-10 in the culture medium, the number of 8 hole strips required for analysis was taken from a collapsible microplate coated with antibodies to IL-10. To increase the reliability of the analysis results, the test and control samples were placed in duplicates using two wells for each sample. 50 μl of each standard, test sample, or control was added to the appropriate wells. 50 μl of standard incubation well buffer and 50 μl of dilution working solution were added to wells containing culture medium. The plate was covered with film and incubated for 2 hours at room temperature. The contents of the wells were completely removed.

Wash all wells 4 times with working buffer, adding 400 μl each time to all wells. At the same time, each time the solution was completely filled and removed from the holes of the strips. 100 μl of biotinylated anti-IL-10 antibodies were added to all wells, mixed by tapping the plate. The plate was covered with film and incubated for 2 hours at room temperature. The contents of the wells were completely removed; all wells were washed 4 times with working buffer solution. 100 μl of streptavidin peroxidase conjugate stock solution was added to each well, except for the chromogenic blank.

The plate was covered with film and incubated for 30 min at room temperature. The contents of the wells were completely removed. The wells were washed 4 times with working buffer solution. Then, 100 μl of a chromogenic solution of tetramethylbenzidine (TMB) were added to all wells.

Incubated for 20-30 minutes at room temperature in the dark until a blue color appeared in the wells with a calibration sample with a maximum content of IL-10. 100 μl of stop solution (1N sulfuric acid solution) was added to each well to stop the enzymatic reaction. The reagents were mixed by gently tapping the strip holder. The color of the solution changed from blue to yellow.

The results were also recorded photometrically on a photometer for enzyme immunoassay at a wavelength of 450 nm immediately after stopping the enzymatic reaction.

To determine the concentration of IL-10 in the studied samples, a calibration curve was constructed in the coordinates: abscissa axis — concentration of IL-10 in calibration samples (rg / ml); the ordinate axis is the corresponding value of the optical density. Using the calibration curve, on the basis of the obtained optical density, the concentration of IL-10 in the studied samples was determined. If the samples were previously diluted, the obtained concentration values should be multiplied by a dilution factor (10, 100, 1000, etc.).

As a result of the studies, it was found that the agent containing TBH, Ig-G and tetrapeptide type H-Tyr-X-Y-Glu-OH has the ability to induce the production of 2 cytokines simultaneously - TGF-B1 and IL-10 by mononuclear cells of peripheral blood. Moreover, the level of IL-10 and TGF-B1 cytokines produced by peripheral blood mononuclear cells under the action of the claimed agent is 1.5 times higher than under the influence of TBH with Ig-G. Moreover, the inventive agent administered to the patient eliminates the exacerbation of autoimmune diseases for a longer period (1 year), while when the patient was introduced with TBH with Ig-G, the effect manifested itself only for six months.

Example 4

We studied the effect of a drug consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH, in a ratio of 1:99:20 on the proliferative activity of peripheral blood mononuclear cells isolated by the Boyum method of 1969. The range of doses used was 0.5 μg / ml - 480 μg / ml.

The research results are shown in table 3.

Table 3 The dose of pure TBH in μg / ml in culture medium (both in the experimental and in the control) n 0.5 1,5 3 7.5 fifteen thirty 60 120 240 480 % Inhibition index in control medium (TBH + IgG + physiological p-p) 10 0 5 ± 0.05 15 ± 0.1 24 ± 0.2 32 ± 0.2 46 ± 0.4 38 ± 0.3 28 ± 0.2 14 ± 0.1 0 The inhibition index in% in the tool (TBH + IgG + tetrapeptide) 10 2.5 ± 0.03 7.2 ± 0.04 21.5 ± 0.04 36.8 ± 0.05 44.8 ± 0.04 64.8 ± 0.3 54.7 ± 0.08 36.6 ± 0.04 20.4 ± 0.03 5.5 ± 0.08

As can be seen from table No. 3, the agent, consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in a ratio of 1:99:20, suppresses the proliferative activity of peripheral blood mononuclear cells caused by phytohemagglutinin to such an extent as well as the claimed agent containing TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in a ratio of 1:19:20.

Example 5

We studied the effect of a drug consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH, in a 1: 1: 20 ratio on the proliferative activity of peripheral blood mononuclear cells isolated by the Boyum method of 1969. The range of doses used was 0.5 μg / ml - 480 μg / ml. The research results are shown in table 4.

Table 4 The dose of pure TBH in μg / ml in the culture medium (both in the experimental and in the control) n 0.5 1,5 3 7.5 fifteen thirty 60 120 240 480 Inhibition index in% of control medium (TBH + Ig G + physiological p-p) 10 0 4 ± 0.03 11 ± 0.04 24 ± 0.6 39 ± 0.8 45 ± 0.1 36 ± 0.07 22 ± 0.04 14 ± 0.5 0 The inhibition index in% in the tool (TBH + IgG + tetrapeptide) 10 2 ± 0.03 6 ± 0.04 16 ± 0.03 36 ± 0.05 44 ± 0.07 67 ± 0.1 52 ± 0.15 33 ± 0.08 21 ± 0.06 9 ± 0.08

As can be seen from table No. 4, the agent, consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in a ratio of 1: 1: 20, inhibits the proliferative activity of peripheral blood mononuclear cells caused by phytohemagglutinin to such an extent as well as the claimed agent containing TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in the ratios of 1:19:20 and 1:99:20.

Example 6

The results of studies of the effect of an agent consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in ratios of 1:99:20 in the ability to induce suppressor activity of MHCs are shown in Table 5. Dose range 0.5-480 mcg / ml.

Table 5 The dose of pure TBH in μg / ml in the culture medium (both in the experimental and in the control) n 0.5 1,5 3 7.5 fifteen thirty 60 120 240 480

Suppression index in% in the control medium (TBH + IgG + physiological p-p) 10 0 3.5 ± 0.03 8 ± 0.05 18 ± 0.03 24 ± 0.07 38 ± 0.09 28 ± 0.04 17 ± 0.05 6 ± 0.03 0 The suppression index in% in the tool (TBH + IgG + tetrapeptide) 10 2 ± 0.03 5 ± 0.05 12 ± 0.04 27 ± 0.06 36 ± 0.08 57 ± 0.1 42 ± 0.06 25 ± 0.04 9 ± 0.05 4 ± 0.03

As can be seen from table No. 5, the tool, consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in a ratio of 1:99:20, induces the suppressor activity of peripheral blood mononuclear cells to the same extent as the tool containing TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in ratios 1:19:20, and induce the production of cytokines-transforming growth factor beta 1 TGF-B1 and interleukin-10 (IL-10) mononuclear peripheral blood cells (MNCs).

Example 7

The results of studies of the effect of an agent consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in ratios 1: 1: 20 on the ability to induce suppressor activity of MHCs are shown in Table 6. Dose range 0.5-480 mcg / ml.

Table 6 The dose of pure TBH in μg / ml in the culture medium (both in the experimental and in the control) n 0.5 1,5 3 7.5 fifteen thirty 60 120 240 480 Suppression index in% in the control medium (TBH + IgG + physiological p-p) 10 0 4 ± 0.03 9 ± 0.05 22 ± 0.06 29 ± 0.04 42 ± 0.01 33 ± 0.08 19 ± 0.04 8 ± 0.02 0

The suppression index in% in the tool (TBH + IgG + tetrapeptide) 10 4 ± 0.03 6 ± 0.05 14 ± 0.06 31 ± 0.07 42 ± 0.08 61 ± 0.09 46 ± 0.08 28 ± 0.06 13 ± 0.05 6 ± 0.03

As can be seen from table No. 6, the tool, consisting of TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in a ratio of 1: 1: 20, induces the suppressor activity of peripheral blood mononuclear cells to the same extent as the tool containing TBH, Ig-G and tetrapeptide type H-Tyr-XY-Glu-OH in ratios of 1: 19: 20.1: 99: 20, and induces the production of cytokines transforming growth factor beta 1 (TGF-β1) and interleukin -10 (IL-10) mononuclear cells (MNCs) of peripheral blood.

It should be noted that the conducted INVITRO studies made it possible to determine the most optimal ratios of the studied drugs for the treatment of autoimmune diseases, the effectiveness of which will be shown below.

Example 8. The effect of an agent consisting of TBH, Ig-G and a tetrapeptide of the type H-Tyr-XY-Glu-OH in ratios of 1:19:20 on the level of interleukin-10 (IL-10) and transforming growth factor beta 1 was investigated. (TGF-β1) when administered intravenously to a healthy person. It turned out that taking the aforementioned agent allowed to increase the level of IL-10 and TGF-β1 cytokines by 8-10 times compared with taking each of the agents separately.

Example 9. EXTRACT from the medical history.

Patient O. 46 years old, was treated in the therapeutic department with a diagnosis of rheumatoid arthritis-polyarthritis, seropositive, with systemic manifestations (fever, lymphadenopathy, anemia, amyotrophy, neuropathy, rheumatoid nodules), activity 3 tbsp., Stage III, insufficiency of function joints III. Upon admission, she complained of severe pain in the small and large joints of the limbs (proximal interphalangeal hands, metacarpophalangeal, wrist, elbow, shoulder, knee, ankle, metatarsophalangeal, sternocleid), their swelling, limited mobility, severe stiffness in the joint all day, tingling sensation at the tips of the fingers and toes, fever up to 37.8 degrees.

She noted arthralgia for 4 years, moderate pain in the joints with their swelling for 2 years, and morning stiffness in the joints. She took non-steroidal anti-inflammatory drugs, often used catadolone to relieve pain, sometimes tram. On the recommendation of a rheumatologist 1.5 years ago, methotrexate was prescribed, which was canceled after 2 months due to intolerance (dyspeptic disorders, a feeling of heaviness in the right hypochondrium were noted, a five-fold increase in the level of liver enzymes in blood tests). Intra-articular administration of corticosteroids was performed three times. The real worsening of the condition 3 weeks before hospitalization - after an acute respiratory infection - the temperature rose to febrile numbers, multiple swelling of the joints developed, the above complaints appeared. She received antibiotics, non-steroidal anti-inflammatory drugs, and analgesics on an outpatient basis. Hospitalized.

Upon receipt, the condition is serious. Body temperature is 37.8 degrees. Immobilized due to pain in the joints and their swelling. The skin is pale. Enlarged (1.5 × 1.5 cm, 1.5 × 1.5 cm) lymph nodes (submandibular, axillary, inguinal) are palpated. In the lungs, vesicular breathing. Heart sounds are moderately muffled, tachycardia up to 89 per minute, rhythmic pulse, satisfactory filling, blood pressure 140/85 mm RT. The abdomen is soft, painless. Liver at the edge of the costal arch. The spleen is not palpable. The symptom of striking (Pasternatsky) is negative.

Amyotrophy of the limbs. Severe swelling, configuration of joints-proximal interphalangeal hands, metacarpophalangeal, wrist, elbow, knee, ankle. Hyperthermia of the skin over the joints. Restriction of active and passive movements in the joints. Unitary deviation of the hands. Contractures of the wrist and elbow joints. Rheumatoid nodule in the area of the left elbow joint 2.5 × 2.0 cm in size. Radiography of the hands - osteoporosis, narrowing of the articular joints, multiple erosions, subluxations. Blood test: Hemoglobin - 92 g / l, L - 12100, ESR - 62 mm / h, CRP - 132 μmol / l, RF - 80, ADC - 84 (normal to 5), HBs - (-), AST - 44 , ALT - 32, total blood protein - 68 g / l.

The suppressor activity of peripheral blood T-lymphocytes upon induction of the claimed drug was 16%. ECG sinus rhythm, diffuse changes in the myocardium. X-ray of the chest organs - there are no focal and infiltrative changes.

The patient was treated with diclofenac 3 ml intramuscularly daily, aertal 100 mg 2 times a day, omez 20 mg 2 times a day, injections of dipyrone with diphenhydramine twice a day. The patient's condition without significant dynamics within 12 days. Anemia, fever up to 37.6-37.4, pronounced exudative changes in the joints persist. The patient is immobilized. In blood tests, leukocytosis 11800, ESR 65, CRP 136 μmol / L. The patient underwent a course of daily intramuscular injections of the claimed drug containing TBG - 1 mg, Ig-G - 19 and tetrapeptide type H-Tyr-X-Y-Glu-OH - 20 mg - (1 mg: 19 mg: 20 mg).

After the injection of the above doses of the claimed drug, the patient's condition improved significantly !!! The pains in all joints, their swelling noticeably decreased, the range of movements increased. Body temperature returned to normal. Stiffness in the joints disappeared. The patient began to walk, was able to serve herself. Blood hemoglobin level increased to 120 g / l, ESR decreased to 24 mm / h, CRP became 12 μmol / l, total blood protein increased to 82 g / l.

When re-analysis revealed an increase in the suppressor activity of peripheral blood T-lymphocytes upon induction of the claimed drug -64.2%. In a patient in the reaction of Ouchterloni on days 7, 14 and 28, antibodies against the claimed drug were not detected. The patient was discharged home in satisfactory condition. It is recommended that the patient continue taking non-steroidal anti-inflammatory drugs (such as diclofenac) as an outpatient, and prescribe basic drugs (such as sulfasalazine). It should be noted that during the year the patient did not have an exacerbation of the disease.

Example 10. Patient M., 42 years old, was treated with a diagnosis of rheumatoid arthritis-polyarthritis, seropositive, with systemic manifestations (amyotrophy, anemia, neuropathy, fever) activity of the II stage, stage II, NFSP. Upon admission, he complained of pain in the joints - proximal interphalangeal, metacarpophalangeal hands, wrist, shoulder, knee, ankle, swelling of these joints, limitation of mobility in them. Complaints of severe stiffness in the joints in the morning, lasting until noon, numbness of the fingertips, fever to subfebrile numbers.

Considers himself ill for 5 years. The onset of the disease with damage to the ankle and knee joints, in the last 2 years joined swelling of the wrist joints and interphalangeal joints of the hands. Since that time, he began to note stiffness in the joints in the morning. The treatment was carried out with non-steroidal anti-inflammatory drugs, methotrexate with a positive effect. Methotrexate was canceled 8 months ago due to side effects (hepatotoxicity). The real deterioration occurred 3 weeks ago - after an acute respiratory illness - a pronounced swelling of the joints developed - proximal interphalangeal, metacarpophalangeal, wrist, knee, ankle, stiffness in the morning increased (continued until lunch), the temperature rose to 37.5. Conducted therapy with non-steroidal anti-inflammatory drugs, antibiotics - without a significant effect, and therefore hospitalized. Upon receipt, immobilized, walks with difficulty, with a wand. Defiguration, swelling of the proximal interphalangeal and metacarpophalangeal joints of the hands, wrist, knee, ankle, restriction of active and passive movements in the joints. Hyperthermia of the knee, wrist and ankle joints. Wrist joint contracture. Pronounced amyotrophy of the muscles of the thighs, shoulder. For organs without a pronounced pathology.

In blood tests: Нb - 103 g / l, L. - 10300, ESR - 46 mm / h, CRP 78, RF 110 μmol / l, total protein 70 g / l, AST-36, ALT-38. Urinalysis: beats. weight 1023, L - 1-2 in the field of view. X-ray of the joints of the hands: osteoporosis, narrowing of the interarticular cracks, multiple erosions. The patient was treated with non-steroidal anti-inflammatory drugs (diclofenac injection, xefocam injection), aertal, physiotherapy.

A condition without pronounced positive dynamics. Subfebrile condition, swelling of the joints persisted, stiffness in the joints increased. ESR increased to 50 mm / h, CRP 92. The suppressor activity of peripheral blood T-lymphocytes during the induction of TBH was 20%.

Injections of the claimed agent containing TBG 1 mg, Ig-G 19 mg and a tetrapeptide of the type H-Tyr-X-Y-Glu-OH 20 mg in ratios of 1:19:20 daily for 7 days were prescribed.

The condition of the patient with significant improvement. Stiffness in the joints disappeared, body temperature returned to normal. The pain in the joints, their swelling significantly decreased, the range of motion in the joints increased. The patient walks freely around the department. Active The compression force of the hands increased from 7 mm Hg to 92 mm Hg (left hand) and to 106 mm Hg (right hand). The swelling of the ankle and knee joints completely disappeared. Blood hemoglobin level increased to 140 g / l. ESR decreased to 22 mm / h, CRP became 12.

When re-analysis revealed an increase in the suppressor activity of peripheral blood T-lymphocytes upon induction of the claimed drug - 60.4%. In a patient in the reaction of Ouchterloni on days 7, 14 and 28, antibodies against the claimed drug were not detected. The patient in satisfactory condition was discharged for outpatient treatment. It should be noted that during the year the patient did not have an exacerbation of the disease.

Example 11

Patient S., 45 years old, with a diagnosis of multiple sclerosis, cerebrospinal form, remitting course, stage of exacerbation of the process. The duration of the disease is 7 years. In neurological status: horizontal nystagmus when viewed to the right, tendon reflexes are alive, 0> 5 on the legs. Abdominal reflexes - upper low, middle and lower absent. Symptom of Babinsky from two sides. Clonuses of the feet, coarser on the right. There are no paresis of the limbs, muscle tone is not changed. Atactic gait. In the Romberg position is unstable. Ataxia when performing a heel-knee test. Urinary retention. Cerebrospinal fluid:

protein - 0.8%, Lange reaction 66644322.

Eye examination: blanching of the temporal halves of the optic nerves. The activity of T-suppressors of peripheral blood during the induction of TBH-12%.

After administration of TBH 1 mg, Ig-G 19 mg and tetrapeptide type H-Tug-X-Y-Glu-OH 20 mg daily for 7 days, the patient showed signs of remission. Normalized gait, urination. In the Romberg position is steady. The heel-knee test performs clearly. Repeated assessment of the immune status revealed an increase in the functional activity of peripheral blood T-suppressors during the induction of TBH - 48%. In a patient in the Ouchterloni reaction on days 7.14 and 28, antibodies against TBH were not found. It should be noted that during the year the patient did not have an exacerbation of the disease.

Example 12

Patient D., 33 years old, was in the hospital with a diagnosis of autoimmune thrombocytopenic purpura. Hospitalized with complaints of a rash, skin hemorrhage, dizziness, weakness, fever up to 37.8 ° C. He became ill 5 days ago after acute respiratory infections - the temperature rose to 37.8 ° C, pain appeared during urination, the color of urine changed (the urine turned red), and hemorrhagic rashes appeared on the skin. Hospitalized. Relatives of the patient have no such diseases. At admission: the skin is pale. Punctate and petechial rashes on the skin of the chest, abdomen, back, limbs, sclera. Moderate (up to 1-1.5 cm) increase in submandibular lymph nodes. The thyroid gland is not palpable, no peripheral edema. Symptom pinch, tourniquet (+). There are no wheezing in the lungs. Heart sounds are muffled, tachycardia up to 100 in one minute. The pulse is rhythmic, satisfactory filling. HELL 105/70 tt Nd. The abdomen is soft, painless. Liver, spleen not enlarged. Symptom of Pasternatsky (-). In a blood test: Hb 94%, E - 2.4, reticulocytes 14%, L-9.1, n - 3, seg. - 65, lymph. - 19, m - 7, EO3 - 4, ESR - 44 mm / h. Platelets 21 thousand. General an. urine: ud. weight 1019, protein - traces, E - completely cover the field of view. L - 2-6 in the field of view. Biochemical analysis of blood: transaminase ACT - 41, ALT - 29, bilirubin 19, cholesterol 4.21, glucose 5.1, urea 4.9, total protein 79 g / l. The coagulation time is 5 minutes (M), the duration of bleeding according to Duke is 30 minutes (normal 5-10). Activated recalcification time 60 s (M), fibrinogen 4.2, prothrombin 84%, blood clot retraction 0.1 '(M 0.3-0.5). HB ₃ - antigen (-), 1_E-cells (-), rheumatic factor (-), ANF (-). Sternal puncture: in the bone marrow, signs of irritation of the erythroid sprout, the number of megakaryocytes is increased. Chest x-ray:

pulmonary fields are transparent. Cor - in N. R-graphy of the bones of the pelvis, skull without pathological abnormalities. Ultrasound of the liver, kidneys, spleen - the size and texture of the organs is not changed. Cup-and-pelvis system without features. There are no calculi.

After the introduction of TBG 1 mg with immunoglobulin G 19 mg and tetrapeptide 20 mg on the 2nd day, the patient showed signs of remission. The condition of the patient with improvement - the temperature returned to normal, arthralgia disappeared, there was no hepaturia, hemorrhages disappeared. The hemoglobin level increased to 136 g / l, the number of platelets increased to 200 thousand. The bleeding time and retraction of the blood clot returned to normal. The patient was discharged home in satisfactory condition. It should be noted that during the year the patient did not have an exacerbation of the disease.

Example 13. Patient C., 45 years old, was hospitalized with a diagnosis of autoimmune thyroiditis, type A chronic gastritis (autoimmune), B ₁₂ deficiency anemia. Received complaints of general weakness, weight loss, palpitations, numbness of the toes, discomfort in the epigastric region after eating, sleep disturbance, arthralgia. A history of chronic gastritis, thyroiditis, worsening during the last month - the above complaints appeared, lost 5 kg in weight.

On admission: the skin is pale with a jaundice. No peripheral edema. Palpable submandibular lymph nodes measuring 1.2 × 1.4 cm, soft, mobile, moderately sensitive. An increase in the thyroid gland of 2-3 degrees - the gland during palpation is moderately dense, mobile, not fused with the surrounding tissues, its surface is smooth. In the lungs, vesicular breathing, no wheezing. Sog-tones are sonorous, tachycardia up to 100 per minute, rhythmic pulse, satisfactory filling, blood pressure 120/80. The abdomen is soft, slightly sensitive in the epigastrium with deep palpation. The tongue is red (raspberry), the papillae are atrophied.

Liver, spleen not enlarged. Symptom of Pasternatsky (-).

Small tremor of the fingers of outstretched arms is determined, slightly bulging eyes. Positive symptoms of Mobius, Delrimple.

Blood test: Нb - 88 g / l, color indicator 1.8, leukocytes 4.3, p - 3, seg. 64, lymph. - 34, monots. - 3. Taurus Jolly and Cabot. Megalocytosis is expressed. Polysegmented neutrophils are determined, ESR - 54 mm / g. Platelets - 120 thousand

Urinalysis - beats. weight 1017, protein - abs, A - 3-4 in s / s.

Biochemical blood test: transaminases ACT - 38, ALT - 43, cholesterol 2.8, bilirubin - 21.7, nep. 17, urea 5.7, glucose 4.1, total protein 84.3, serum iron 16 (normal). Albumins - 43%, Glob .: a ₁ - 3.6, a ₂ - 15.1, p - 12.1, y - 26.2%, Rheumatoid factor (-), 1E cells are not detected. Antibodies to thyroglobulin in high titer are determined. T ₃ and T ₄ levels are moderately elevated. HB ₈ antigen (-).

Sterile puncture: in the bone marrow, mixed normo- and megaloblastic hematopoiesis; red sprout annoyed. Pattern B ₁₂ deficiency anemia. Thyroid biopsy: diffuse infiltration of the gland tissue with lymphocytes and plasma cells, fields of fibrosis - a picture of autoimmune thyroiditis.

Gastroduodenoscopy is a picture of atrophic gastritis. Gastrobiopsy: atrophy of the glands, reduced the number of parietal cells, infiltration of the mucous membrane by plasma cells and neutrophilic leukocytes. Given the above data, the patient was diagnosed with autoimmune thyroiditis, type A chronic gastritis (autoimmune), exacerbation, B ₁₂ deficiency anemia.

After the introduction of TBG 1 mg with immunoglobulin G 19 mg and tetrapeptide 20 mg on the 2nd day, the patient showed signs of remission. The condition of the patient with improvement. The temperature returned to normal. There is no tachycardia. The hemoglobin level increased to 128 g / l. The size of the thyroid gland decreased to 1-2 tbsp. The patient gained 1.5 kg in weight. The titer of antibodies to thyroglobulin decreased by 4 times. The number of leukocytes increased to 6.8, platelets to 220 thousand. The patient was discharged for outpatient treatment. It should be noted that during the year the patient did not have an exacerbation of the disease.

To study the safety of the claimed drug, consisting of 1 mg TBH, 19 mg Ig-G, and H-Tyr-XY-Glu-OH 20 mg tetrapeptide in ratios of 1:19:20, its acute toxicity was studied before its clinical use. The study of acute toxicity was carried out in accordance with the Methodological recommendations of the Pharmacological Committee of the Russian Federation “Requirements for the preclinical study of the general toxic effect of new pharmacological substances”, Moscow, 2001. The results of the study showed that with the intraperitoneal administration of a 1000-fold dose of the claimed drug, there was no acute toxic effect At these doses, it was not possible to reach its LD ₅₀ .

Thus, the proposed tool, consisting of TBH 1 mg, Ig-G 19 mg and tetrapeptide type H-Tyr-X-Y-Glu-OH 20 mg in a ratio of 1:19:20, is not toxic.

Although the above inventions have been described in sufficient detail, it will be quite clear to a person skilled in the art, in light of the description of this invention, that certain changes and modifications of the invention can be made without departing from the idea or scope of the proposed claims.

Industrial applicability

The stated advantages of the proposed agent containing TBH, Ig and tetrapeptide type H-Tyr-XY-Glu-OH, where X is Gln and / or Glu, Y is Cys (acm) and / or Cys in the above doses of its use provide the possibility of its wide applications both in scientific research and in experimental biochemistry, and in practical medicine and veterinary medicine.

Claims (2)

1. An agent for the treatment of autoimmune diseases containing trophoblastic β-1-glycoprotein (TBH) and immunoglobulin (Ig), characterized in that it further comprises a tetrapeptide of the type H-Tyr-XY-Glu-OH, where X is Gln and / or Glu, Y - Cys (acm) and / or Cys, moreover, a single dose of the drug is TBG 1 mg, Ig 19 mg and tetrapeptide 20 mg. 2. The tool according to claim 1, characterized in that as the immunoglobulin use immunoglobulin class G (Ig-G), or class A (Ig-A), or class M (Ig-M).