RU2252760C1 - Method for treating autoimmune diseases - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: it is suggested to apply tris-(2-hydroxyethyl)ammonium salt of 1-benzylindolyl-3-thioacetic acid earlier known as a stabilizer of cell membrane as preparation to treat autoimmune diseases. The property of the above-mentioned salt to inhibit T-dependent activation of B-lymphocytes, under conditions of decreased medullary function and body leukopenia should enable to develop new pharmacological preparation for treating autoimmune diseases, such as, for example, systemic lupus, rheumatoid polyarthritis, transplant's detachment at transplanting either organs or bony marrow.

EFFECT: higher efficiency of application.

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Description

The invention relates to new biologically active substances of their class of arylheteroalkanecarboxylic acids and can be used in pharmacology and medicine as the basis for creating a drug for the treatment of autoimmune diseases (ulcerative colitis, rheumatoid arthritis, lupus erythematosus, lupus nephritis, chronic hepatitis, etc.) as well as for the prevention and treatment of transplant rejection during organ transplantation, to suppress the reaction of the transplant against the host during bone marrow transplantation ozga.

Modern immunopharmacology pays close attention to the creation of new drugs that can effectively reduce the manifestations of autoimmune reactions of the body (underlying the pathogenesis of widespread diseases), since the nomenclature of such drugs is very limited.

Currently, in medicine, for the prevention of transplant rejection during transplantation of a kidney, heart, and other organs, as well as for some autoimmune diseases, the drug Azathioprine is used, which has antitumor and immunosuppressive properties (1). This drug has a number of serious side effects (inhibits bone marrow function, causes leukopenia and agranulocytosis, which requires weekly monitoring of peripheral blood composition; induces the development of toxic hepatitis and allergic reactions, etc.).

In order to expand the arsenal of synthetic drugs that suppress the severity of immune responses, it is proposed to use tris (2-hydroxyethyl) ammonium salt of 1-benzylindolyl-3-thioacetic acid as a new drug for the treatment of autoimmune diseases, as well as to prevent the development of a transplant rejection reaction when transplanting various organs to the recipient.

Derivatives of alkane carboxylic acids have a variety of biological effects. In particular, the tris- (2-hydroxyethyl) ammonium salt of 1-benzylindolyl-3-thioacetic acid has the properties of stabilizing cell membranes (“Clinical efficacy and safety of cephalosporin antibiotics manufactured by ABOLmed LLC. Proceedings of NIIKI, ASMU and ICD. Novosibirsk, 2002. - p. 179; www. Abolmed.ru/pages/article33.htm). It is known to use a number of drugs containing alkane carboxylic acid salts as local anti-inflammatory and / or analgesic agents (2, 3).

The essence of the invention lies in the fact that the immunomodulating properties of tris (2-hydroxyethyl) ammonium salt of 1-benzylindolyl-3-thioacetic acid were studied by the ability of this compound to affect the amount of protein in the urine in mice with immunocomplex glomerulonephritis, the number of antibody-forming cells (IgM AOK) in vivo (primary humoral immune response).

We used healthy sexually mature female mice - hybrids (C57BL / 6 × DBA / 2) F1 (B6D2F1), mice of hybrids (CBA × C57Bl / 6) F1, female mice of the DBA / 2 line, weighing 18-20 g., From nursery ("Dawn", Tomsk). Before and during the experiment, control and experimental animals were kept in a vivarium under the same conditions: standard plastic cages with small wood shavings (no more than 10 individuals) on a standard diet. All studies were carried out at the same time of the day (in the morning).

The model of immunocomplex glomerulonephritis (autoimmune disease) was induced in B6D2F1 females twice with a weekly interval of intravenous lymphoid cells from females of the parental DBA / 2 line. Urine protein content was determined calorimetrically with a Kumsai brilliant blue dye (Loba Feinchemie) on a Titertec Multiscan, wavelength λ 570 nm. In the experiments, mice with stable proteinuria and a protein content of 3 mg / ml or more were used (protein in the urine was determined repeatedly) (4).

The data obtained were processed according to the nonparametric criterion U of Wilcoxon-Mann-Whitney.

Example 1. The effect of the compound at a dose of 300 mg / kg on the level of protein in the urine.

In mice with stable proteinuria for 2 months and a protein content in the urine of from 3.0 to 6.5 mg / ml, a course administration of the compound was carried out. The course was 5 daily intraperitoneal injections at a dose of 300 mg / kg. 5 days after the last administration of the compound, proteinuria was measured. It was found that in 83% of cases (5 mice out of 6 mice used in the experiment) showed a decrease in proteinuria by 10; 18.5; 30.8; 45.2 and 55.6%, one mouse has practically no effect. On average, in the group before treatment, proteinuria was 5.1 mg / ml, after treatment - 3.6 mg / ml, which was significantly lower (P <0.05), the decrease in the group averaged 32.0%.

Example 2. The effect of the compound at a dose of 5 mg / kg on the level of protein in the urine.

The compound at a dose of 5 mg / kg was administered to mice per os daily (once a day), the course was 10 injections. In a similar manner and at the same dose (5 mg / kg), mice were injected with the azathioprine comparison drug. Measurement of proteinuria was performed one day after the last injection. Table 1 presents data on the effect of intragastric administration of the compound at a dose of 5 mg / kg on the amount of protein in the urine compared with azathioprine.

Table 1. Azathioprine Compound Protein in the urine before treatment
(mg / ml) Protein in the urine after treatment
(mg / ml) % inhibition Protein in the urine before treatment
(mg / ml) Protein in the urine after treatment
(mg / ml) % inhibition 5.9 5.2 12 11.2 4.8 58 9.5 13.6 -43 8.9 6.4 28 6.5 6.6 -1.5 5.9 8.1 -37 14.6 5.2 64 6.5 5,6 14 7.6 10.1 - 33 8.1 3.0 63

As can be seen from table 1, under the influence of azathioprine, a decrease in the level of protein in the urine was observed only in 2 out of 5 mice (i.e. in 40% of cases), while the compound caused a decrease in proteinuria in 4 of 5 mice (i.e. in 80% of cases). In other words, the test compound used at a dose of 5 mg / kg turned out to be twice as effective as compared with the well-known drug azathioprine. The average percent inhibition for the compound was 41%.

Example 3. The effect of the compound at a dose of 10 mg / kg on the level of protein in the urine.

The compound at a dose of 10 mg / kg was administered to mice per os daily (once a day). The course was 10 introductions. In a similar manner, the reference drug azathioprine (at a dose of 10 mg / kg) was administered. Measurement of proteinuria was performed one day after the last injection.

Table 2 presents data on the effect of intragastric administration of the compound at a dose of 10 mg / kg on the protein content in urine in comparison with azathioprine.

Table 2. Azathioprine Compound Protein in the urine before treatment (mg / ml) Protein in the urine after treatment (mg / ml) % inhibition Protein in the urine before treatment (mg / ml) Protein in the urine after treatment
(mg / ml) % inhibition 2.7 0.7 84 3.0 1.8 60 1.9 5,6 -290 4.0 5,0 -25 3.9 1.8 54 2,4 1.9 21 4.6 3,5 24 3.9 2.7 31 4.1 2.1 49 2,3 1.0 57 3.9 1,5 62

As can be seen from table 2, under the influence of azathioprine, 5 out of 6 mice (in 83% of cases) showed a decrease in proteinuria (on average, the decrease was 55%). Under the influence of the compound, 4 out of 5 mice (in 80% of cases) showed a decrease in proteinuria (on average, the decrease was 42%).

Analyzing the data obtained, we can conclude that the compound used in doses of 5 and 10 mg / kg in 80% of cases leads to a decrease in protein in the urine. Using the compound in smaller doses (5 and 10 mg / kg) is more effective than in a large dose (300 mg / kg). Doses of 5 and 10 mg / kg reduce proteinuria by 41 and 42%, respectively, while a dose of 300 mg / kg by 32%. In general, the data obtained indicate that the compound used at a dose of 5 and 10 mg / kg is not inferior in effectiveness to the drug azathioprine widely used in clinical practice (at a dose of 5 mg / kg, it is 2 times more effective). However, when comparing the compound and azathioprine in terms of toxicity parameters, it can be noted that the compound (LD ₅₀ = 500 mg / kg) is twenty times less toxic than azathioprine (LD ₅₀ = 25 mg / kg). According to the literature, azathioprine already at a dose of 10 mg / kg inhibits bone marrow function, causes leukopenia and agranulocytosis (1).

In our mouse model of glomerulonephritis, the immunological substrate that triggers a cascade of disorders in the kidneys is T-dependent polyclonal activation of B cells, accompanied by increased synthesis of anti-DNA antibodies, the formation of immune complexes, which leads to a systemic immunocomplex inflammatory process in the kidneys - nephritis and proteinuria. This type of kidney damage is morphologically and functionally comparable to a serious human disease - systemic lupus erythematosus (5). Confirmation of the immunomodulatory effect of the compound, i.e. its ability to suppress T-dependent activation of B cells are the data set forth in example 4.

Example 4. The effect of the compound on the synthesis of IgM antibodies in vivo upon activation of B cells with a T-dependent antigen - sheep erythrocytes.

Animals were immunized intravenously with sheep erythrocytes (EB) at a dose of 0.5 × 10 ⁷ / mouse. From the day of immunization, the compound was administered intraperitoneally or per os daily (for 4 days, once a day) at a dose of 5 and 10 mg / kg. The amount of IgM AOK in the spleen of mice was evaluated on the 4th day after immunization by the number of local hemolysis zones in a semi-liquid medium using the modified Cunningham method (1968). The results were expressed as the absolute amount of IgM AOK in the spleen. Data on the effect of the compound on the number of antibody-forming cells (AOKs) are presented in table 3.

Table 3. Groups The number of AOK / spleen Method for administering a compound Intragastric Intraperitoneally The control 4023 (n = 15) 6227 (n = 12) Compound at a dose of 5 mg / kg
10 mg / kg 730 * (n = 16)
585 * (n = 15) 1129 * (n = 14)
752 * (n = 15)

* significantly, P <0.05; n is the number of animals

As can be seen from table 3, the compound, regardless of the method of administration, several times reduces the number of IgM antibody-forming cells in the spleen, i.e. effectively suppresses T-dependent activation of B-lymphocytes.

Thus, our studies suggest that the tris (2-hydroxyethyl) ammonium salt of 1-benzylindolyl-3-thioacetic acid, having a pronounced property to suppress the T-dependent activation of B-lymphocytes (i.e., inhibit the main pathogenetic link the mechanism of formation and development of the autoimmune process, for example, glomerulonephritis), is a promising chemical compound for the creation of new drugs with the aim of effective treatment of autoimmune diseases.

Literature

1. Mashkovsky M.D. // Medicines.-2001.-S.197-200.

2. UK patent No. 2093693.

3. US patent No. 4551475.

4. Kimura M., Gleichmann E. Depressed antibody responses to exogenous antigens in mice with lupus-like graft-versus-host diseases. // Clin. Immunol. and Immunopathol.-1987.-V.43.-N.1.-P.97-109.

5. Appleby P., Webber D.G., Bowen J.G. Murine chronic graft-versus-host disease as model of systemic lupus erythematosus .: Effect of immunosuppressive drugs on disease development.//Clin. and Exp. Immunol.-1989.-V.78.-N3.-P.449-453.

Claims (1)

The use of tris- (2-hydroxyethyl) ammonium salt of 1-benzylindolyl-3-thioacetic acid as a treatment for autoimmune diseases.