RU2254861C1 - Gamma-interferon inductor - Google Patents

Abstract

FIELD: medicine, pharmacology.

SUBSTANCE: invention relates to study for effect of niglisine representing a derivative of glycyrrhizic acid. Invention proposes an agent for induction of gamma-interferon under conditions in vitro and in vivo. Invention proposes glycyrrhizic acid penta-O-nicotinate as such agent that is known early as an anti-inflammatory agent and inhibitor of HIV reproduction. Niglisine in the range of concentrations 0.167-6 mcg/ml enhances significantly the secretion of gamma-interferon by blood mononuclear cells that will allow its using for non-specific protection of body.

EFFECT: valuable properties of gamma-interferon inductor.

1 tbl, 3 dwg

Description

The invention relates to medicine and pharmacology, in particular to the study of the effect of the derivative of glycyrrhizic acid - niglizine on the secretion of gamma-interferon in vitro and in vivo.

The basis for the normal functioning of the immune system is the balanced production of cytokines. One of the main endogenous immunomodulators that affect the function of T cells, macrophages is gamma interferon. In addition to the immunostimulating effect, gamma interferon is characterized by antiproliferative, antiviral and antiparasitic effects. The main sources of gamma interferon are type 1 helper T cells, cytotoxic lymphocytes and normal killer cells. In clinical practice, gamma-interferon is used to treat tumor diseases in patients with AIDS in the treatment of opportunistic infections. It has also been shown that the administration of gamma interferon improves the survival of patients with sepsis.

It is known that niglizine is used to inhibit the reproduction of Marburg viruses and human immunodeficiency [1, 2] as an anti-rheumatic, hepatoprotective and antidote drug [3], but this substance was not used as an interferon inducer.

Closest to the claimed technical solution (prototype) is glycyrrhizin (a drug manufactured by the Japanese pharmaceutical company "Minophagen"), which consists of one molecule of glycyrrhizic acid and two molecules of glucuronic acid [4]. This drug is able to stimulate the production of gamma interferon in a cell culture of monocytes and macrophages [5] and T cells [6]. It is used to treat HIV infection and viral hepatitis in a daily dose of up to 1.6 grams with intravenous administration [7, 8].

Niglizine (penta-o-nicotinate glycyrrhizic acid) is used in tablets (per os) and in a daily dose of 0.3 grams does not affect the functional state of the cardiovascular and hematopoietic systems, liver and kidney function, does not cause pathological changes in the brain, heart , lungs, liver, spleen, stomach and intestines, and also does not have teratogenic and carcinogenic properties [9].

The objective of the invention is the search for a new drug that increases the secretion of gamma interferon in the body and does not have side effects.

The problem is solved using a derivative of glycyrrhizic acid - (penta-o-nicotinate glycyrrhizic acid) as an inducer of gamma-interferon in the body.

Niglizine is dissolved in DMSO, and then the FSB solution is adjusted to the required concentration. Groups of 3 mice were injected with a 0.5 ml niglizine solution with a concentration of 400 μg / ml, 40 μl / ml and 4 μg / ml intraperitoneally once, after 12, 24 and 48 hours, the spleens were taken and gamma-interferon secretion was evaluated. As a control, 4% DMSO was administered to mice.

The proposed inducer of interferon niglizine is not described in the literature, therefore, we can conclude that the technical solution meets the criteria of the invention of "novelty" and "inventive step".

The list of figures of graphic images.

Figure 1 - The effect of niglizine on the secretion of gamma-interferon by blood mononuclear cells. Control is the cellular environment; 0.167 μg / ml; 1.0 μg / ml; 6.0 μg / ml - niglizine concentration; positive controls: I - ionomycin with PMA; II - monoclonal antibodies anti-CD3.

Figure 2 - dependence of the number of gamma-interferon-producing cells on the concentration of niglizine.

Figure 3 - dose-dependent effect of niglizine on the number of IF-gamma-secreting cells 48 hours after a single intraperitoneal administration to mice (* - p <0.01; ** - not statistically significant).

The following is an example of the method.

1. Blood donors, sampling and isolation of mononuclear cells.

10 volunteers aged 22 to 52 years are selected, the average age is 37 years. In the study group, 9 women and 1 man. Blood with a volume of 4-5 ml is taken by venipuncture into tubes containing EDTA as an anticoagulant. The blood is then diluted with phosphate buffer in a 1: 1 ratio and the diluted blood is applied to a Ficoll-Hypaque solution. Separation is carried out by centrifugation at 1500 rpm for 40 minutes Interphase cells are harvested, resuspended in 3 ml of phosphate buffer and washed twice by centrifugation for 5 min at 1500 rpm. The third washing is carried out in RPMI 1640 medium in a volume of 2 ml. After the third wash, the mononuclear cells are resuspended in 1 ml of medium.

2. Reagents.

Niglizine (penta-o-nicotinate glycyrrhizic acid) is dissolved in DMSO and then adjusted to a concentration with a solution of phosphate buffer (PBS). Phorbol-12-myristate-13-acetate (PMA) and ionomycin, as well as anti-CD3 monoclonal antibodies, are used as a positive control. Cells are incubated in RPMI 1640 medium with 10% fetal serum supplemented with antibiotics.

3. Determination of gamma-interferon-secreting mononuclear cells by ELISPOT.

For sorption of antibodies using a 96-well plate with nitrocellulose bottom (Milliliter, Millipore). Sorption of murine anti-gamma-interferon monoclonal antibodies was carried out in phosphate buffer overnight at 4 ° C, while 100 μl of a solution with an antibody concentration of 5 μg / ml was added to the well. The next day, the wells are washed with phosphate buffer and 150 μl / well of RPMI 1640 medium with 10% fetal serum is added to block free sites. The plate is incubated for 2 hours at 37 ° C. Then, mononuclear cells are introduced into the wells so that 200,000 cells are contained in a final volume of 200 μl. Niglizine is added to the wells to a final concentration of 0.167, 1 and 6 μg / ml. As a positive control, phorbol-12-myristate-13-acetate with ionomycin in concentrations of 30 ng / ml and 500 ng / ml, respectively, and anti-CD3 monoclonal antibodies at a dilution of 1:30 are used. The plate is incubated for 36-40 hours at 37 ° C and 5% CO ₂ . After the incubation period, the cells are removed and the wells washed with a solution of phosphate buffer with tween (FSB-T). Then add mouse anti-gamma-interferon monoclonal antibodies at a concentration of 2.5 μg / ml at 37 ° C for 2 hours. After washing with FSB-T solution three times, rabbit anti-mouse IgG labeled horseradish peroxidase diluted 1: 800 was added to the wells and incubated 1 h at 37 ° C. After removing unbound components and washing the wells, the resulting complexes (spots) appear with a freshly prepared solution of 4-chloro-1-naphthol. After 20-30 minutes, the substrate is removed, the wells are washed with distilled water and allowed to dry.

4. Animals

In the experiment, linear BALB / C mice weighing 18-20 g of both sexes were used, a total of 36 individuals.

5. Injection of niglizine

Niglizine is dissolved in DMSO, and then the FSB solution is adjusted to the required concentration. Groups of three mice are injected with a solution of niglizine in a volume of 0.5 ml with a concentration of 400 μg / ml; 40 μg / ml and 4 μg / ml intraperitoneally once, after 12, 24 and 48 hours, the spleens are taken and the secretion of gamma-interferon is evaluated. As a control, 4% DMSO solution was administered to mice.

6. Isolation of the spleen and preparation of splenocytes.

After 12, 24, and 48 hours after niglizine injection, the mice were sacrificed by cervical translocation and the spleens were removed under aseptic conditions. Then the spleens are wiped through a metal sieve, suspended in RPMI 1640 medium, then washed three times in the same medium, each time centrifuged at 1500 rpm for 10 minutes.

7. Determination of production of gamma interferon by splenocytes of mice by ELISPOT method

Anti-mouse gamma-interferon binding monoclonal antibodies at a concentration of 5 μg / ml in a volume of 100 μl are sorbed on a nitrocellulose bottom plate. The next day, unbound sites block 150 μl / well of RPMI 1640 solution with 10% fetal serum. After washing, a suspension of splenocytes with a volume of 200 μl with a cell concentration of 1 million / ml is added to the wells. The plate is incubated for 36-40 hours at 37 ° C, 5% CO ₂ . After the incubation period, the cells are removed and the wells are washed with FSB-T solution. Then, second anti-gamma-interferon detectors detecting biotin-labeled monoclonal antibodies at a concentration of 0.5 μg / ml are added and incubated at 37 ° C for 2 hours. After washing with FSB-T solution three times, the horseradish peroxidase conjugate diluted 1 is added to the wells : 100 and incubated for 1 h at 37 ° C. Then, unbound components are removed and the wells are washed, the resulting complexes (spots) appear with a freshly prepared solution of 4-chloro-1-naphthol. After 20-30 minutes, the substrate is removed, the wells are washed with distilled water and allowed to dry.

8. Counting the number of spots formed

The resulting spots are counted under a binocular at a 50x magnification. The average number of spots is determined by two repetitions.

9. Statistical analysis

Comparison of the experimental and control groups is carried out using a two-sample t-test with different variances (Excel). The results are considered statistically significant for p <0.05.

RESULTS

1. The effect of niglizine on the production of gamma interferon in healthy volunteers in vitro

As shown in figure 1, niglizine in the concentration range of 0.167-6 μg / ml statistically significantly (p <0.05) increases the secretion of gamma-interferon by blood mononuclear cells.

Investigating the effect of different concentrations of niglizine on the secretion of gamma-interferon, we can conclude that the maximum stimulation is observed when the amount of niglizine in the medium is equal to 1 μg / ml (figure 2). At the same time, on average, a more than twofold increase in the number of spots is observed (from 1.2 to 5.5 times).

2. The effect of niglizine on the production of gamma interferon by splenocytes of mice in vivo

The results of an in vivo experiment conducted in mice are shown in Table 1. An increase in the secretion of gamma-interferon by splenocytes is observed only 48 hours after a single injection of niglizine at a dose of 1 and 10 μg / kg (Fig. 3). 12 and 24 hours after a single injection of niglizine in the studied doses, the number of detected spots does not differ from the control.

From the foregoing, it can be concluded that the use of niglizine increases the secretion of gamma-interferon, which allows it to be used as an antiviral drug for nonspecific protection of the body.

Table 1
The effect of niglizine on the production of gamma interferon by splenocytes of mice Niglizine mg / kg / mouse The average number of spots ± standard deviation 12 hours 24 hours 48 hours The control 18.33 ± 6.17 36.50 ± 8.23 29.17 ± 6.62 0.1 19.50 ± 5.24 39.50 ± 15.50 26.33 ± 7.66 1 19.33 ± 4.50 31.83 ± 5.04 44.50 ± 19.98 10 18.33 ± 6.01 34.50 ± 7.09 50.60 ± 12.28

Literature

1. USSR author's certificate No. 1804848 "Inhibitor of the reproduction of the human immunodeficiency virus", A 61 K 31/705, published BI No. 12, 1993.

2. RF patent No. 2088232 "Marburg virus reproduction inhibitor", A 61 K 31/70, published BI No. 24, 1997.

3. "Glycyrrhizic acid." Tolstyakov G.A., Baltina L.A., Schulz E.E., Pokrovsky A.G., Bioorganic Chemistry, 1997, Volume 23, No. 9, pp. 691-709.

4. Utsunomiya T., Kobayshi M., Pollard R. B., Suzuki F., Glycyrrhizin, an Active Component of Licorice Roots, Reduces Moriditily and Mortality of Mice Infected with Lethal Doses of Influenza Virus, Antimicrobial Agents and Chemotherapy. 1997, v.41, N3, p.551-556.

5. Shinada M., Azuma M., Kawai H., Sazaki K., Yoshida T., Suzutani T., Enhancement of interferon-γ prodaction in glycyrrhizin-treated human peripheral lymphocytes in responce to concavalin A and to surfase antigen of Hepatitis In virus (42241), Proc. Soc. Exp. Biol. Med., 1986, v. 181, p. 205-210.

6. Abe N., Ebina T., Ishida N. Interferon induction by glycyrrhizin and glycyrretinic acid in mice. Microbiol. Immunol., 1982, v. 26, p. 535-539.

7. Rossum T.G.J., Vulto A.G., Man R.A., Brouwer J.T., Schalm S.W., Review article: glycyrrhizin as potential treatment for chronic hepatitis C., Alymentary pharmacology and therapeutics, 1998, v. 12, No. 3, p. 199-205.

8. Hattori T., Ikematsu S., Matsushita S., Maeda Y., Hada M., Fujimaki M., Takatsuki K., Prelimenary evidence for inhibitory effect of glycyrrhizin on replication in patients with AIDS, Antiviral Res., 1989, v. 11 (5-6), p. 255-261.

9. Nasyrov H.M., The mechanism of action of antiphlogists as the basis for the search for new anti-inflammatory drugs. Thesis of the doctor honey. Sciences, Kazan: Honey. Institute, 1986, p. 365.

Claims (1)

The use of penta-O-nicotinate glycyrrhizic acid as an inducer of gamma interferon.

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