RU2092177C1 - Agent showing effect with respect to infectious agents - Google Patents

Abstract

FIELD: medicine. SUBSTANCE: invention relates to biologically active substance showing antiviral and antibacterial effects. Invention proposes the preparation alpizarin that shows effects with respect to cytomegalovirus, human immunodeficiency virus, tuberculosis mycobacteria and Protozoa. Preparation shows new combination of traits - antiviral, antibacterial, immunostimulating, immunomodulating and interferon-inducing properties. EFFECT: enhanced effectiveness of new preparation. 6 tbl

Description

 The invention relates to medicine, namely to the development and search for new antiviral and bacterial drugs. It is known that the drug alpizarin has antiviral activity (Japan patent N 1518445 for "Method for producing mangiferin", England patent N 2108383 for "Alpizarin drug", US patent N 4436732 for "Alpizarin drug", US patent N 4518592 for "Method for obtaining mangiferin ", the patent of Germany N 31411970 for the" Drug for the treatment of viral diseases ", French patent N 8119820 for the drug Alpizarin, French patent N 2486941 for" Method for producing mangiferin ", ed. St. USSR N 1336301 for" Antiviral agent Alpizarin ", for patent turnout of the Russian Federation N 5014502 on "Method for producing mangiferin."

 Alpizarin 2-C-β-D-glucopyranosyl-1,3,6,7-tetraoxyxanthone is obtained from the alpine lychee herb Hedysarum alpinum L. and the yellowish lycophene, Hedysarum flavescens Rgl. et Schmalh. the legume family Fabaceae (Leguminosae) and from the leaves of the mango Mangifera indica L. sumac family Anacardiaceae.

Known antiherpetic drug VIRU-MERZ (ointment 1% by 5 g in tubes). Effective for herpes simplex skin and mucous membranes in the initial stages of the disease. The disadvantage of the drug is:
narrow spectrum of action;
does not prevent the development of relapses;
does not affect the formation of antibodies;
hyperergic reactions on the skin (contact allergy) caused by the use of the drug (prospectus for the drug "VIRU-MERZ" company "Pharmacos", Yugoslavia).

Known antiherpetic drug acyclovir (Zovirax) (200 mg tablets, cream 5% solution for intravenous infusion). Effective for skin diseases caused by the herpes simplex virus, including primary and recurrent genital herpes and lip herpes. The disadvantage of this drug is:
soreness of injection;
damage to the renal tubules with prolonged administration;
sustainability development;
narrow spectrum of action.

 Known methods and formulations using polysaturated fatty acids for the treatment of malaria and other diseases (WO 93/0008, CL A 61 K 31/20, 1993).

Stable forms of biologically active proteins for parenteral injection are known, containing recombinant interleukin-2 or interferon-b, having biological activity against bacteria and viruses (US 4816440, class A 61 K 37/02, 1989). This technical solution is taken as a prototype [2]
Not all drugs that have an inhibitory effect against herpes virus inhibit the development of cytomegalovirus. Oxolin preparations (Oksolin Avenue, TsBNTImedprom), Bonafton (Bonaphton Avenue, TsBNTImedprom), Arbidol (Arbidol Avenue, S. Ordzhonikidze All-Union Scientific Research Chemical-Pharmaceutical Institute), Virazol (Virazol Avenue ICN Pharmaceuticals, Juc) have antiherpetic activity, but nevertheless, there is no evidence of their activity against cytomegalovirus.

 The aim of the invention is to expand the scope of alpizarin by identifying new properties of the drug.

 This goal is ensured by the fact that the treatment of viral and bacterial infections, as well as an immunostimulant, immunomodulator and inducer of interferon, offers alpizarin 2-C-b-D-glucopyranosyl-1,3,6,7-tetraoxyxantone.

 It was experimentally established that alpizarin, along with the previously known action on the herpes virus, has antiviral activity against cytomegalovirus, immunodeficiency virus and antimicrobial activity against gram-positive and gram-negative bacteria (Mycobacterium tuberculosis, Entamoeba histolitica, Trichomonas vaginalis). Its immunostimulating, immunomodulating and interferon-inducing properties were also identified.

 The novelty and inventive step consists in identifying a new combination of features (spectrum of antiviral activity), as well as in identifying antibacterial activity against gram-positive and gram-negative bacteria.

A method of producing alpizarin is described in patent application N5014502 of September 6, 91 MKI ⁵ A 61 K 35/78 "Method for producing mangiferin."

 A distinctive feature of alpizarin is the presence along with antiviral activity of antimicrobial activity, as well as immunostimulating, immunomodulating and interferon-inducing properties. Low toxicity, complete absence of side effects.

In terms of toxicity with a single intraperitoneal, intragastric methods of administration to laboratory animals, alpizarin is a low-toxic substance. With the intraperitoneal administration of LD ₅₀ for male mice, rats and guinea pigs are equal, respectively: 1385 ± 121; 780 ± 84 and 650 ± 39 mg / kg. With the introduction of alpizarin into the stomach in mice to males, males and females LD ₅₀ increase 10-12 times, to rats males and females 18-20 times and amount to 13000-15900 mg / kg.

 Alpizarin does not have an allergenic, mutagenic or locally irritating effect.

 Alpizarin is approved for use in adults and children as an antiviral agent for acute and recurrent forms of herpes simplex of genital and extragenital localization, Kaposi's eczema, for viral diseases of the oral mucosa, herpes zoster, chickenpox (decision of the Pharmacological Committee M3 of the Russian Federation of March 29, 1993 g.).

 Example 1. The study of the immunomodulatory properties of alpizarin.

 The effect of alpizarin on the humoral immunity.

 In the experiment, 200 CBA mice (males, weight 20-22 g) and 60 outbred rats (males, weight 260-280 g) were used. The model for the immune response to heterogeneous red blood cells was fundamental to the study of the immunomodulatory effect of alpizarin. The results were evaluated by the titer of antibodies to sheep erythrocytes (EB) in the hemagglutination microreaction and the number of antibody-forming cells (AOK) of the spleen. The number of AOKs in the spleen was determined by local hemolysis and Cunningham modification (1965). Animals were immunized with an optimal dose of EB. Alpizarin was administered at doses of 10 and 100 mg / kg into the stomach of CBA mice for 5 days, and to rats for 4 weeks. As a solvent, 40% dimethyl sulfoxide (DMSO) was used. As a result of the studies, it was found that 5-fold administration of alpizarin at doses of 10 and 100 mg / kg into the stomach of CBA mice significantly enhanced the transformation of B-lymphocytes into plasma cells. The number of AOKs in the spleen increased 2.5-4 times. In addition, the number of antibodies to sheep erythrocytes increased 3–7 times (Table 1).

 The data obtained are confirmed in experiments on outbred rats treated with the drug at doses of 10, 100, 500 mg / kg in the stomach for 4 weeks. A clear dose-dependent effect was revealed. Alpizarin 1.5-2.5 times increased the amount of AOK in the spleen, stimulated the formation of antibodies (Table 2).

 Thus, in all animal doses, alpizarin in experiments on animals stimulated the formation of antibodies to the thymus-dependent antigen and increased the activity of humoral immunity effectors.

 The study of the immunostimulating properties of alpizarin.

 The immunostimulatory activity of alpizarin was studied in a model system for the formation of specific cytolytic t-lymphocytes (t-killers) in a mixed culture of lymphocytes (Mixed Lympgocyte Culture, MLC) from spleens of allogeneic mice.

где максимальный выход 51 Cr выход изотопа в надосадочную жидкость после обработки клеток-мишеней 2% раствором додецилсульфата Na в 0,05 М растворе боратного буфера pH 9,9.Methodology In order to obtain cytolytic lymphocytes of the spleen of BALB mice, SPB was extracted aseptically, carefully homogenized in Potter glass homogenizers in RPMI-1640 medium, filtered through several layers of gauze and the number of living cells was counted using zozontrypanovoy blue. In vitro stimulation was carried out according to the previously described method with some modification. The lymphocytes of BALB / c mice (H-2 ^d at a concentration of 5 • 10 ⁶ in 1 ml were used as reacting cells), as spleen cells of spleen mice of CZN (H-2 ^k ) irradiated prior to incubation with a dose of 1000 P. The ratio of reacting and stimulating the cell count in most experiments was 5: 1. The control was monoculture of BALS / c lymphocytes or irradiated lymphocytes of mice of SZN.The culture medium contained 100 ml of DMEM medium, 20% inactivated fetal calf serum (TES), 2 • 10 ³ m glutamine, 5 • 10 ³ HEPES, ^{5 October} 3 • 2-mercaptoethanol, penicillin and streptomits to 100 U per 1 ml of medium. Incubation was performed in a 24 well flat boards 76-03 (Linbro) in an oven "Napco" at 5% CO ₂ for 5 days. The drug alpizarin dissolved in Hank's solution, sterilized by passage through a filter "Millipore "and introduced into the culture in a volume of 10 μl for up to 2 days. Each series of experiments was repeated at least 3 times. To determine the activity of specific T-killers formed in MLC, lymphocytes were incubated with target cells in a ratio of 0.2: 1, 1: 1, 5: 1, 20: 1 in 0.2 ml of RPMI-1640 medium with 5% TES in 96-well flat-bottom boards (FLOW LAB) for 18 h at 37 ^o C. As the targets used a monolayer of lymphocytes (H-2 ^k , which on the eve of the experiment were incubated with 100 µCi Na ₂ 51CrO ₄ in 1 ml of RPMI-1640 medium with 5% TES for 1 h at 37 ^o C. Then the cells washed three times with medium 199 and poured into the wells of microplates (FLOW LAB) in the amount of 10 ⁴ cells per well. Each determination (one ratio of lymphocyte: target cell) was performed in 6 parallel samples. To determine spontaneous lysis, target cells were incubated with the same medium without lymphocytes. In our experiments, spontaneous lysis, as a rule, was 15–20%. After incubation, 0.1 ml of supernatant was taken from each well and transferred to plastic tubes. Radiometry of the samples was carried out on a gamma counter firm Nucléar Chicago. The percentage of specific cytolysis was determined by the formula:

where the maximum yield of 51 Cr isotope yield in the supernatant after treatment of the target cells with a 2% solution of Na dodecyl sulfate in a 0.05 M solution of borate buffer pH 9.9.

 The results of the study are shown in tables 3 and 4.

 It was found that alpizarin, added to the in vitro culture on day 0-2 of incubation at a concentration of 1 μg / ml, increased the formation of T-killers by 1.5-2.5 times. Thus, we can say that alpizarin has immunostimulating properties in relation to cellular immunity.

 Example 2. The study of interferon-inducing properties of alpizarin.

 Alpizarin was tested for interferon-producing activity in donors and patients with recurrent herpes.

 In the work, a method was used to determine the ability of cells to produce alpha and gamma interferons in whole blood and, in comparison with conventional inducers of gamma interferon, staphylococcal endotoxin type A and alpha interferon virus, New Castle disease.

 It was found that the drug has the ability to cause the production of interferon, in quantitative terms comparable to the production of gamma interferon under the action of CEA in the studied individuals. Moreover, the interferon-inducing activity of alpizarin was manifested on blood cells and was not detected on human diploid fibloblasts M-19, which proves the effect of the drug on blood cells.

 In addition, the dependence on the dose of the drug on the interferon-producing activity of blood cells was revealed. In donors, doses of 10 and 100 μg / ml were equally effective, and in patients, 50 μg / ml. When typing interferons by adjusting the pH to 2 and carrying out a neutralization reaction with monoclonal antibodies to human alpha and gamma interferons, it was found that acid-stable gamma-type interferon is induced.

 Thus, alpizarin is a promising drug for inclusion in the course of antiviral and immunocorrective therapy, since it has, in addition to the well-known antiviral activity, the ability to induce the production of gamma-interferon in blood cells.

 Example 3. The study of the effectiveness of alpizarin against human immunodeficiency virus (AIDS).

 Materials and methods.

 Viruses. Used human immunodeficiency virus type I, strain HTLV-III, produced by the cell line H9 / IIIB.

Cells. We used a primary trypsinized culture of chicken embryo fibroblasts (FEC), transplantable cultures of human T-lymphocytes H9 and H9 / IIIB, and primary cultures of splenocytes from C ₅₇ BL / 6 mice.

 Animals. Non-linear white mice of both sexes.

 Immune serum. Used to control viral strains: sera of AIDS patients (B.F. Vestergaard, State Serum Institute, Copenhagen, Denmark).

 Infection of H9 cells with HIV-I. H9 cells infected HIV-I by inoculating live H9 / IIIB culture cells. The drug was added to the H9 + H9 / IIIB culture immediately after infection. The experiments were accompanied by control of the culture without the drug and control of the toxicity of doses of alpizarin for H9 cells.

The level of production of HIV-I infected cultures was evaluated:
by the number of cells (%) expressing viral antigens on the membranes, determined by the method of indirect immunofluorescence;
by HIV-I reverse transcriptase activity in the supernatant.

 The effect of alpizarin on the expression of the CD4 receptor was evaluated after 24-hour incubation of alpizarin with H9 cells by flow fluocytometry on a laser sorter using monoclonal antibodies labeled with FTC-OCT4 (as a control of OCT8).

A study of the effectiveness of alpizarin was carried out in in vitro experiments in a mixed cell culture of H9 / IIIB and H9. A mixture (1: 1) of cells was prepared immediately before the experiment and poured into 2 ml in each well in Coster 24-well plates. At the same time, alpizarin was added to the wells in order to obtain final concentrations of 50, 25, 5 and 1 μg / ml in the wells. The plates were incubated at 37 ^° C and 5% CO ₂ for 7 days. Cell viability was assessed daily using an inverted microscope. The results of the experiment were taken into account for 7 days by indirect immunofluorescence using serum of an AIDS patient in a 1:40 dilution.

 As a result of the study, it was found (Table 5) that alpizarin at a concentration of 50 μg / ml inhibits almost completely viral production in the studied system of H9 / IIIB and H9 cells. At lower concentrations (25, 5, and 1 μg / ml), the inhibitory effect of alpizarin was not detected under these experimental conditions.

 The data obtained indicate the ability of alpizarin to protect H9 cells not infected with the virus from being infected with the virus under conditions of co-culture with H9 / IIIB cells.

 Example 4. The effect of alpizarin on cytomegalovirus in cell culture.

 The aim of the study was to study alpizarin for the reproduction of cytomegalovirus (CMV) in cell culture.

 Materials and methods.

 The effect of alpizarin on the reproduction of cytomegalovirus was studied on diploid cells of human M-19.

 Diploid cells were obtained from the skin and muscles of 7-10 week old human embryos.

 Tissue disaggregation was carried out with 0.25% trypsin solution. A similar enzyme solution was used to separate cells from glass during the passage. As the growth medium, MEMA Needle medium with 10% fetal calf serum was used. During morphological study of the lines, it was noted that the cultures consist of fibroblast-like cells. Karyological analysis showed that the cells belong to the group of diploid lines.

The reference strain of human CMV Ad-169 was used in the work. In cell culture, the virus caused specific cytopathic lesions on day 7 with typical intranuclear inclusions. The infectious titer was 10 ⁵ TCD ₅₀ / ml.

 Alpizarin was used at concentrations of 250, 125, 50, and 25 μg / ml.

Cells were cultured in test tubes on coverslips using Eagle's medium in 10% fetal calf serum. 0.2 ml of virus with a titer of 10 ⁵ TCD ₅₀ / ml was added to test tubes with grown cultures. After adsorption for 1 h, the virus was removed and a growth support medium was introduced: a mixture of Eagle medium and 199 medium in equal volumes (50%) with 2% fetal calf serum.

 After 48 hours, as well as within two weeks, the coverslips were removed from the tubes, stained with hematoxylin and eosin and the number of lesions of the cell layer was determined under a light microscope.

 The results of the study.

 As a result of the studies, it was found that alpizarin at concentrations of 250, 125, 50 and 25 μg / ml did not have a toxic effect on uninfected cell cultures during the entire observation period.

Alpizarin at a concentration of 100 μg / ml completely suppressed the reproduction of the cytomegalovirus strain Ad-169 by 5 lg TCD ₅₀ / ml, which is 10,000 infectious doses of the virus.

 Example 5. The study of the antimicrobial spectrum of alpizarin.

 Research Methodology.

 The following test microorganisms were used to determine the antimicrobial spectrum of alpizarin: Staphylococcus aureus 209-P, Escherichia coli 675, Mycobacterium tuberculosis H-37 RV, Mycobacterium tuberculosis "Academia", Candida albicans 1755, Microsporum canis, Entamoebaicholicolytica.

 When determining the bacteriostatic activity of alpizarin, meat-peptone broth (MPB) was used.

 The fungistatic action was studied in Saburo's liquid medium.

 The tuberculostatic effect was studied in Soton's liquid medium.

 A sample of at least 10 mg was introduced into a sterile tube and sterilized for 1 h with 95% ethanol (ethanol at a concentration of 2% did not have an antimicrobial effect) at the rate of 0.1 ml of alcohol per 5 mg of the drug. Then, a sterile nutrient medium corresponding to each microorganism was added in an amount necessary to create an initial drug concentration of 1000 μg / ml. From this tube, a series of decreasing concentrations were prepared by double serial dilutions. The last test tube with a clean medium (without adding a drug) served as a control. After that, all tubes (experimental and control) were seeded with cultures of microorganisms.

Suspensions of gram-positive and gram-negative bacteria were prepared in an isotonic solution of sodium chloride according to the bacterial standard turbidity of 10 units (10 ⁹ microbial bodies / ml), then 0.2 ml of a suspension containing 10 ⁴ microbial bodies / ml was added to each tube. Crops were incubated in a thermostat at a temperature of 37-38 ^o C for 24 hours

A suspension of mushrooms was prepared in an isotonic solution of sodium chloride according to the bacterial standard turbidity of 10 units, then diluted with isotonic solution 20 times. 0.2 ml of the obtained suspension were inoculated into experimental and control tubes. Crops were incubated at a temperature of 30 ^o C: yeast-like fungi for 48 hours, mycelial fungi for 10-14 days.

Suspensions of tuberculosis mycobacteria were prepared in an isotonic solution according to BCG standard containing 5 mg of bacilli in 1 ml. The resulting suspension was diluted with Soton's 1:10 medium and 0.2 ml of diluted suspension containing 0.1 mg of bacilli in 1 ml was added to each tube and incubated in an incubator at a temperature of 37 ^o C for 7-10 days.

 The experiments were performed in triplicate.

 Accounting for results.

 The antibacterial and antifungal effect was determined by the minimum concentration of the drug (μg / ml), at which the growth of microorganisms was not visually observed.

 The results of the study.

 As a result of the study, it was found (Table 6) that alpizarin inhibits the growth of gram-positive and gram-negative bacteria at a concentration of 500 μg / ml; Mycobacterium tuberculosis at a concentration of 15.6-62.5 μg / ml, protozoa at a concentration of 15.6-62.5 μg / ml. With respect to yeast and mycelial fungi, alpizarin is not active.

Claims (1)

 The use of alpizarin as an agent with action against infectious agents, such as human immunodeficiency virus, cytomegalovirus, tuberculous mycobacteria and protozoa.

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