RU2390343C1 - Water-soluble antiviral and immunomodulatiory drug based on ionic silver and methylene blue compound and method for preparing thereof - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention covers a water-soluble drug based on ionic silver and methylene blue compound, and also a method for preparing thereof. Said method implies synthesis when heating to 90-95C by mixing the following components: methylene blue - silver chloride - ammonia in the molar ratio 1.06:1:58 to be cooled; a by-product is filtered, steamed in vacuum, washed with acetone and dried in vacuum at room temperature. ^ EFFECT: prepared drug meets the composition C16H18CI2N3SAg and exhibits antiviral and immunomodulatory action. ^ 2 cl, 2 dwg, 3 tbl

Description

The invention relates to water-soluble antiviral and immunomodulatory drugs, to the field of pharmaceutical chemistry and medicine. The invention also relates to the field of production of these agents. The invention can be used to create finished dosage forms for the treatment of viral, bacterial and other infections.

The goal is the creation of highly effective antiviral, immunomodulatory drugs, especially for the treatment of viral hepatitis and antibiotic-resistant infections, including HIV infection.

SUMMARY OF THE INVENTION A new antiviral agent based on a compound of silver with methylene blue and corresponding to the composition of C ₁₆ H ₁₈ Cl ₂ N ₃ SAg has been proposed (Table 1).

Known drugs are called Argochrom by Merck (produced in Germany from about 1916 to 1945 and imported by the USSR in the pre-war period), the composition of which falls under the Austrian Patent No. 69476 effective from 1914 and was called Silber until 1920 by the German methylen blau, as well as the preparation Gegonon, manufactured according to German patent No. 268968, published on January 7, 1914, class 12p, group 16 - prototype. (See the reference Votchal B.E. et al. Pharmaceuticals. M.-L.: ONTI, 1934).

These preparations contain silver in ionic form, firmly bound to organic molecules - ligands. Argochrome is distinguished by increased toxicity and fire hazard of the finished product due to the presence of nitrate ions in the structure. The disadvantage of Hegonon is the low bioavailability of the silver contained in them, since the ligand in this preparation is a high molecular weight carrier - albumose.

In recent years, information has appeared in the scientific literature that silver is a powerful immunomodulator. Under the influence of silver, the amount of immunoglobulins of classes A, M, G increases, the formation of interferon is induced, the percentage of the absolute amount increases

T-lymphocytes, that is, immunity is activated. In addition, silver ions themselves actively act on pathogenic bacteria and viruses.

In the late 90s of the last century, it was convincingly proved that methylene blue destroys viruses, including the viruses of hepatitis, herpes, AIDS, etc. Already at blood transfusion stations in the production of preparations from human plasma, plasma is treated with a solution of methylene blue and exposed to light to inactivate various viruses, followed by removal of methylene blue.

Methylene blue is a phenothiazine dye. Dyes of this class are able to invade the structure of the nucleic acids of viruses and bind closely to DNA / RNA guanidine residues.

The objective of the invention was the creation of a fundamentally new form of the drug compound, which allows you to combine the advantages of each of the components together and obtain a drug that has pronounced virus-inhibiting and immunomodulating properties due to the effective penetration of the drug through biological membranes, starting from penetration into the bloodstream in the case of oral or rectal methods the introduction of the drug, and neutralize the virus outside the cell, as well as the effective penetration of the complex through the cell membrane of the affected cell in order to stop the activity of infectious agents while triggering the body's defenses.

The problem is solved by the fact that a water-soluble, photostable silver solution is obtained in solutions, which is a durable silver metal complex with methylene blue.

A method is also proposed for producing a new water-soluble antiviral drug (substance), which consists in the fact that the synthesis is carried out by heating to 90-95 ° C by mixing the following components: methylene blue - silver chloride - ammonia in a molar ratio of 1.06: 1: 58, in this case, methylene blue is first dissolved in water, a prepared solution of the ammonia complex of silver chloride is added to the resulting solution, obtained by mixing calculated amounts of a suspension of silver chloride and a 25% aqueous solution of ammonia aka, the reaction mixture is heated to 90-95 ° C and held for 1 h, the reaction mixture is cooled, the by-product is filtered off, evaporated in vacuo, acetone is added to the solid residue and triturated to a homogeneous state, the precipitate is filtered, washed with acetone and dried to constant weight. vacuum at room temperature.

The proposed tool meets the chemical composition of C ₁₆ H ₁₈ Cl ₂ N ₃ SAg.

An example implementation of the method.

Methylene blue (25 g, 66.9 mmol) is dissolved by heating to 60-70 ° C in 500 ml of distilled water. The solution was cooled to room temperature, and a solution of silver chloride (9 g, 62.8 mmol) in concentrated (25%) ammonia solution (250 ml) was added to the solution with vigorous stirring. With stirring, the reaction mixture is heated to 90-95 ° C and kept under stirring at this temperature for 1 hour. The reaction mixture is cooled, the precipitate formed is filtered off and the resulting solution is evaporated on a rotary evaporator. 0.5 L of acetone is added to the solid residue, the precipitate is removed from the walls with a spatula and left on a magnetic stirrer for 3 hours. The precipitate is filtered off, washed with several portions of acetone (total volume of acetone is 1.2 L). The precipitate is dried on a filter, then in a vacuum desiccator to constant weight. Yield 24.3 g (78.4%).

The data of elemental analysis of the claimed funds are presented in table 1, obtained using an automatic analyzer of elemental composition. The silver content was determined by the atomic adsorption method.

Table 1 Elemental Analysis Data Found (%) FROM N N Cl Ag 41.12 4.11 9.14 15.22 23.34 41.46 4.04 9.64 15.23 23.20 41.40 3.83 9.24 15.10 22.91 Calculated (%) for C ₁₆ H ₁₈ Cl ₂ N ₃ SAg 41.49 3.92 9.07 15.31 23.29

The results of laboratory studies on the biological activity of the claimed funds with the code name Argotiazine.

As a result of the experiments in the laboratory, the cytotoxicity and antiviral and interferonogenic activity of the claimed drug with the conditional name Argotiazine was studied.

Research methodology and results.

In vitro toxicity determination

Determination of the toxicity of drugs in vitro was carried out in accordance with the "Guidelines for the experimental (preclinical) study of new pharmacological substances" (under the general editorship of R.U. Khabriev, 2005).

We used a transplantable cell line SPEV (transplantable cell line of pig kidney embryos), FEC (chicken embryo fibroblasts) and transplantable cell culture line L-929.

All experiments were performed in triplicate. Statistical processing - by generally accepted methods (I.P. Ashmarin et al., 1974).

To determine the toxicity in vitro, various dilutions of the preparation in a culture medium were prepared, which were introduced in a two-three-day monolayer of cell culture grown by a micromethod in 96-well plates.

For one dilution of the drug used 4 wells with a monolayer of cell culture. The cytotoxic effect of the drug and the cells were taken into account by the degree of change in the monolayer and controlled for 3 days.

The cyto-destructive effect of the drug was noted according to the 4-cross system, according to the Finter method, where 100% cell destruction is indicated by four crosses + + + +, 75% is indicated by three crosses + + +, 50% is indicated by two crosses + + and 25% is indicated one cross +. The minimum concentration of the drug, causing a cytotoxic effect of 50%, is two crosses + +, is considered as a cytopathogenic dose (TCD ₅₀ ).

Studies have shown that Argotiazine was non-toxic in the culture of SPEV cells at a dose of up to 18 ± 2.0 μg / ml, and in the culture of cells of FEC up to 19 ± 3.0 μg / ml.

Studies of the virus-inhibiting activity of the drug Argotiazine in cell cultures of SPEV and FEC

Virus-inhibiting activity of the drugs was determined using a micromethod (Guide edited by Khabriev R.U. - Moscow. - 2005. - P.536). As a test virus, vesicular stomatitis virus (BBC) was used, the Indiana vaccine strain at a dose of 100 CPV _{50 of the} BBC. Inhibition of the virus by drugs was evaluated after 48 hours by determining the titer of the virus in the supernatant.

table 2 Virus-inhibiting activity of the drug Argotiazine against the BBC virus in cell culture SPEV Drug name The used dose of the drug The amount of air force virus (M ± m), lg
TCD _{50 / 0.1 ml} Experience The control Argotiazine 16 mcg / ml Not determined 4.46 ± 0.16 8 mcg / ml 2.32 ± 0.16 4 mcg / ml 3.82 ± 0.12

Studies have shown that in the cell culture of SPEV, the drug Argotiazine completely blocks the multiplication of the virus in a dose of 16 μg / ml, i.e. the virus was not detected in the supernatant, while in the control, where there was no drug, the accumulation of the virus reaches up to 4.461g ± 0.16 TTCD _{50 / 0.1 ml} .

Table 3 Virus-inhibiting activity of the drug Argotiazine against the BBC virus in cell culture FEC Drug name The used dose of the drug The amount of air force virus (M ± m), lg
TCD _{50 / 0.1 ml} Experience The control Argotiazine 16 mcg / ml Not determined 4.88 ± 0.16 8 mcg / ml 3.46 ± 0.022 4 mcg / ml 3.98 ± 0.28

Studies have shown that in the cell culture of the FEC, the drug Argotiazine completely blocks the multiplication of the virus in a dose of 16 μg / ml, i.e. the virus is not detected in the supernatant, while in the control, where there was no drug, the accumulation of the virus reaches up to 4.88 lg ± 0.16 TTCD _{50 / 0.1 ml} .

Determination of interferon-inducing activity of the drug in vitro.

To determine the interferon-inducing activity of the drug Argotiazine in vitro, it was introduced in non-toxic doses: 10; 100 μg / ml in a 48-hour cell culture

L-929 (toxic dose for L-929 cells was higher than 125 μg / ml). The cell culture contact with the preparation was 6 (for determining early interferon) and 24 hours (late interferon), respectively, after which it was removed, the cells were thoroughly washed and poured with fresh nutrient medium. After 24 hours of incubation, the culture fluid (supernatant) was collected and used to determine the activity of interferon. The determination of interferon in samples of supernatants and in the blood serum of mice was determined by titration in L-929 cell culture. To this end, cells at a concentration of 3 × 10 ^{5 were} grown in 96-well flat-bottomed plates at 37 ° C in an incubator with 5% CO ₂ in a medium of Needle MEM with a double set of amino acids, 5% fetal blood serum and 100 μg gentamicin for 24- 48 hours to form a dense monolayer.

Determination of interferon-inducing activity of the drug in vivo.

To determine the interferon-inducing activity of the drug in vivo, nonlinear white mice weighing 16-18 g were used, selected into groups according to the analogue principle, 3 for each dose of the drug (10; 100 μg / kg body weight) administered intraperitoneally. The drug was administered to animals in the same volume, control - in the same volume of saline.

All experiments were performed in triplicate; the results were statistically processed by conventional methods.

RESEARCH RESULTS

The results of studies to determine the interferon-inducing activity of the drug Argotiazine in vitro in cell culture are presented in figure 1.

The contact of the drug with cells for 6 and 24 hours led to a slight induction of the formation of interferon 5.33 ± 1.09 - 9.33 ± 2.88 IU / ml and 9.33 ± 2.88 - 10.67 ± 2.18 IU / ml, respectively.

The results of studies to determine the interferon-inducing activity of various doses of the drug Argotiazine in vivo in non-linear white mice after intraperitoneal administration are presented in figure 2.

The results obtained indicate that the intraperitoneal administration of various doses of the drug Argotiazine led to the production of both early and late interferon in non-linear white mice. 6 hours after a single injection of the drug, the indicators of serum interferon in them were: 9.33 ± 2.88 - 10.67 ± 2.17 IU / ml, and after 24 - 13.33 ± 2.3 - 18.67 ± 4 , 46 IU / ml. Within three days, a decrease in serum interferon induced by Argotiazine was noted to 9.33 ± 2.88 and 13.33 ± 2.3 IU / ml, respectively, the administered dose (10; 100 μg / kg of animal body weight).

As a result of the studies, it was found that the drug Argotiazine after a single injection causes the production of interferon in cell culture (in vitro), as well as in the organism of outbred white mice (in vivo).

The results obtained indicate interferon-inducing activity in vitro and in vivo in the drug Argotiazine.

As a result of the work, a non-toxic dose of the drug was found for the transplanted cell line SPEV (transplanted cell line of pig kidney embryos), FEC (chicken embryo fibroblasts) in vitro. This dose showed pronounced virus-inhibiting properties and completely blocked the multiplication of the virus in cell culture.

Studies have shown that the drug also has immunomodulatory properties, induces the formation of interferon in vitro and in vivo. Thus, in the inventive preparation combines both antiviral and immunomodulating properties. The use of this drug as the basis of antiviral and immunomodulating drugs will make it possible to obtain new effective drugs with a different mechanism of action compared to the currently used drugs.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 presents data on the activity of interferon in samples of supernatants of cell culture induced by various doses of the drug (1 - before drug administration; 2 - after 6 hours and 3 - 24 hours after drug administration).

Figure 2 presents data on the activity of interferon in serum samples of nonlinear white mice induced by different doses of the drug (4 - 10 μg / kg body weight; 5 - 100 μg / kg body weight; 6 - control).

Claims (2)

1. The method of obtaining a water-soluble agent with antiviral and immunomodulating activity, which consists in the fact that the synthesis is carried out by heating to 90-95 ° C by mixing the following components: methylene blue-silver chloride-ammonia in a molar ratio of 1.06: 1: 58, in this case, methylene blue is first dissolved in water, a pre-prepared solution of the ammonia complex of silver chloride is added to the resulting solution, obtained by mixing calculated amounts of a suspension of silver chloride and a 25% aqueous solution of ammonia ka, the reaction mixture was heated to 90-95 ° C and held for 1 h, the reaction mixture was cooled, the by-product was filtered off, evaporated in vacuo, acetone was added to the solid residue and triturated to a homogeneous state, the precipitate was filtered, washed with acetone and dried to constant weight. vacuum at room temperature. 2. A water-soluble agent with antiviral and immunomodulating activity, based on a compound of ionic silver with methylene blue, obtained by the method according to claim 1 and corresponding to the composition C ₁₆ H ₁₈ Cl ₂ N ₃ SAg.

Images