WATER-SOLUBLE ANTIVIRAL AGENT BASED ON SILVER-CISTINE COMPOUND AND METHOD OF PREPARING THEREOF

The invention relates to water-soluble antiviral agents, to the field of pharmaceutical chemistry and medicine. The invention also relates to the field of preparing such agents. The invention may be used for preparing drug dosage forms for the treatment of viral, bacterial and other infections.

The object of the invention is the provision of effective antiviral pharmaceutical preparations, in particular, for the treatment of viral hepatitis, antibiotic-resistant infections, including HIV infection.

Essence of the invention. A new antiviral compound based on silver and a natural amino acid L-cistine of the formula C ₆ H ₁₉ N ₄ O ₆ S ₂ LiAg ₂ is proposed (Table 1).

There is known a preparation "Argochrom" of the firm Merck (produced in Germany approximately from 1916 to 1945 and imported into the USSR in the pre-war period), which composition falls within an Austrian patent No 69476 being in force from 1914 and up to 1920 called in German "Silber methylene blau", as well as a preparation "Gegonon" produced according to the German patent No. 268968, published on 07 January, 1914, class 12p, group 16 - a prototype (see reference book BoTπaji B.E. H pp. «φapMa^BTHHecκHe πpeπapaτi>i», M.-JL: OHTH, 1934).

The above-mentioned preparations comprise silver in ionic form firmly bonded to organic molecules — ligands. The drawback of these preparations is a poor bioavailability of silver contained in them, since in the case of "Argochrom" the ligand is a synthetic dye - methylene blue, and in the case of "Gegonon" — a high molecular carrier — albumose.

It is an object of the present invention to improve the efficiency of the penetration of silver ions through biological membranes, beginning with the penetration into bloodstream in the case oral or rectal mode of administration, and also the effective delivery of silver ions to diseased cells and penetration through the cell membranes for the purpose of ceasing the vital activity of infectious agents.

The problem is solved by means of a water-soluble, photostable in solutions silver preparation representing a strong complex of silver and a natural amino acid (L-cistine), wherein one cistine molecule binds two silver ions.

Also a method of preparing the new water-soluble antiviral pharmaceutical agent (substance) is proposed, wherein the synthesis is carried out at room temperature and a molar ratio of cistine, lithium hydroxide, silver nitrate and ammonia of 1:2:2:8. Firstly, cistine is dissolved in water and transformed into the lithium salt by adding lithium hydroxide. Then the ammonia complex of silver nitrate [Ag(NH ₃ ) ₂ ]NO ₃ previously obtained by mixing the calculated amounts of silver nitrate solution and 25% aqueous ammonia solution is added, and the reaction mixture is held at room temperature for 6 hours, then evaporated, in vacuum up to ¹ A of the initial volume at a temperature of 40-50°C, during which an intense precipitation of the target compound in the form of yellow precipitate takes place. The obtained suspension is mixed with an equivalent volume of ethanol, which is added potionwise by four equal portions, the suspension is cooled up to +4 - +6°C and held for 12 hours. The obtained yellow precipitate is filtered, washed with ethanol and dried under vacuum at room temperature until a constant weight.

The proposed structural formula corresponding to the empirical formula C ₆ Hi ₉ N ₄ O ₆ S ₂ LiAg ₂ .

Example of carrying out the method.

Cystine (24.00 g), lithium hydroxide monohydrate (8.4Og) and distilled water (100 ml) are placed into a round-bottom flask of volume 1 liter. The reaction mixture is stirred with a magnetic stirrer at room temperature until starting compounds are completely dissolved.

Silver nitrate (33.97 g) and distilled water (50 ml) are placed into a glass of volume 300 ml. Once the salt is dissolved, 54.4 ml of 25% aqueous ammonia is added under vigorous stirring.

The solution of ammonia complex of silver nitrate is poured under stirring with a magnetic stirrer at room temperature to the homogeneous solution of cystine dilithium salt. The reaction mixture gradually turns yellow; the color becomes stronger during 1-2 hours. After 6 hours the reaction mixture is evaporated on rotary evaporator at a temperature of 40- 5O ⁰ C up to volume of 50 ml. During evaporation an intensive precipitation of a fine yellow precipitate takes place. The suspension is then placed into a glass of volume 500 ml, and 200 ml of ethanol portionwise by 50 ml is added during 30 minutes under vigorous stirring with a mechanical stirrer. The suspension is stirred for 30 minutes and then placed into a refrigerator (+4 - +6 ⁰ C) for 12 hours.

The obtained yellow precipitate is filtered, thoroughly washed with 300 ml of ethanol and dried in vacuum at room temperature until a constant weight.

The elemental analysis data given in Table 1 are obtained using automatic element composition analyzer. The metal content is determined by the atomic adsorption method.

The results of laboratory studies on biological activity.

The laboratory studies on cytotoxicity and antiviral activity of the claimed silver- cistine complex under working title "Argobiocin-2S" were carried out.

The determination of cytotoxicity was carried out in accordance with «MeτoflHHecκκe peKOMeimaijHH no Hcnojπ>3θBaHHio κyjπ>τyp κjieτoκ M^BK H KCT λJW ouemcH AHTOTOKCHHecKoro fleiicTBHa JieκapcτBeHHBix cpeλCTB» (C.F.KoJiecHHKOBa H jψ., 2002).

All experiments were repeated three times. The statistical treatment was carried out by conventional methods (H.lT.AπiMapHH H flp., 1974).

The determination of cytotoxicity for cell culture.

For the determination of cytotoxicity the test preparations were introduced into a two- day monolayer of cell culture of cow embryo coronary vessels (CECV) grown by micromethod on a 96-well microtiter plate in the following doses: 10 mg/ml; 1 mg/ml; 100 μg/ml; 10 μg/ml; 1 μg/ml; 0.1 μg/ml; 0.001 μg/ml. Four wells with culture monolayer were used for one dilution of each preparation. The cytotoxic action of the preparation on cells were determined by the degree of monolayer change and controlled for 3 days. The results are given in Table 2.

The toxic dose of the preparation "Argobiocin 2S" is 10 mg/ml. As a result of the investigation it was established that a cytotoxic dose of the preparation "Argobiocin 2S" for CECV cell culture is 10 mg/ml. Non-toxic dose of "Argobiocin 2S" for CECV cell culture is 1 mg/ml.

For the determination of minimal cytotoxic dose the tested preparation was introduced into a two-day monolayer of CECV cell culture grown by micromethod on a 96-well microtiter plate in the following doses: 10 mg/ml; 5 mg/ml; 2.5 mg/ml; 1.25 mg/ml. Four wells with a culture monolayer were used for one dilution of each preparation. The cytotoxic action of the preparation were determined by the degree of monolayer change and controlled for 3 days. The tests were repeated three times. The results are given in Table 3.

The minimal cytotoxic dose of the preparation "Argobiocin 2S" for CECV cell culture is 6.67± 1.36 mg/ml, and the maximal non-toxic dose - is 3.33±0.68 mg/ml.

Hence, the maximal cytotoxic dose of the preparation "Argobiocin 2S" for CECV cell culture is 10 mg/ml, and a minimal dose - 6.67± 1.36 mg/ml. The maximal non-toxic dose of "Argobiocin 2S" preparation for CECV cell culture is 3.33±0.68 mg/ml.

The determination of antiviral activity of the preparations against bovine viral diarrhea (mucosal disease) virus (BVDV) was carried out as follows. The monolayer of the cell culture in use was infected by BVDV in a dose of not less than 1-10 TDC/cell; 1.5 hour later cells were washed with a nutrient medium without serum and the preparations in maximal nontoxic for CECV cell culture doses (test) or a dilution nutrient medium (control) were introduced. After 72 hours of cultivation of the virus in such biosystems, the culture liquid was titrated.

The antiviral effect of the preparations was calculated based on the difference in infectious activity of viruses in control and test samples (Bapycojiorna. MeτoflBi τiop, pefl B. MeiixH, M.: Mπp, 1988). The antiviral effect of preparations was expressed in Ig TCD _SO AU mi of the reduction in virus titers H Ap. Boπpoc&i BHpycojioriϊH, 1986, ¹ T).

The preparation was considered effective, when the infectious titer of the virus reduces in at least 2 Ig TCDso _/ cu _m i (100 times) as compared to the control (cell cultures not treated with the preparations). In each test experiment the additional control of the toxicity of the tested preparation dose was carried out.

The titration of viruses was carried out as follows. The determination of infectious titer of BVDV was carried out by micromethod (BnpycojiorHH. MeTOflH. no^ pe^ B. Meπxπ, M.: Mπp, 1988) in on a 96-well microtiter plate with CECV cell culture using at least 4 parallel series. The infectious titer of the virus was expressed in TCD _50A u _m i (50% tissue cytopathogenic dose). The results of the investigations are given in Table 4.

As a result of the investigation is it established that the preparation "Argobiocin 2S" exhibits antiviral action by reducing the infectious activity of BVDV by 2.01 Ig TCD ₅ o _/ o.i _m i as compared to the control.

As a result of the investigation, the nontoxic dose of the preparation in vitro for CECV cell culture on which the BVDV was cultivated was determined. This dose exhibits pronounced antiviral properties by reducing the infectious activity of the virus a 100 times. Hence, the use of this preparation as a base for antiviral pharmaceutical agents will make it possible to prepare new effective pharmaceutical agents having different mechanism of action

as compared to the presently used pharmaceutical preparations, which do not contained metal ions in their structure.