RU2573977C9 - 4,6-di(3,12-diaza-6,9-diazoniadispiro[5,2,5,2]hexadecan-1-yl)-2-methyl-5-nitropyrimidine tetrachloride hydrochloride hexahydarate for treating herpetic infection, pharmaceutical composition for local application - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and represents a pharmaceutical composition in the form of a cream or gel for treating a herpes viral infection, containing 4,6-di(3,12-diaza-6,9-diazoniadispiro[5,2,5,2]hexadecan-1-yl)-2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate. The invention consists in a method of treating or preventing a herpetic infection in a human or animal.

EFFECT: providing a pharmaceutical composition of 4,6-di(3,12-diaza-6,9-diazoniadispiro[5,2,5,2]hexadecan-1-yl)-2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate in the form of a dosage form for local application possessing a high level of therapeutic efficacy.

4 cl, 2 dwg, 2 tbl, 5 ex

Description

The invention relates to the use of 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyryllidine tetrachloride dihydrochloride hexahydrate having activity against the herpes virus , for the prevention and treatment of herpetic infection in humans and animals using a local method of application.

Despite numerous studies in the field of virology, the problems of herpes virus infections (GVI), especially those associated with the herpes simplex virus (Herpes simplex) type 1 and 2 (HSV-1 and HSV-2), do not lose their relevance and medical and social significance . In the structure of human infectious-viral lesions, GVI occupy the 2nd place, second only to influenza. GVI can affect almost all organs and systems of a macroorganism, causing latent, acute and chronic infections.

The data on the prevalence of BBVI presented in the literature vary significantly, which is most likely due to differences in the demographic and clinical characteristics of the studied groups of patients used by diagnostic methods. On the one hand, according to a number of researchers, GVI is clinically manifested in 20-25% of infected people, on the other hand, the results of scientific studies show that the clinical symptoms of GVI are manifested in 60-70% of cases. In the Russian Federation, large-scale epidemiological studies of the prevalence of HBV have not been conducted. However, the data of a sample epidemiological study among people in the age group from 14 to 64 years indicate that the prevalence of HSV type 1 reaches 99.4% [Khryanin A.A. et al. Long-term trends in the prevalence of the herpes simplex virus in Siberia. To help a practitioner. 2010. No. 5] Official statistics on the prevalence of HBV caused by HSV-2 have been recorded in Russia only since 1993. The results of the analysis of the data of the annual state statistical observation indicate that against the background of a decrease in the incidence of sexually transmitted infections (STIs), there is an increase in the incidence of HBV of the urogenital tract. The same situation, according to WHO, is also observed for causes of death from viral infections: 35.8% - influenza virus and 15.8% - HSV. According to various estimates, up to 98 million cases of labial herpes and 20 million cases of recurrent genital herpes, 500 thousand cases of ophthalmic herpes and more than 5 thousand cases of herpes encephalitis, about 1 thousand cases of herpes simplex are registered in the United States annually. Compared with the 80s, by the end of the millennium, the number of registered herpes patients increased in the United States by 13-40%, in Europe by 7-16%, and in Africa by 30-40%. A comparative analysis of the NHANES I and NHANES II data showed that the incidence increased in equal proportions in all age groups.

Currently, the following groups of antiherpetic drugs are known [De Clercq E. The discovery of antiviral agents: Ten different compounds, ten different stories. Med Res Rev 2008; 28: 929-953; De Clercq E. Antiviral drug discovery: Ten more compounds, and ten more stories (part B). Med Res Rev 2009; 29: 571-610; De Clercq E. Another ten stories in antiviral drug discovery (part C): "Old" and "new" antivirals, strategies and perspectives. Med Res Rev 2009; 29: 611-645; De Clercq E. Yet another ten stories on antiviral drug discovery (part D): Paradigms, paradoxes and paraductions. Med Res Rev 2010; 30: 667-707; De Clercq E. The next ten stories on antiviral drug discovery (part E): Advents, advances andadventures. Med Res Rev 2011; 31: 118-160]: a class of abnormal nucleosides, derivatives of phosphonoacetic and phosphonoformic acids, derivatives of adamantane hydrochloride, heterocyclic derivatives, herbal preparations, interferon and its inducers, vaccines.

The class of abnormal nucleosides is the most studied and extensive class of antiherpetic compounds and includes pyrimidine analogs of nucleosides, which include idoxuridine (IMU) for chemotherapy of herpes virus infections caused by HSV in ophthalmology (for the treatment and prevention of herpetic keratitis); purine nucleoside analogues, with the most famous representative Vidarabine, which was recommended for systemic use in herpes encephalitis, however, lost its importance due to its high toxicity: in high doses, it had a carcinogenic, mutagenic and teratogenic effect; derivatives of nitrogen heterocycles - this group includes the chemotherapy drug virazole (ribavirin) [Smith DB, Martin JA, Klumpp K, Baker SJ, Blomgren PA, Devos R, Granycome C, Hang J, Hobbs CJ, Jiang WR, Laxton C, Le Pogam S , Leveque V, Ma H, Maile G, Merrett JH, Pichota A, Sarma K, Smith M, Swallow S, Symons J, Vesey D, Najera I, Cammack N. Design, synthesis, and antiviral properties of 4_-substituted ribonucleosides as inhibitors of hepatitis C virus replication: The discovery of R1479. Bioorg Med Chem Lett 2007; 17: 2570-2576] - active against DNA and RNA-containing viruses (influenza A and B viruses, parainfluenza, measles, HSV types I and II, varicella zoster, etc.). It has a number of serious toxic effects: it has a negative effect on the bone marrow.

Acyclic analogues of purine nucleosides - the most prominent representative is acyclovir and its derivatives valaciclovir, penciclovir, famciclovir, ganciclovir, cidofovir. This is a class of compounds, analogues of guanosine and adenosine, in which a fragment of the nucleic base is stored. Antiherpetic drugs of this class (for local and systemic action) are highly specific and low toxic. In an infected cell, in the process of copying maternal viral DNA, acyclovir after three-step phosphorylation performed in the first stage by viral thymidine kinase (monophosphorylation), and in the second and third stages of cell thymidine kinase, is picked up by viral DNA polymerase and integrates into the chain of DNA under construction for daughter virions. Due to the fact that acyclovir lacks, unlike natural guanosine, the 3-hydroxyl group necessary to attach the next nucleoside to it, when it is included in DNA synthesis early, this process breaks off at the very beginning, the assembly of daughter viral DNA stops and new viral particles simply do not form. It is very important that, when included in any part of the new DNA, acyclovir is never replaced by natural guanosine. The use of nucleosides interrupts the replication of herpes viruses at any stage, new virus generations are not formed, and the actual spread of the infection is stopped. The limited number of herpes virus antigens remaining in the body allows the immune system to understand their types and repertoire without interference, which leads to the formation of full-fledged immunity in all people with a normally functioning immune system. Other acyclic nucleosides [Sofia MJ, Bao D, Chang W, Du J, Nagarathnam D, Rachakonda S, Reddy PG, Ross BS, Wang P, Zhang HR, Bansal S, Espiritu C, Keilman M, Lam AM, Steuer HM, Niu C, Otto MJ, Furman PA. Discovery of a-d-2-deoxy-2-fluoro-2-C-methyluridine nucleotide prodrug (PSI-7977) for the treatment of hepatitis C virus. J Med Chem 2010; 53: 7202-7218; Furman PA, Murakami E, Niu C, Lam AM, Espiritu C, Bansal S, Bao H, Tolstykh T, Micolochick Steuer H, Keilman M, Zennou V, Bourne N, Veselenak RL, Chang W, Ross BS, Du J, Otto MJ, Sofia MJ. Activity and themetabolic activation pathway of the potent and selective hepatitis C virus pronucleotide inhibitor PSI-353661. Antiviral Res 2011; 91: 120-132; Reddy PG, Chun BK, Zhang HR, Rachakonda S, Ross BS, Sofia MJ. Stereoselective synthesis of PSI-352938: A.-D-2-deoxy-2-fluoro-2-C-methyl-3,5-cyclic phosphate nucleotide prodrug for the treatment of HCV. J Org Chem 2011; 76: 3782-3790; Lam AM, Espiritu C, Murakami E, Zennou V, Bansal S, Micolochick Steuer HM, Niu C, Keilman M, Bao H, Bourne N, Veselenak RL, Reddy PG, Chang W, Du J, Nagarathnam D, Sofia MJ, Otto MJ, Furman PA. Inhibition of hepatitis C virus replicon RNA synthesis by PSI-352938, a cyclic phosphate prodrug of D-2-deoxy-2-fluoro-2-C-methylguanosine. Antimicrob Agents Chemother 2011; 55: 2566-2575] use the same principle of action, but are focused on other enzyme groups of herpes viruses. However, it turned out that some chemotherapeutic drugs have strong side effects and unsatisfactory pharmacokinetic properties, which limits the indications and / or the possibility of their use either by age or by clinical criteria.

Derivatives of pyrimidinedispirotipiperazinium (4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate) detected as a result of target -directional search for new drugs, have the ability to specifically block heparan sulfate (HS) containing receptors on the cell wall and thus prevent the adsorption of viruses to host cells. The most famous pathogenic viruses using such a mechanism of adsorption to the cell wall are herpes viruses type 1 and 2, papillomas of the virus, cytomegalovirus, some types of HIV, respiratory syncytial virus and others.

The mechanism of action of diazoniadispiro [5.2.5.2] hexadecane derivatives was studied using the herpes type 1 virus (HSV-1) and green monkey kidney cells as an example, and it was proved to be associated with the specific property of the compounds to bind to HS, which leads to a dramatic decrease virus replication counts. It has been shown that the addition of the test substances is antagonized by heparin. The target of diazoniadispiro [5.2.5.2] hexadecanes is two sulfate groups located in neighboring saccharide residues, for example, for GlcA2S-GlcNS6S, GlcA2S-GlcNS3S, IdoA2S-GIcNAc6S, IdoA2S-GlcNDSNS2S2S2S2S2S2S2S2S2S2S2S2S2 negative charge on the sulfate group and positively charged nitrogen atoms of 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate. It was also shown that a similar kind of interaction can occur with the carbonyl group of the ΔUA-GlcNSldoUA2S-GlcNAc-UA2S-GlcNS-IdoUA2S-GlcNH23S octasaccharide, which is the necessary HS site for HSV-1 to enter the host cell. Thus, diazoniadispiro [5.2.5.2] hexadecans block the key functional groups of HS, preventing the replication of the virus, providing high antiviral activity [Schmidtke M., Riabova O., Dahse N.-M., Stelzner A., Makarov V., Synthesis, Cytotoxicity and Antiviral Activity of N, N′-bis-5-nitropyrimidyl Derivatives of Dispirotripiperazrazine, Antiviral Research, 2002, 55, 117-127; Schmidtke M., Karger A., Meerbach A., Egerer R., Stelzner A., Makarov V., Binding of a N, N′-bisheteryl derivative of dispirotripiperazine to heparan sulfate residues on the cell surface specifically prevents infection of viruses from different families, Virology, 2003, V. 311, p. 134-143; Schmidtke, M., Wutzler, P., Makarov, V. Novel opportunities to study and block interactions between viruses and cell surface heparan sulfates, Lett. Drug Design Discov., 2004, 1, p. 293-300].

With systemic, mainly oral administration of an antiherpetic agent, long-term treatment is required for treatment, as well as the administration of high doses (up to 1000 mg / day) of an antiviral agent. The disadvantage of local dosage forms, such as gels, creams, solutions, lotions, varnishes, etc., is that the pharmaceutically active agent is not in close contact with the affected area. The correct conduct of complex treatment with the use of antiviral drugs that differ in the mechanism and spectrum of action is one of the most important tasks associated with herpetic human pathology.

The objective of the present invention was to find a dosage form containing 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate , which would have high activity against herpes infection, including strains resistant to currently existing drugs. The result of solving this problem was the development of a cream containing 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, which has antiherpetic activity.

This task and result are achieved through the use of a pharmaceutical composition for the treatment of herpes virus infection in the form of a cream containing 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane-1 as an active substance -yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate from 0.5 to 10.0%. Another option provides that the claimed pharmaceutical composition for the treatment of herpes virus infection in the form of a gel containing 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl as the active substance ) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate from 0.5 to 10.0%. The claimed use of a pharmaceutical composition in the form of the above cream, according to which the cream has anti-herpes activity against strains of HSV-1 and HSV-2, including those resistant to currently existing drugs.

Also claimed is a method of treating or preventing a herpes infection in a human or animal, comprising topically administering to a patient a pharmaceutical composition according to claim 1 and / or claim 2 in an effective amount.

Administration of 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate in the form of a pharmaceutical composition or a medicine based on it in the treatment or prophylaxis of humans or animals, it can be carried out by conventional methods in the form of forms suitable for external use, for example in the form of ointments, creams, pastes, lotions and linimentes placed in a package, or plasters.

As pharmaceutically acceptable excipients in the pharmaceutical composition according to the invention, any pharmaceutically acceptable components that are compatible with the active ingredient can be used, do not harm patients and are traditionally used for the preparation of dosage forms. These include, for example, fillers, binders, plasticizing agents such as camphor, triphenyl phosphate or pentanyl diisobutyrate, diisobutyl phthalate, etc., pigments, solubilizing agents, stabilizers, diluents, adjuvants, preservatives, components of buffer systems, solvents, dispersing agents, preservatives, thickeners, food coloring, emulsifiers, film-forming substances, such as hydroxyethyl or propyl cellulose, hydroxypropylchitosan, prolongators, substances that improve the penetration of drugs a pharmaceutical agent, for example benzyl alcohol, etc. Pharmaceutically acceptable excipients are, for example, gum arabic, polyvinyl pyrrolidone, polyvinyl alcohol, alginic acid, sodium alginate, anhydrous silicic acid, magnesium stearate, talc, carboxyvinyl polymer, titanium oxide and ether fat sorbitol, sodium lauryl sulfate, glycerin, glycerol fatty acid ester, lanolin, glycerol gelatin, polysorbate, macrogol, vegetable oil, wax, paraffins, propylene glycol, polyethylene glycol, water, this nol, polyalcohols, polyoxyethylene hydrogenated castor oil, sodium chloride, sodium hydroxide, hydrochloric acid, dibasic sodium phosphate, monobasic sodium phosphate, citric acid, glutamic acid, benzyl alcohol, methyl p-hydroxybenzoate, ethyl p-hydroxybenzoate, etc.

The topical composition may also contain as an adjuvant vegetable oil such as jojoba oil, olive oil, sunflower oil, soybean oil, peanut oil, rapeseed oil, almond oil, palm oil, coconut oil or palm kernel oil. The composition may contain as emulsifiers, for example, glycerin, possibly mixed with sucrose laurates or sorbitol, an M / B emulsifier (oil / water), for example, a vegetable protein hydrolyzate.

The composition can also be used in the form of liniment (lat. Linimentum) - a thick or gelatinous dosage form for external use, which is rubbed into the skin. As auxiliary substances, liniment may contain, for example, propylene glycols, benzalkonium chloride. Emulsion fat liniments may have an oil-in-water or water-in-oil emulsion structure. Emulsion liniments, as well as suspension ones, need stabilization, which can be increased with an emulsifier, for example, T-2 emulsifier (T-2 emulsifier is a fatty acid ester of glycerol polymer), Tween 80 and other surfactants. In some cases, emulsifiers can be formed as a result of the interaction of the ingredients that make up the liniment.

Pharmaceutically acceptable excipients for the preparation of a pharmaceutical composition for external use are, for example, natural or rejected oils, for example cocoa butter, waxes, fats, glycerol and saturated fatty acid esters, glycerogelatin, macrogol, semi-liquid or liquid polyols, triglycerides, etc. Pharmaceutical base compositions for external use may also include a surfactant or stabilizer.

Brief Description of the Drawings

In FIG. Figure 1 shows the course of an experimental herpes virus infection in guinea pigs during treatment with cream when applied topically.

In FIG. Figure 2 shows the course of experimental herpes virus infection in guinea pigs during treatment with a gel preparation for topical application.

The implementation of the invention

The compound 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate can be prepared according to the following synthesis protocol.

Obtaining N-benzoylpiperazine hydrochloride

45 ml of acetic acid are added to 10 g (0.116 mol) of anhydrous piperazine under cooling and vigorous stirring. Then add 20 ml of acetone and 20 ml of water. After 20 minutes of stirring, 6.7 ml (0.058 mol) of benzoyl chloride are added. The solution was stirred for 3 hours. Then the reaction mass was evaporated by half. 450 ml of a 40% NaOH solution are added. The precipitate formed is filtered off, the aqueous mother liquor is extracted with chloroform 3 times, 100 ml each. Dried. It is evaporated to dryness. 13.4 g of a yellow oil are obtained containing about 60% of the basic substance.

The resulting oil is dissolved in 5 ml of water, the solution is heated to 45 ° C and 0.78 ml of formalin is added in 0.25 ml portions every 10 minutes with stirring. After the addition of formalin was stirred for 40 min at 55 ° C. It is cooled to 7 ° C and maintained at this temperature for 1.5 hours. The precipitate is filtered off and washed with water 3 times. Dried at 80 ° C for 15 hours. 6.7 g of methylene bis-4 (1-benzoylpiperazine) are obtained. Before the next step, the product is ground. _Mp = 125-127 ° C.

To a suspension of 6.6 g of crushed methylene bis-4 (1-benzoylpiperazine) in 35 ml of ethyl alcohol, with intensive stirring and cooling to 10-15 ° C, a 12% alcohol solution of hydrogen chloride was added dropwise to pH = 1-2. Then stand the reaction mass for 3 hours at 10 ° C. The precipitate is filtered off and washed with 50 ml of ethanol 4 times. Dried at 80 ° C for 10 hours. 5.9 g of product are obtained. _Mp = 274 ° C with decomposition. Preparation of N-benzoyl-N ′ - (2-hydroxyethyl) piperazine hydrochloride.

To a solution of KOH (0.024 mol) in 14 ml of ethyl alcohol, N-benzoylpiperazine hydrochloride (0.02 mol) was added with stirring and stirred for 1 hour. Ethylene chlorohydrin (0.05 mol) is added to the resulting suspension and a solution of KOH (0.052 mol) in 25 ml of ethanol is added dropwise. In this case, the temperature in the reaction mass is maintained no more than 20 ° C. After adding a solution of KOH, the reaction mass is stirred for 20 hours. KCl is filtered off, a 12% alcoholic solution of hydrogen chloride is added to the mother liquor to pH = 1-2 and cooled at -5 ° C for 5 hours. The precipitate formed is filtered off and washed with ethyl alcohol. Dried at 50 ° C for 5 hours. The product yield is 70%. _Mp = 207-210 ° C.

Obtaining N-benzoyl-N ′ - (2-chloroethyl) piperazine hydrochloride

To a suspension of N-benzoyl-N ′ - (2-hydroxyethyl) piperazine hydrochloride (0.013 mol) in 22 ml of chloroform, thionyl chloride (0.028 mol) is added dropwise with stirring and slowly heated. Stand 30 minutes at 45 ° C, 30 minutes at 45 ° C and 3 hours at 70 ° C. This precipitates first in the form of oil, and then solidifies into a crystalline mass. The reaction mass is cooled to 6-8 ° C and stirred at this temperature for 1 hour. The precipitate is filtered off, washed with chloroform 3 times in 10 ml. Dried at 70 ° C for 10 hours. Product yield 72%. _Mp = 226-227 ° C.

Obtaining dihydrochloride N, N-dibenzoyl-N ′, N ′ ′ - dispirotripiperazinia

N-benzoyl-N ′ - (2-hydroxyethyl) piperazine hydrochloride (0.0156 mol) is added to a solution of NaOH (0.017 mol) in 25 ml of ethyl alcohol. Stir the suspension for 1.5 hours. NaCl is filtered off. The mother liquor is boiled for 1 hour. Then it is evaporated and the dry residue is heated in an oil bath for 10 hours. The temperature of the bath is 120 ° C. It is cooled to room temperature, ethyl alcohol is added and the precipitate is filtered off. Washed with ethyl alcohol. Dried at 100 ° C for 3 hours. Product yield 60%. _Mp over 300 ° C with decomposition.

Preparation of 3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane dichloride dihydrochloride

A mixture of dihydrochloride N, N ″ ″ - dibenzoyl-N ’, N ″ - dispirotripiperazinia (0.002 mol) and 7 ml of 10% hydrochloric acid are boiled for 4 hours. It is cooled to 15 ° C and the benzoic acid is filtered off, washed with water. The mother liquor is evaporated to dryness, methyl alcohol is added. The precipitate was filtered off and washed with methanol. Dried at 90 ° C for 5 hours. Product yield 85%. _Mp over 300 ° C with decomposition.

Preparation of 3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane dichloride

To a suspension of 3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane dihydrochloride dichloride (0.0017 mol) in 3 ml of water, LiOH (0.0033 mol) is added. At the same time control pH = 8.5-9. After 30 minutes, activated carbon is added to the reaction mass, and stirred for 20 minutes. Charcoal is filtered off, 25 ml of methyl alcohol are added to the filtrate and left for 16 hours at 5-10 ° C. The precipitate formed is filtered off, washed with methanol and dried at 90 ° C for 3 hours. The yield is 90%. _Mp over 320 ° C with decomposition.

Preparation of 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine dihydrochloride tetrachloride

To a solution of 4,6-dichloro-2-methyl-5-nitropyrimidine (0.12 mol) in 940 ml of ethyl alcohol is added a solution of 3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane dichloride (0.24 mol) in 220 ml of water with vigorous stirring. The suspension is heated for 4 hours at 70 ° C. It is cooled to room temperature, the precipitate is filtered off, washed with ethanol. It is dried at 110 ° C for 18 hours and then in air for 4 days. The yield of 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine dihydrochloride tetrachloride is 87%.

_Mp = 216-220 ° C with decomposition.

MS (m / z): 694 (M +) C ₂₉ H ₅₃ Cl ₃ N ₁₁ O ₂ (M +)

Microelement analysis,%

Calculated: C ₂₉ H ₆₇ Cl ₆ N ₁₁ O ₈ : C, 38.25; H, 7.42; Cl 23.36; N, 16.92

Found: C, 38.44; H, 7.38; Cl 22.98; N, 16.78

Fisher water analysis

Calculated: 11.86%

Found: 11.72%

Examples of the preparation of pharmaceutical compositions in the form of various forms suitable for use as an external medicine and containing 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] -hexadecan-1-yl) -2 methyl 5-nitropyrimidine tetrachloride dihydrochloride

Example 1. Preparation of a pharmaceutical composition in the form of a cream

Active ingredient 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate 10.0 g Petroleum jelly 10.0 g Poloxamer (Emuxol 268) 8.0 g Emulsion Wax 3.0 g Sorbitan Monostearate (Span-60) 0.5 g Sorbic acid 0.125 g Purified water up to 100.0 ml

The active substance, poloxamer and sorbic acid are dissolved in 50 ml of water, emulsion wax and sorbitan monostearate are added with vigorous stirring, then they are kept. A cream is obtained, which can then be used as an antiviral agent.

Example 2. The pharmaceutical composition in the form of a gel

Compound 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate 10.0 g Cellulose-35 7.5 g Sorbic acid 0.150 g Water up to 100.0 g

The active substance is dissolved in 50 ml of water, methylcellulose-35 and sorbic acid are added and the remaining water is added with vigorous stirring and then allowed to swell. Get a gel, which can then be used as an antiviral agent.

Example 3. Preparation of a pharmaceutical composition in the form of an ointment

Ointment can be made as follows: 0.5-3% weight. from the total mass of the active substance is melted in a small amount of ointment, cream or candle base. The resulting melt is thoroughly mixed with the required amount of base, heated to (35-80) ° C. The resulting mixture is homogenized. The melted mixture is poured into tubes.

Example 4. Preparation of a pharmaceutical composition in the form of liniment

Liniment is obtained in the form of a 5% transparent yellow liquid with a faint specific odor, containing: 1-2 mg of the active compound of formula (I), 50 mg of benzalkonium chloride and 1,2-propylene glycol. Propylene glycol is used as a moisturizer.

Example 5. The pharmaceutical composition in the form of a fat emulsion liniment

3 g of active substance, colloidal silicon dioxide (Aerosil) 5 g, castor oil 89 g. It is possible to add an emulsifier, for example T-2 or Tween 80 emulsifier.

Description of antiviral activity of the cream of the invention

MATERIALS AND METHODS

Study drugs

In the studies, 10% cream (Example A) and 10% gel (Example B) were used, prepared on the basis of the substance of the compound 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane-1- il) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, further substances.

Acyclovir (9- (2-hydroxyethoxymethyl) guanine), a topical cream preparation Zovirax containing 5% acyclovir (GlaxoSmithKline, GlaxoSmithKline Consumer Helsker, Great Britain) was used as a comparison drug. As a placebo in experiments with guinea pigs, a cream and gel of the same composition were used without the substance of the active substance.

Animals

Male guinea pigs weighing 250-300 g were obtained from the nursery RAMS "Andreevka" (Moscow region). The keeping of animals corresponded to the rules for the design, equipment and maintenance of experimental biological clinics (vivariums). The animals were fed with briquetted feed in accordance with the approved standards. Marking of animals in groups was carried out using staining with dyes (eosin) of the body surface. The studies were conducted in accordance with ethical regulatory documents in force on the territory of the Russian Federation, after the approval of the Ethics Committee of the FSBI NIIIVS im. I.I. Mechnikov, as well as taking into account the recommendations of international licensing authorities (1-4).

Viruses and cells

Cell culture. We used a transplantable Vero green monkey kidney cell culture obtained from ATTS (American Collection of Cell Cultures and Viruses). Cells were cultured in growth medium, which was DMEM (Minimum IgLA medium, PanEco, Russia) supplemented with 10% heat inactivated calf serum (ETS, PanEco, Russia), 2 mM L-glutamine (Sigma, USA) and antibiotics (100 U / ml penicillin and 100 μg / ml streptomycin). The support medium contained all of the above ingredients and 2% ETS. Cells were incubated in a thermostat in an atmosphere of 5% CO ₂ at + 37 ° C.

Viruses. The experiments used the herpes simplex virus HSV-2 antigenic type, strain BH, obtained from the State collection of viruses of the Research Institute of Virology. DI. Ivanovsky. The virus was maintained in serial passages and titrated on a transplanted culture of green monkey kidney cells VERO. Virus titers were evaluated according to standard methods (according to Reed and Mench) using a micromethod in 96-well culture plates using cell culture and expressed in Ig TCD ₅₀ / ml. Minimal virus dilution was taken for TCD ₅₀ of the virus, causing 50% damage to the cell monolayer in the absence of degeneration of the cell monolayer in the control without infection (50% tissue cytopathic dose of TCD ₅₀ virus). In the experiments, a virus was used in the form of a virus-containing suspension with an infectious titer of 6.0 Ig TCD ₅₀ / ml. The resulting viruses were stored in aliquots frozen.

Evaluation of the effectiveness of drugs on the model of genital herpes

Male guinea pigs were infected with culture, virus-containing liquid (HSV-2, "VN"). Virus-containing liquid (at a dose of 100 TCD ₅₀ ) was applied using a pipette (followed by rubbing) onto the previously scarified penis skin. Scarification was performed using a surgical lancet after the animals were anesthetized with anesthetic lidocaine (1%). The size of the scarification area was 4-7 mm ² . Each group contained 4-5 animals.

A placebo was also used in the experiments, representing a cream and gel of the same composition, only without the addition of the substance PDSTP. In addition, as a control preparation, a dosage form in the form of a 5% ointment of Zovirax (acyclovir) was used. Treatment was carried out locally according to the treatment regimen: 3 or 48 hours after infection (with severe manifestations of the disease). The preparations were applied by thin-layer application on the lesions 2 times a day, daily, for 5 days. The area of application of the drug was 1.3 cm ² .

The severity of the infection process was assessed daily before treatment and was monitored throughout the period of the disease according to the following parameters: the presence and degree of specific lesions (vesicles, pustules, ulcerations, erosion, crusts); edema, hyperemia, orchitis. The maximum severity of each trait was 4 points. The total duration of observation of animals was 21 days.

The effectiveness of the drugs was evaluated at the peak of the severity of the pathological process according to standard methods. The criteria for evaluating the therapeutic effect of the drugs were: a decrease in the intensity of clinical manifestations, an index of therapeutic effect (IPD), a reduction in the duration of the disease in the experimental groups compared to the control.

ILD (in%) = (total score in the control group - total score in the group of animals treated with the drug) × 100 / total score in the control group

In guinea pigs, 5 days after infection, swabs were obtained from the mucosa of the urogenital tract in sterile tubes with 1.8 ml of DMEM culture medium.

Determination of virus titer isolated from urogenital swabs of guinea pigs

Titration of the virus from the contents of swabs from the mucosa of the urogenital tract in guinea pigs was carried out in vitro on a VERO cell culture using standard virological research methods.

Samples in the experiment after sampling before the study were stored at 70 ° C.

VERO cells were grown in 96 well Costar cell culture plates. Cells in a complete culture medium were added 100 μl to each well of the plate at a concentration of 10 ⁴ cells / ml, incubated in an incubator at 37 ° C in an atmosphere of 5% CO ₂ . After the formation of a complete monolayer (after 24 hours), the medium was removed, washed three times with nutrient-free medium without serum, and used in further experiments.

Before introducing samples, 90 μl of serum-free culture medium was added to all wells of the plate (in complete DMEM culture medium (PanEco, RF) with antibiotics, a 0.008% solution of gentamicin sulfate (FEREYN, RF), 300 mg of glutamine (M PiVE GU .P. Chumakova RAMS, RF) per 500 ml of medium). The test samples were added at 10 μl / well (1: 100 dilution). Then, by serial dilutions in tablets, titration was performed in the range of 10 ¹ -10 ⁵ TICD ₅₀ / ml. In parallel, the virus was titrated in the range of 10 ³ -10 ⁹ TICD ₅₀ / ml.

An intact cell culture (negative control) served as a control. Then the cells were incubated in an incubator at 37 ° C in an atmosphere of 5% CO ₂ for 5 days. The cell monolayer was examined daily under a light microscope in order to assess the degree of its intactness and to detect the specific cytopathic effect of the virus (CPD). Analysis of results was evaluated visually using an inverted microscope LABOVERT FS (Leitz). Cytopathic changes, which were expressed as grouped, increased in the size of rounded cells, were evaluated on the following scale:

+ - minor changes in the monolayer (25%);

++ - the presence of small discontinuities in the monolayer (50);

+++ - destruction of about 50% of the monolayer (75%);

++++ - almost complete destruction of the monolayer (100%).

To quantify the cytopathic effect of the samples, a modified method of counting living and dead cells using the Neutral Red (NR) dye was used. After washing (3 times), an NR solution (concentration of 1.11 μg / ml) was added 50 μl to each well of the plate, after incubation for 2 hours at 37 ° C in an atmosphere of 5% CO _{2, the} tablet was washed three times and 100 μl was added a solution of 50% EtOH / 50% 0.1 M NH ₄ H ₂ PO ₄ (pH 3.5). After 30 minutes of incubation, counting was performed on a spectrophotometer at a wavelength of 490 nm.

Each test sample was tested at 4 points (wells), and each value represents the arithmetic mean.

Statistical processing of the obtained data was carried out by generally accepted methods of variation statistics using the Ecxell-5.0 application programs, estimating the confidence interval for 95% probability.

RESULTS

1. Study Design

Treatment with topical preparations - cream, gel, containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, and the reference drug Zovirax cream were carried out according to the treatment scheme in two versions: a) 3 hours after infection; b) 48 hours after infection (with severe manifestations of the disease). Preparations for external use were applied (by the method of application) with a thin layer on the lesions 2 times a day, daily, for 5 days.

Description of study groups

Taking into account the purpose of the experiment, all animals were divided into 7 groups, 4 pigs each, the eighth group contained 6 animals.

The first group contained animals infected with HSV-2 and treated with cream for external use according to the treatment regimen 3 hours after infection daily 2 times a day for 5 days.

The second group contained animals infected with HSV-2 and treated with cream for external use according to the treatment regimen 48 hours after infection daily 2 times a day for 5 days.

The third group contained infected animals treated with gel for external use according to the treatment regimen 3 hours after infection daily 2 times a day for 5 days.

The fourth group contained infected animals treated with gel for external use according to the treatment regimen 48 hours after infection daily 2 times a day for 5 days.

The fifth group contained infected animals treated with Zovirax in the form of a cream for external use according to the treatment regimen 3 hours after infection daily 2 times a day for 5 days.

The sixth group contained infected animals treated with Zovirax according to the treatment schedule 48 hours after infection daily 2 times a day for 5 days.

The seventh group (positive control) contained only infected HSV-2 animals that did not receive treatment.

The eighth group contained 6 animals that were only scarified, while 2 pigs were not further treated, and 2 pigs were treated with placebo, the appropriate cream and gel according to the scheme 3 hours after infection daily 2 times a day for 5 days. These animals were considered as a negative control, in relation to which the severity of the virus-caused pathological process and the local irritating effect of the drugs were compared.

Characterization of the infectious process in infected animals

In the control group of infected animals (group No. 7) not receiving treatment, typical local manifestations of genital herpes developed 24-48 hours after infection. Initially, on the mucous membrane of the penis and adjacent areas of the skin, the appearance of single vesicular eruptions on an erythematous background was noted, then, as the disease progressed, confluent foci with hemorrhagic contents appeared, bleeding erosion, ulceration, and moderate orchitis. In the following days, erosion and ulcers epithelized either under the crust or without its formation. The specificity of local manifestations of GH was confirmed by us by the method of isolating the virus from the contents of the vesicles, swabs taken from the urogenital area of infected animals.

The process was distinguished by the presence of pronounced edematous-inflammatory phenomena, prolonged preservation of hyperemia, “sluggish" healing of erosive-ulcerative elements. The maximum development of symptoms was observed on the 5th day after infection: the total symptom severity index (SIWS) in this group was 34.74 points, the average disease duration (SDR) was 15.0 ± 2.46 days.

2. The results of the treatment of experimental genital herpes in guinea pigs using a cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl -5-nitropyrimidine tetrachloride dihydrochloride hexahydrate for topical application

The results of the treatment of experimental genital herpes in guinea pigs using a cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5 -nitropyrimidine tetrachloride dihydrochloride hexahydrate, and the reference preparation Zovirax for topical use are presented in table 1, in figures 1 and 2.

As follows from the data presented in table 1, in figures 1 and 2, the use of cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecane-1- sludge) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate for topical application, had an effect of varying severity on the course of genital herpes in male guinea pigs.

The most significant treatment results were obtained in the group of infected animals that received the cream according to the treatment regimen - 3 hours after infection.

It was established that the use of the drug in the form of a cream at an early stage of the infectious process (3 hours after infection) led to a statistically significant therapeutic effect, a 1.5-fold decrease in the severity of symptoms and a significant reduction in the average duration of the disease compared with similar indicators in the group of infected animals not receiving treatment (positive control). The IPD amounted to 32.64%, SDR - 9.0 ± 1.0 days (see table. 1, Fig. 1). Animals treated with the cream recovered an average of 6.0 days earlier than those that did not receive treatment. The infectious process was characterized by the presence of moderately expressed exudative-inflammatory phenomena, a reduction in the duration of exudative-inflammatory phenomena and resolution of elements. The studies did not reveal significant differences between the effectiveness of the cream and the reference drug Zovirax for all the studied parameters (according to the effect on the parameters of IPD, SIWS, SDR, on the nature of the course of the infectious process), (p> 0.05). Against the background of treatment with the reference drug Zovirax (sixth group), the SDR was - 8.33 ± 0.57 days; IPD - 35.23%.

The use of cream in infected animals against the background of pronounced manifestations of the pathological process (group No. 2) also led to a statistically significant therapeutic effect and a reduction in the duration of the disease compared with infected animals that did not receive treatment. At the same time, the drug in terms of therapeutic effect (26.59%) was inferior to that in the group of animals that received the cream early after infection (32.64%) and in the group of animals that received the reference drug Zovirax (30, 05%), but was comparable with the first group in terms of SDR. So, SDR in animals in the second group was 9.33 ± 1.52 days, in animals in the first group - 9.0 ± 1.0 days, p> 0.5. When comparing the SDR in animals of the second group and in animals treated with the reference drug, it was found that the infected animals receiving the test cream recovered 1.33 days later, p <0.5. It should also be noted that in the group of the late cream treatment (beginning after 48 hours), exudative inflammatory phenomena were more pronounced and lasting than in animals in the first group who received early treatment with the same drug and in animals that received the reference drug Zovirax.

Analysis of the use of the gel for external use according to the treatment regimen on the model of experimental genital herpes in guinea pigs showed, as in the case of cream, that this form of the drug showed more pronounced activity when used in the early stages after infection.

So, the use of the gel 3 hours after infection (group No. 3) led to a reliable IPD of 25.73% and a significant decrease in SDR by 3.34 days compared with the group of animals that did not receive treatment. According to the effectiveness of the application, the gel used according to the scheme - 3 hours after infection, was comparable to the effectiveness of the cream used against the background of pronounced manifestations of the infectious process (group No. 2, the start of treatment 48 hours after infection), but was 2.4 times less on average days according to the effect on the duration of the disease compared with the action of the cream used according to the scheme 3 after infection, and with the action of the reference drug (see table. 1).

Studies have shown that the use of gel in infected animals 48 hours after infection for 5 days (group No. 4) led to a therapeutic effect of 22.01% and a decrease in SDR of 2.66 days compared with infected animals, not receiving treatment. Nevertheless, the use of the gel according to the +48 h schedule was less effective in all assessment parameters compared to using both cream at different times of the infection process and the reference drug Zovirax (group No. 6). Observation of animals from group 8 showed that 2 animals, which were subjected to scarification only, experienced rapid (within 1-2 days) healing, after which no pronounced phenomena were observed in pigs in this group. In addition, in 4 pigs treated with a placebo corresponding to a cream and gel according to the scheme 3 hours after infection daily 2 times a day for 5 days, there was no swelling, visible local irritating effect of the drugs and general side effects.

Thus, according to the degree of decrease in effectiveness, the studied modifications of preparations containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, for external use, can be positioned as follows: group No. 1 (cream +3 hours after infection)> group No. 3 (gel +3 hours after infection) / group No. 2 (cream +48 hours after infection)> group No. 4 (gel +48 h after infection).

3. The study of the effect of cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, for external use on the frequency and level of isolation of herpes simplex virus type 2 in male guinea pigs

To determine the effect of cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, on the frequency of HSV-2 isolation and its replication level in infected animals, we studied samples of swabs from lesions collected from animals on the 5th day after infection using a standard technique for virus isolation in a VERO cell culture.

table 2

Effect of cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, for external use on the frequency of isolation and the level of virus replication from lesions in male guinea pigs infected with HSV-2 (according to the virological method)

As can be seen from the data in table 2, on the 5th day after infection, the frequency of virus isolation in infected animals both in the experimental groups and in the control groups was 100%. The obtained results, in turn, confirmed the specificity of the symptoms of genital herpes observed by us in experimentally infected animals.

It was found that the use of a cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, in infected animals (groups No. 1-4) led to a decrease in the level of replication and infectious activity of the virus isolated from swab samples from the affected area, compared with the infectious activity of the virus isolated from infected animals that were not treated (see table. 2) . So, the titers of the virus isolated from animals during treatment with cream were 1.9 ± 0.52 Ig TCD ₅₀ / ml and 2.41 ± 0.4 Ig TCD ₅₀ / ml, respectively; during gel treatment, 2.25 ± 0.5 Ig TCD ₅₀ / ml and 3.0 ± 0.11 Ig TCD ₅₀ / ml, respectively.

The most pronounced decrease in the infectious activity of the virus - 100 times - was noted when using the cream in the early stages of the infectious process, while the use of the cream against the background of pronounced manifestations of herpes virus infection led to a decrease in the infectious activity of the virus by 30 times.

The use of the gel, depending on the application, led to a decrease in the infectious activity of the virus by 50 and 10 times.

Virus titers in infected animals under the influence of the reference drug Zovirax statistically significantly decreased by more than 100 times compared to the virus titers in the control (p <0.05) and amounted to 1.66-1.75 Ig TCD ₅₀ / ml.

Thus, in experiments on the isolation and titration of the virus from biomaterial samples in infected animals with GH in a VERO cell culture, we obtained data indicating the presence of selective antiviral activity in ion cream and gel containing the substance 4,6-di (3,12- diaz-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, when applied topically. Data obtained using the virological method correlate with data on the effectiveness of the drugs obtained using the characteristics of the infectious process caused by HSV-2. Thus: a cream and gel containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate, when local application according to the treatment regimen (two-time application for 5 days) on the model of genital herpes in guinea pigs have a reliable therapeutic effect, which allows to suppress the development of herpes virus infection caused by HSV-2. The most effective was the treatment with a cream containing the substance 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate the scheme in the early stages (3 hours) after infection (group No. 1), which had a pronounced therapeutic effect on the course of infection in animals, contributing to a significant decrease in the severity of symptoms, a reduction in the resolution of elements, the average duration of the disease, and the infection activity of HSV-2. The effect of cream treatment in the early stages (3 hours) after infection is confirmed by the results of the virological research method, which revealed a significant decrease in titer in the treated animals compared with the viral control group. The effectiveness of the cream when used according to the treatment schedule in the early stages after infection, both in terms of the characteristics of the infection process and in reducing the titer of the virus in the treated animals compared with the virus control group, is comparable to the reference drug Zovirax used according to a similar scheme.

Claims (4)

1. The pharmaceutical composition in the form of a cream for the treatment of herpes virus infection, containing as an active substance from 0.5 to 10.0% wt./about. 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate. 2. The pharmaceutical composition in the form of a gel for the treatment of herpes virus infection, containing as an active substance from 0.5 to 10.0% of the mass. 4,6-di (3,12-diaza-6,9-diazoniadispiro [5.2.5.2] hexadecan-1-yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate. 3. The pharmaceutical composition according to claim 1, where the cream has anti-herpes activity against strains of HSV-1 and HSV-2, including those resistant to currently existing drugs. 4. A method of treating or preventing a herpes infection in a human or animal, comprising topically administering to the patient a pharmaceutical composition according to claims 1 and 2 in an effective amount.

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