Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0014—Skin, i.e. galenical aspects of topical compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/20—Antivirals for DNA viruses
  • A61P31/22—Antivirals for DNA viruses for herpes viruses

Definitions

• HVI herpes virus infections
• Herpes simplex herpes simplex virus
• Type 1 and Type 2 HSV-1 and HSV-2
• HVIs rank second, behind only the flu.
• HVI-causing herpes viruses can affect almost all organs and systems of a macroorganism, causing latent, acute and chronic infection.
• the most studied class of abnormal nucleosides is a large class of antiherpetic compounds and includes pyrimidine nucleoside analogs, such as idoxuridine (IDU) used in ophthalmology for chemotherapy of herpesvirus infections caused by HSV (in treatment and prevention of herpetic keratitis); purine nucleoside analogs, among which the most famous vidarabine was recommended in the systemic drug therapy of herpes encephalitis, but has lost its importance due to a high toxicity: in high doses the drug has cancerogenic , mutagenic and teratogenic action; derivatives of nitrogen heterocycles such as virazole (ribavirin) used in chemotherapy [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, S
• acyclic analogues of purine nucleosides are acyclovir and its derivatives, valacyclovir , penciclovir, famciclovir, ganciclovir, and cidofovir. They belong to the class of compounds being guanosine and adenosine analogs comprising a nucleic base moiety. Antiherpetic drugs of this class (for local and systemic action) are highly specific and have low toxicity.
• acyclovir In an infected cell, during the amplification of maternal viral DNA, acyclovir, after three-step phosphorylation (by viral thymidine kinase at the first stage (monophosphorylation) , and by the cell thymidine kinase at the second and the third stages) , is picked up by viral DNA polymerase and incorporated into the DNA chains of produced daughter virions. Since acyclovir, unlike the natural guanosine, does not comprise a 3- hydroxyl group required for attachment of the next nucleoside thereto in the chain, its early introduction into the synthesis of DNA terminates the process at the beginning, assembly of daughter viral DNAs is ceased, and new viral particles are not produced.
• nucleosides breaks the replication of herpes viruses at any stage, and new generations of viruses are not formed, which actually prevents the infection from spreading.
• the limited number of herpes virus antigens remaining in the body allows the immune system to determine, without hindrance, the types and repertoire thereof, providing adequate immunity in humans with normally functioning immune system.
• pyrimidine-dispirotripiperazinium (4 , 6-di (3 , 12 -diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-1- yl) -2-methyl-5-nitropyrimidine tetrachloride dihydrochloride hexahydrate
• HS heparan sulfate
• the most known pathogenic viruses using this mechanism of adsorption to the cell wall are herpes viruses type 1 and 2, papilloma virus, cytomegalovirus, some species of HIV, respiratory syncytial virus, and others.
• the invention relates to the use of 4 , 6-di (3 , 12-diaza-
• diazoniadispiro [5.2.5.2] hexadecane derivatives has been studied on the example of herpes virus first type (HSV-1) and kidney cells of green monkey, and it has been shown that said mechanism of action is associated with a specific property of the compounds to bind to HS, dramatically decreasing the number of viral replications. It has also been shown that the addition of the studied compounds is antagonized by ⁇ heparin.
• the target of diazoniadispiro [5.2.5.2] hexadecane compounds is two sulfate groups in adjacent sugar residue.
• GlcA2S-GlcNS6S, GlcA2S-GlcNS3S , IdoA2S-GlcNAc6S, IdoA2S- GlcNH23SS6S, IdoA2S-GlcNS6S , and IdoA2SGlcNS3S demonstrate a good electrostatic interaction between the negative charge on the sulfate group and the positively charged nitrogen atoms of 4 , 6 -di (3 , 12 -diaza- 6 , 9 - diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl-5- nitropyrimidine tetrachloride dihydrochloride hexahydrate .
• 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] .
• Systemic mainly oral, administration of an antiherpetic agent used for the treatment requires a long- term therapy and administration of high doses (up to 1000 mg/day) of an antiviral drug.
• a drawback of topical dosage forms such as gels, creams, solutions, lotions, lacquers, and the like, is in that a pharmaceutically active agent is in insufficiently close contact with an affected areas.
• the objective of the present invention is to find a
• Pharmaceutically acceptable additives used in the pharmaceutical composition according to the invention include pharmaceutically acceptable components which are compatible with the active ingredient and do not harm patients, and which are conventionally used for preparing dosage forms.
• Such components include, for example, fillers, binders, plasticizing agents (such as camphor, triphenyl phosphate or fentanyl diisobutyrate , diisobutyl phthalate et al.) , pigments, solubilizing agents, stabilizers, diluents, adjuvants, preservatives, components of buffer systems, solvents, dispersants, preservatives, thickeners, colorants, emulsifiers, film- forming agents (such as hydroxyethyl or propylcellulose, and hydroxypropyl chitosan) , prolongators , substances improving drug penetration (for example, benzyl alcohol) , and others.
• plasticizing agents such as camphor, triphenyl phosphate or fentanyl diisobutyrate
• compositions include, for example, gum arabic, polyvinylpyrrolidone, polyvinyl alcohol, alginic acid, sodium alginate, anhydrous silicic acid, magnesium stearate, talc, carboxyvinyl polymer, titanium oxide, sorbitol fatty acid ester, sodium laurylsulfate, glycerol, glycerol fatty acid ester, lanolin, glycerogelatin, polysorbate, macrogol, vegetable oil, wax, paraffin, propylene glycol, polyethylene glycol, water, ethanol, polyols, 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 comprise a vegetable oil such as jo
• the composition can comprise glycerol, optionally in admixture with sucrose laurates, or sorbitol, o/w (oil/water) emulsifier, for example, a vegetable protein hydrolyzate.
• the composition can also be used in the form of liniment (lat. Linimentum) which is a thick or jelly topic dosage form rubbed into the skin.
• Liniment can comprise additives, such as, for example, propylene glycols and benzalkonium chloride.
• Emulsion fatty liniments can has a structure of "oil-in-water” or “water-in-oil” emulsions.
• Emulsion liniments, as well as suspension liniments should be stabilized with an emulsifier, for example, emulsifier T-2 (emulsifier T-2 is a polymeric glycerol fatty acid ester), Tween 80, and other surfactants.
• emulsifiers can be formed in a reaction between ingredients constituting the liniment.
• compositions include, for example, natural or hardened oils, such as cocoa butter, waxes, fats, glycerol saturated fatty acid ester, glycerogelatin, macrogol, semiliquid or liquid polyols, triglycerides, etc.
• the basis of the topical pharmaceutical composition can also include a surfactant or a stabilizer.
• Acetic acid 45 mL is added to 10 g (0.116 mol) of anhydrous piperazine under cooling and vigorous stirring. Then, acetone (20 mL) and water (20 mL) are added. After 20 minutes of stirring, 6.7 mL (0.058 mol) of benzoyl chloride are added. The solution is stirred for additional 3 hours. Then the reaction mixture is concentrated by half. 40% NaOH is added in an amount of 450 mL. The precipitated residue is filtered off, and the aqueous stock solution is extracted with 100 mL of chloroform three times, dried, and stripped off to dryness. The obtained yellow oil in an amount of 13.4 g comprises about 60% of the main substance.
• N-benzoylpiperazine hydrochloride (0.02 mol) is added to a solution of KOH (0.024 mol) in 14 mL of ethanol under stirring and further stirred for an additional hour.
• Ethylene chlorohydrin (0.05 mol) is added to the resulting suspension, followed by the addition of a solution of KOH (0.052 mol) in 25 mL of ethanol, while maintaining the temperature in the reaction mass of not higher than 20°C.
• the reaction mixture is stirred for 20 hours.
• KC1 is filtered off, and a 12% solution of hydrogen chloride in alcohol is added to the stock solution until the pH value reaches 2-1, then the solution is cooled at -5°C for 5 hours.
• the precipitated residue is filtered off, washed with alcohol, and dried at 50°C for 5 hours.
• the yield is 70%.
• MP is 207-210°C. 5000281
• N-benzoyl-N- (2-chloroethyl) piperazine (0.0156 mol) is added to a solution of NaOH (0,017 mol) in 25 mL of ethanol. The suspension is stirred for 1.5 hours. NaCl is filtered off. The stock solution in alcohol is refluxed for 1 hour and then stripped off, and the dry residue is heated on an oil bath for 10 hours. The bath temperature is 120°C. Then the stock solution is cooled to room temperature, ethyl alcohol is added, and the residue is filtered off, washed with ethanol, and dried at 100 °C for 3 hours. The yield is 60%.
• MP 300°C (decomposition)
• Examples of preparing pharmaceutical compositions in various dosage forms suitable to be used as a topical medicament comprising 4 , 6-di (3 , 12-diaza-6, 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride.
• Example 1 Preparation of pharmaceutical composition in the form of cream, comprising:
• the active agent, poloxamer and sorbic acid are dissolved in 50 mL of water under vigorous stirring, then emulsifying wax and sorbitan monostearate are added, and the mixture is allowed to be kept.
• the obtained cream can be further used as an antiviral drug.
• Example 2 Preparation of pharmaceutical composition in the form of gel, comprising:
• active agent 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidine tetrachloride dihydrochloride (10 g) , methylcellulose-35 (7.5 g) , sorbic acid (0.150 g) , water - up to 100 g.
• Active agent is dissolved in 50 mL of water, methylcellulose-35 and sorbic acid are added thereto, and then the remaining water is added under vigorous stirring. The mixture is kept for swelling.
• the obtained gel can be further used as an antiviral drug.
• Ointment can be prepared as follows: the active agent in an amount of 0.5-3 wt.% based on the total weight is melted in a small amount of an ointment, cream or suppository base. The obtained melt is carefully mixed with a required amount of the base heated to 35-80 °C. The obtained mixture is homogenized. The melted resulting mixture is poured into tubes .
• Liniment is prepared in the form of 5% transparent yellow liquid with a slight specific odor.
• the liniment consists of: the active compound of formula (I) (1-2 mg) , benzalkonium chloride (50 mg) , and 1, 2-propylene glycol.
• Propylene glycol is used as a humectant.
• Example 5 Preparation of pharmaceutical composition in the form of emylsifying liniment, comprising:
• Example A 10% cream (Example A) and 10% gel (Example B) prepared on the basis of 4 , 6-Di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidine tetrachloride dihydrochloride (further the substance) were studied.
• Aciclovir [9 - ( 2 -hydroxyethoxymethyl) guanine] was used as a reference drug in the form of topical cream Zovirax comprising 5% aciclovir (5%) (from GlaxoSmithKline Consumer Healthcare, GB) .
• Placebo used in in the experiments with guinea pig was cream and gel of the same composition, but without of the substance of the active compound.
• the cells were cultured in a DMEM growth medium (minimum Eagle's medium “PanEco” , Russia) supplemented with 10% heat- inactivated fetal bovine serum (FBS "PanEco", Russia), 2 mM L-glutamine (Sigma, USA), and antibiotics (100 g/mL penicillin and 100 g/mL streptomycin) .
• a support medium contained all the above ingredients and 2% FBS.
• the cells were incubated in a 5% C0 2 atmosphere at 37°C.
• Viruses Experiments were conducted with antigenic type of herpes simplex virus HSV-2, strain BH, obtained from the State Collection of Viruses of the Russian Federation, D.I. Ivanovsky Research Institute of Virology. The virus was maintained in the serial passages and titrated in a continuous cell culture of the green monkey kidney-derived Vero cell line. Virus titers were assessed by standard (Reed-Mench) micromethod in 96 -well tissue- culture plates by using the cell culture, and expressed as lg TCD 50 /mL.
• TCD 50 tissue cytopathic dose of the virus TCD 50
• TCD 50 of virus The minimum dilution of the virus causing 50% destruction of the cell monolayer, while the cell monolayer free of infection (control) remained undamaged (50% tissue cytopathic dose of the virus TCD 50 ) was taken as TCD 50 of virus.
• the virus was used in the form of a virus-containing suspension with an infectious titer of 6,0 lg TCD 50 /mL. The resulting virus was stored in frozen aliquots.
• HSV-2 culture virus - containing liquid
• the virus-containing liquid (at a dose of 100 TCD 50 ) was applied with a pipette (followed by rubbing) on the previously scarified skin of the penis. Scarification was performed by a surgical lance, 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.
• Placebo used in the experiments in the form of cream and a gel had the same composition, but was free of the substance PDSTP. Furthermore, 5% ointment Zovirax (acyclovir) was used as a reference dosage form.
• the topical treatment was conducted according to the following scheme: 3 or 48 hours after infection (in case of pronounced manifestations of the disease) . Preparations were applied by application of a thin layer on the lesions twice a day, every day for 5 days. The area of the drug application was 1.3 cm 2 .
• the severity of the infection was assessed every day before the treatment, and monitored throughout the whole period of the disease by the following parameters: the existence and degree of specific lesions (vesicles, pustules, ulcers, erosions, crusts) ; edema, hyperemia, and orchitis.
• the maximum severity of each symptom was 4 scores.
• the total duration of the monitoring period was 21 days .
• the efficacy of the preparations was assessed in the maximum manifestation of the pathological process, by standard methods. Criteria for the assessment of the therapeutic effect of the preparations were: a reduction in the intensity of clinical symptoms, index of therapeutic action (ITA) , a reduction of the disease duration in the experimental groups, compared with the control.
• ITA index of therapeutic action
• the total score of the control group the total score in the group of animals treated with drug the total score in the control group
• mucosal swabs from the urogenital tract were taken in sterile tubes with 1.8 mL of the DMEM culture medium.
• the virus was titrated in vitro from the contents of the mucosal swabs from the guinea pig urogenital tract in a VERO cell culture by standard virological methods.
• the taken samples were stored at -70°C, before analysis .
• VERO cells were grown in "Costar" 96 -well cell culture plates.
• the cells in the complete culture medium were added to each well of the plate in an amount of 10 mL at a concentration of 10 4 cells/mL, and incubated in 5% C0 2 at 37°C. After the formation of a complete monolayer (24 hours) , the medium was removed, and the monolayer was washed three times with the serum- free culture medium and used in further experiments.
• Intact cell culture was used as a control (negative control) . Then the cells were incubated in a thermostat at
• the cytopathic effect of the samples was quantitatively measured by a modified method of counting live and dead cells, using Neutral Red (NR) dye. After washing (3 times) , 50 L of the NR solution (concentration of 1.11 pg/mL) was added to each well of the plate, and after incubation for 2 hours at 37 °C in 5% of C0 2 , the plate was washed three times, and 100 L of a solution of 50% of EtOH/50% of 0.1M NH 4 H 2 P0 4 was added to each well (pH 3.5). After 30 min incubation, the cells were counted in a spectrophotometer at a wavelength of 490 nm.
• NR Neutral Red
• Topical preparations comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2 -methyl- 5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, and with the reference drug (cream Zovirax) was carried out by the following two variants of the treatment scheme: a) 3 hours after infection; b) 48 hours after infection (in case of expressed manifestations of the disease) . Topical drugs were applied (by the method of application) in a thin layer to lesions twice a day, every day for 5 days.
• the first group comprised animals infected with HSV-2 and treated with the topical cream according to the treatment scheme of 3 hours after infection, twice a day, every day for 5 days .
• the second group comprised animals infected with HSV-2 and treated with the topical cream according to the treatment scheme of 48 hours after infection twice a day, every day for 5 days .
• the third group comprised infected animals treated with the topical gel according to the treatment scheme of 3 hours after infection twice a day, every day for 5 days.
• the fourth group comprised infected animals treated with the topical gel according to the treatment scheme of 48 hours after infection twice a day, every day for 5 days.
• the fifth group comprised infected animals treated with drug "Zovirax” in the form of a topical cream according to the treatment scheme of 3 hours after infection twice a day, every day for 5 days.
• the six group comprised infected animals treated with drug "Zovirax" according to the treatment scheme of 48 hours after infection twice a day, every day for 5 days.
• the seventh group (positive control) comprised only HSV-2 -infected animals which did not receive treatment.
• the eighth group comprised 6 animals which were subjected to only scarification, wherein two pigs were not subjected to further treatment, and 2 pigs were treated with corresponding cream and gel placebo, according to the treatment scheme of 3 hours after infection twice a day, every day for 5 days. These animals were considered as a negative control, against which the severity of virus - caused pathological process and local irritating action of the drugs were assessed.
• TSSS total symptom severity score
• ADD average duration of disease
• the studied modifications of the topical preparations comprising the substance, , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, can be arranged, in terms of reduction in the efficiency, as follows: Group 1 (cream + 3 hours after infection) > Group 3 (gel + 3 hours after infection) > group 2 (cream + 48 hours after infection) > Group 4 (gel + 48 hours after infection) .
• virus titers isolated from the animals treated with the cream were 1.9+0.52 lg TCD 50 /mL and 2,41+0,4 lg TCD 50 /mL, respectively; and treated with the gel were 2,25 ⁇ 0,5 lg TCD 50 /mL and 3.0+0.11 lg TCD 50 /mL, respectively.
• Virus titers in infected animals treated with the reference drug Zovirax were significantly decreased, more than 100 times, as compared with the virus titers in the control (p ⁇ 0.05) and were 1,66-1,75 lg TCD 50 /mL.
• the cream and gel comprising the substance, 4,6- di (3 , 12-diaza-6 , 9-diazoniadispiro [5.2.5.2] hexadecan-l-yl) - 2-methyl-5-nitropyrimidin tetrachloride dihydrochloride hexahydrate, in topical administration according to the treatment scheme (two time administration for 5 days) in the guinea pig model of genital herpes, have a significant therapeutic effect that can suppress the development of herpes virus infection caused by HSV-2.
• the most effective treatment was the treatment with the cream comprising the substance, 4 , 6-di (3 , 12-diaza-6 , 9- diazoniadispiro [5.2.5.2] hexadecan-l-yl) -2-methyl-5- nitropyrimidin tetrachloride dihydrochloride hexahydrate, according to the treatment scheme at early stages (after 3 hours) after infection (group 1) , which has a pronounced therapeutic effect on the course of infection in animals, providing a significant reduction in the severity of symptoms, a reduction in the terms of resolution of elements, a reduction in the average duration of the disease, and infectious activity of HSV-2.

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• 2014
  • 2014-04-30 RU RU2014117294/15A patent/RU2573977C9/ru active
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