RU2716709C1 - Oral dosage form of the preparation in capsules for treating and preventing orthopoxvirus-related diseases - Google Patents

Abstract

FIELD: medicine; pharmaceutics.

SUBSTANCE: invention refers to an oral dosage form of a capsule formulation effective against VV (variola virus) and other human and animal pathogenic orthopoxviruses, and can be used in pharmaceutics, virology, medicine and veterinary science. Ready dosage form contains as an active compound having anti-orthopoxvirus activity 7-[N-(4-trifluoromethylbenzoyl)-hydrazinocarbonyl]-tricyclo[3.2.2.02,4]non-8-ene-6-carboxylic acid (NIOH-14), lactose monohydrate, amorphous silicon dioxide anhydrous (aerosil), magnesium stearate and microcrystalline cellulose (MCC), potato starch, sodium chloride and purified water in the amounts specified in the claim.

EFFECT: technical result of the invention consists in expanding the range of ready dosage forms used in medicine and veterinary science for treating and preventing orthopoxvirus-mediated diseases.

1 cl, 2 dwg, 4 tbl, 9 ex

Description

The invention relates to an oral finished dosage form of the drug in capsules for the treatment and prevention of diseases caused by orthopoxviruses, and can be used in the field of pharmaceuticals, virology, medicine and veterinary medicine.

Although the program for global eradication of smallpox was successfully completed more than 30 years ago, variola virus (VNO) remains a public health problem. The human population is currently at risk of using natural or recombinant strains of VNO and monkeypox virus (BOO) as a biological weapon [Parker S., Chen N.G., Foster S. et al. Evaluation of disease and viral biomarkers as triggers for therapeutic intervention in respiratory mousepox - an animal model of smallpox. Antiviral Res. 2012; 94 (1): 44-53]. The effects of an outbreak of smallpox in the human population can now be even more catastrophic than in the previous century. This is because the vaccination program was discontinued worldwide in 1980. As a result of this, to date, more than half of the world's population does not have immunity against orthopoxvirus infections, and the increased mobility of people (including intercontinental flights) has increased the spread of the virus across the planet.

Moreover, in the context of the constant growth of population groups with immunodeficiency conditions, vaccination with the smallpox vaccine virus becomes problematic due to the possibility of serious post-vaccination complications. In addition, outbreaks of smallpox monkeys in the population of people in Africa, and cases of its spread constitute a serious threat to the population of the whole world [Borisevich SV, Marennikova SS, Makhlay AA and other Smallpox monkeys: features of the spread after the abolition of compulsory ospoprivaniya. Journal of Microbiology, Epidemiology and Immunobiology. 2012; 2: 69-73]. In this regard, the problem of successfully eliminating post-vaccination complications and outbreaks of orthopoxvirus diseases with the help of highly effective chemotherapeutic drugs is of particular importance. That is why large government investments are being made in the development of improved control measures for smallpox, including new vaccines and antiviral drugs [LeDuc J.W., Jahrling P.V. Strengthening national preparedness for smallpox: an update. Emerging Infectious Diseases. 2001; 7: 155-157].

Currently, the range of therapeutic and prophylactic drugs used for emergency prevention and treatment of diseases caused by orthopoxviruses, including VNO, is extremely limited. These agents include thiosemicarbazones, nucleoside and nucleotide analogs, acyclic nucleosides and nucleotides [Parker S., Handley L., Buller M. Therapeutic and prophylactic drugs to treat orthopoxvirus infections. Future Virol. 2008; 3 (6): 595-612]. Thus, the development of highly effective antiviral drugs that have a therapeutic and prophylactic effect upon infection of VNO is an important task for maintaining the health and life of people.

State of the art

The development of drugs against smallpox is a complex, lengthy and costly process. First, serial large-scale testing of the anti-orthopoxvirus activity of the obtained compounds in vitro is carried out in order to identify the most potent, so-called leading compound. Most of the published works describe precisely these initial stages. However, only a small fraction of the chemical compounds that suppress the replication of surrogate orthopoxviruses (smallpox vaccines, smallpox cows, ectromelia, etc.) in cell cultures are effective against VNO in vitro, and even more so in experiments on smallpox models in laboratory animals .

So, ViroPharma and USAMRIID, when screening more than a million compounds, identified several classes of active agents. Further, active compounds were evaluated against BOO, a limited number of candidates were selected for testing using VNO. As a result, for development at a higher level, including for testing on laboratory animals, only one ST-246 was selected [Yang G., Pevear D.C., Davies M.N. et al. An Orally Bioavailable Antipoxvirus Compound (ST-246) Inhibits Extracellular Virus Formation and Protects Mice from Lethal orthopoxvirus Challenge. J. Virol. 2005; 79 (20): 13139-13149; Smee D.F. Progress in the discovery of compounds inhibiting orthopoxviruses in animal models. Antiviral Chemistry & Chemotherapy. 2008; 19: 115-124]. Further progress in the development of this compound as a prototype of the invention will be described below.

Known drug Metisazon (Marboran ^® ), which showed moderate antiviral activity against VNO when demonstrating 50% inhibition, but to cause 80% inhibition of replication was able only in the maximum tolerated concentration [Baker RO, Bray M., Huggins JW Potential antiviral therapeutics for smallpox, monkeypox and other orthopoxvirus infections. Antiviral Research. 2003; 57: 13-23]. A large number of closely related compounds of the thiosemicarbazone class were evaluated in vitro, and several compounds were tested in mouse models, showing only moderate prophylactic activity, but none of these compounds were selected for subsequent primate trials.

Two other potential anti-arsenic compounds with activity at the DNA replication stage are currently being investigated: Cidofovir (Vistide ^® ), a medicine that can be used in the treatment of herpes retinitis, and CMX001, a lipid prodrug of cidofovir. These compounds are active against surrogate orthopoxviruses in laboratory models using small rodents. However, the data showed that CMX001 does not provide significant protection against lethal infection of mice with the ectromelia virus [Parker S., Chen NG, Foster S. et al. Evaluation of disease and viral biomarkers as triggers for therapeutic intervention in respiratory mousepox - an animal model of smallpox. Antiviral Res. 2012; 94 (1): 44-53].

It was also found that the administration of Zidofovir to monkeys infected with VNO before the onset of the disease, but not after, could prevent their death. However, to obtain optimal results, a very high dose of Tsidofovir (20 mg / kg) was needed, which caused the development of acute nephrotoxicity. Moreover, the intervention after the appearance of smallpox lesions in these models was unsuccessful [World Health Organization. Scientific review of smallpox virus research, 1999-2010 http://whqlibdoc.who.int/hq/2010/WHO_HSE_GAR_BDP_2010.3_eng.pdf. Date of contact 06.05.2019].

It is known that hydrazones of azidoketones of alicyclic and aliphatic series showed weak and moderate activity in vitro against ectromelia, smallpox vaccine, smallpox, cow, smallpox monkeys [RF Patent No. 2376283, IPC С07С 251/76, publ. 12/20/2009]. The disadvantage of the above analogues is their low anti-orthopoxvirus activity, as well as the lack of studies of their activity against VNO.

Also known is the chemical compound 7- [N '- (4-trifluoro-methylbenzoyl) -hydrazinocarbonyl] -tricyclo [3.2.2.0 ^2,4 ] non-8-en-6-carboxylic acid, which has activity against ectromelia viruses, smallpox vaccines and smallpox of cows in vitro [RF Patent No. 2412160, IPC С07С 243/38, publ. 02/20/2011]. The fundamental disadvantage of the synthesized chemical compound is that methanol is used in its production technology, the toxicity of which is approximately 10,000 times greater than that of ethanol [Physiological effect and toxicity of alcohols. http://alcogol.su/page.php?al=fiziologicheskoe_deistvi. Date of contact 06.05.2019]. The use of methanol in the production of medical supplies creates a danger to workers and the environment, which makes it almost impossible to license an enterprise for the production of an oral finished dosage form against UPE due to the need to comply with all points of the current Sanitary Rules [SP 2.3.3.2892-11 “Sanitary and Hygienic requirements for the organization and conduct of work with methanol ”dated 12.07.2011; SP 4079-86 "Sanitary rules for enterprises manufacturing pharmaceuticals" dated 03/14/1986]. Moreover, according to clause 3.1 of SP 2.3.3.2892-11, "the use of methanol is allowed only in those production processes where it cannot be replaced by other less toxic substances."

In addition, the activity of this compound was studied only against surrogate orthopoxviruses and only in cell culture, and not against VNO in laboratory model animals infected with VNO, which is not evidence of its anti-arsenic effect when introduced into animals or humans. To convincingly substantiate and recommend this chemical compound as an anti-fire remedy, it is necessary to obtain the results of its high efficiency with respect to VNO not only in vitro, but also in animal experiments. Thus, the report of the United States Institute of Medicine emphasizes that “the most compelling reason for the long-term preservation of live UPE stocks is their crucial role in testing and developing antiviral drugs for use in the event of smallpox outbreaks” [US Institute of Medicine. Live variola virus: considerations for continuing research. National Academy Press, Washington, DC, 2009]. Despite the fact that numerous compounds that inhibit the replication of surrogate orthopoxviruses in many in vitro test systems have been identified in the world, only a small part of them is able to exhibit antiviral activity against VNO in vitro, and even less in vivo. In this regard, the demonstration that this chemical compound is highly active against VNO in vivo is an indispensable and necessary condition in order to position and recommend it as an anti-inflammatory remedy for the treatment and prevention of smallpox.

Known drug (substance) ST-246 is a chemical compound 4-trifluoromethyl-N- (3,3a, 4,4a, 5,5a, 6,6a-octahydro-1,3-dioxo-4,6-ethenocycloprop [f] isoindole-2 (1H) -yl) benzamide having antiviral activity not only against surrogate orthopoxviruses, but also against VNO [Huggins J., Goff A., Hens ley L. et al. Nonhuman Primates Are Protected from Smallpox Virus or Monkeypox Virus Challenges by the Antiviral Drug ST-246. Antimicrobial Agents and Chemotherapy. 2009; 53 (6): 2620-2625]. This publication describes the therapeutic and prophylactic activity of orally administered ST-246 at a dose of 300 mg / kg / day for 14 days for Cynomolgus monkeys (weighing 3.5 kg) who were infected by intravenous administration of the Harper VNO strain at a dose of 1 × 10 ⁸ PFU (plaque forming units) to each animal. Three monkeys began to receive ST-246 immediately after infection, and three monkeys received ST-246 after 1 day. after infection, two control monkeys received a placebo. Both placebo monkeys were euthanized due to the severity of the disease, while all monkeys receiving ST-246 remained alive in the absence of any significant clinical or laboratory signs of the disease.

However, the above analogue has significant drawbacks: firstly, VNO infection was carried out not through the respiratory organs, which are the “entrance gates” of VNO when people were infected, but by the intravenous method, as a result of which artificial viremia was created in monkeys, and severe smallpox developed significantly faster than humans; secondly, the dose of VNO used for infection of monkeys, equal to 10 ⁸ PFU / animal, is several orders of magnitude higher than the dose of VNO, which can infect and cause human disease; thirdly, for bioethical and economic reasons, and also taking into account the complexity and danger of working with VNO infected monkeys, the protective effectiveness of ST-246 was demonstrated only in a small number of these animals (3 monkeys in the treated groups, 2 in the placebo group).

Known therapeutic agent based on the chemical compound 7- [N '- (4-trifluoromethylbenzoyl) -hydrazinocarbonyl] -tricyclo [3.2.2.0 ^2,4 ] non-8-en-6-carboxylic acid (NIOX-14) in a dose from 4 to 60 mg / kg body weight, having activity against the variola virus (RF patent No. 2543338, IPC AK61/16, published on 02.27.2013). A method of obtaining a therapeutic and prophylactic agent against variola virus, comprising sequential dissolution and interaction of 4-trifluorobenzoic acid hydrazide and 3.3a, 4.4a, 5.5a, 6.6a-octahydro-1,3-dioxo-4,6-etheno -cycloprop [f] -furan in a 1: 1 molar ratio in a solvent, followed by stirring of the resulting suspension to obtain a precipitate that is removed from the solution, filtered and dried to obtain the final product of the chemical compound 7- [N '- (4-trifluoromethylbenzoyl) - hydrazinocarbonyl] tricyclo [3.2.2.0 ^2,4 ] non-8-en-6-carbonyl hydrochloric acid (NIOX-14). Ethyl or isopropyl alcohol is used as a solvent, the processes for preparing a suspension of reagents, separating and filtering the precipitate are carried out at a temperature of + 2-10 ° C, and the yield of dry product is 96.0%.

However, the aforementioned therapeutic and prophylactic agent against VNO is a chemically synthesized compound, which is a finely divided powder, and is unsuitable for use in this form in clinical practice, since it is not a dosage unit dosage form suitable for oral administration and providing proven necessary therapeutic and preventive effect .

The closest technical solution (prototype) is the finished dosage form (commercial name "TROXX") against VNO for oral administration (US patent No. 9744154, IPC A61K 31/404, publ. 08/29/2017), containing in one capsule (390 mg): polymorphic form II 4-trifluoromethyl-N- (3,3a, 4,4a, 5,5a, 6,6a-octahydro-1,3-dioxo-4,6-ethenocilloprop- [f] isoindole-2 ( 1H) -yl) benzamide (ST-246), which on the X-ray powder diffraction pattern has characteristic peaks at a reflection angle of 2-theta. 12.74, 16.03, 16.99, 19.64, 21.96, 23.80 and 25.39 degrees (200 mg), lactose monohydrate (33.15 mg), croscarmellose sodium - thickener (42.9 mg), colloidal silicon dioxide (1.95 mg), hydroxypropyl methyl cellulose - dietary fiber (13.65 mg), sodium lauryl sulfate (7.8 mg), magnesium stearate (1.95 mg), microcrystalline cellulose (88.60 mg) water (rest).

However, the prototype preparation contains sodium lauryl sulfate, which belongs to the emulsifier E487 and is excluded from SanPiN and the national standard of the Russian Federation for food additives, because when ingested and in contact with the mucous membrane of the digestive tract, it causes ulcers, and also has the ability to accumulate in the body and is poorly excreted from it (https://vkusologia.ru/dobavki/stabilizatory-emulgatory/e487.html). The content of the additive E487 should not exceed 1-2 wt. %, and in the GLF prototype sodium lauryl sulfate is contained in an amount of 4.1%. The use of croscarmellose sodium in GFL is also undesirable. It refers to the poorly studied food supplement E468, which acts as a stabilizer, gelling agent and super disintegrator, which causes diarrhea in the intestines of the body (http://findfood.ru/component/pishhevoj-stabilizator-E468-kroskaramelloza) or (https://vkusologia.ru /dobavki/stabilizatory-emulgatory/e468.html). This component should not exceed 1-3 wt. %, and in the ELF of the prototype drug it contains 22.5%, which is an order of magnitude higher than recommended.

The technical result of the claimed invention is to expand the range of finished dosage forms (GLF) used in medicine and veterinary medicine, effective against VNO and other orthopoxviruses pathogenic for humans and animals, as well as to improve the composition of target additives of GLF drug by reducing their number and using harmless and tested components .

The specified technical result is achieved in that in oral GLF in capsules effective against VNO and other orthopoxviruses pathogenic for humans and animals, containing a chemical compound with antiorthopoxvirus activity, lactose monohydrate, silicon dioxide, magnesium stearate and microcrystalline cellulose (MCC), according to the invention additionally contains potato starch, sodium chloride and purified water, as a chemical compound with anti-orthopoxvirus activity, contains 7- [N- (4-t rifluoromethylbenzoyl) -hydrazinocarbonyl] -tricyclo- [3.2.2.0 ^2,4 ] non-8-en-6-carboxylic acid (substance NIOX-14), as silicon dioxide - amorphous silica anhydrous (aerosil) with the following quantitative content of components in one capsule with a total weight of 400 mg:

Potato starch, sodium chloride and purified water are used in the form of a paste, which is introduced into the above capsule composition.

The mass of the contents of the capsule (granulate, substance NIOX-14 with excipients) is 400 mg. The active substance (NIOX-14 substance) is part of the capsule in an amount of 180-220 mg. In order to obtain a high-quality solid dosage form and add weight to the capsule, fillers (excipients) were introduced into the encapsulated mass: microcrystalline cellulose, lactose, sodium chloride, starch, magnesium stearate, anhydrous anhydrous silicon dioxide (aerosil).

The selected fillers affect not only the technological properties of the mass, but also the release rate, the rate and completeness of absorption of the drug substance, as well as its storage stability.

In wet granulation, starch paste is used as a binder to ensure the strength of the granules. The binder in the capsule is 2.25% of the contents of the capsule.

For rapid mechanical destruction of the granules in a liquid medium (water or gastric juice), which is necessary for the release and subsequent absorption of the drug, microcrystalline cellulose, a substance that breaks the granules after swelling in contact with the liquid, was introduced into the encapsulated mass.

To improve the wettability and water permeability of the granules, lactose and sodium chloride were introduced into the encapsulated mass, which contributes to the rapid disintegration and dissolution of the granules.

To eliminate the problem of poor granular flowability, to prevent the granules from sticking together and sticking to the equipment when filling the capsules, magnesium stearate was used as a sliding substance and a substance that prevents adhesion. Starch has the same property.

Good granular flowability guarantees the accuracy and consistency of the dosage of the drug substance in the capsule.

Magnesium stearate is also used as a lubricant to reduce the formation of bumps on the granules.

To remove the electrostatic charge from the granulate, which also improves the flowability of the encapsulated mass, aerosil was used (as a sliding substance). Magnesium stearate also has this property.

Compared with the prototype, the composition of auxiliary GLF additives of the inventive preparation is improved by reducing their list and using harmless and proven components such as potato starch and sodium chloride.

In FIG. 1. presents a diagram of the cytoxicity (circle) and antiviral activity of the substance of the NIOX-14 preparation against variola virus (square) in the Vero cell culture (SoftMax 4.0 program). The abscissa axis is the concentration in μg / ml, the ordinate axis is the optical density (OD). TC ₅₀ ≈300 μg / ml. IC ₅₀ ≈0.004 μg / ml.

In FIG. 2. A diagram of the cytoxicity (circle) and antiviral activity of the finished dosage form (GLF, capsule) of the NIOX-14 drug against variola virus (VNO, square) in Vero cell culture (SoftMax 4.0 program) is shown. The abscissa axis is the concentration in μg / ml, the ordinate axis is the optical density (OD). TC ₅₀ ≈300 μg / ml. IC ₅₀ ≈0.004 μg / ml.

The following are the compositions (examples 1-3) of an oral finished dosage form (GLF) in capsules of a NIOX-14 preparation having anti-orthopoxvirus activity.

Testing of antiviral activity against smallpox vaccine virus (BOB) and GLF cytotoxicity in capsules of the NIOX-14 preparation with a minimum (example 1), optimal (example 2) and maximum (example 3) amount of NIOX-14 substance (using DMSO to obtain solution) in Vero cell culture. We evaluated the 50% virus inhibitory concentration (IC ₅₀ , μg / ml) in relation to BOB (Copenhagen strain) and 50% cytotoxic concentration (TC ₅₀ , μg / ml) of the GLF capsule of the NIOX-14 preparation in Vero cell culture.

Example 1. Oral GLF in capsules of a NIOX-14 preparation with anti-orthopoxvirus activity (with a minimum amount of NIOX-14 substance), containing in one capsule with a total weight of 400 mg:

When statistically processing the indicators of antiviral activity against BOB and cytotoxicity of GLF of the NIOX-14 preparation with a minimum amount of NIOX-14 substance (n = 5), it was shown that IC ₅₀ = 0.004 ± 0.001 μg / ml; TC ₅₀ = 430 ± 79 μg / ml. This is consistent with our previous data on the study of the activity of the NIOX-14 substance against orthopoxviruses [Kabanov A.S., Sergeev A.A., Bulychev L.E., Bormotov N.I., Shishkina L.N. et al. Study of the antiviral activity of chemically synthesized compounds against orthopoxviruses in in vitro experiments // Problems of especially dangerous infections. - 2013. - issue. 2. - S. 54-59].

Example 2. Oral GLF in capsules of a NIOX-14 preparation with anti-orthopoxvirus activity (with an optimal amount of NIOX-14 substance), containing in one capsule with a total weight of 400 mg:

When statistically processing the indicators of antiviral activity against BOB and cytotoxicity of GLF of the NIOX-14 preparation with the optimal amount of NIOX-14 substance (n = 5), it was shown that IC ₅₀ = 0.004 ± 0.001 μg / ml; TC ₅₀ = 480 ± 76 μg / ml. This is consistent with our previous data on the study of the activity of the substance NIOX-14 against orthopoxviruses [see link for Example 1].

Example 3. Oral GLF in capsules of the NIOX-14 preparation with anti-orthopoxvirus activity (with the maximum amount of NIOX-14 substance), containing in one capsule with a total weight of 400 mg:

When statistical analysis of the indicators of antiviral activity against BOB and cytotoxicity of GLF of the NIOX-14 preparation with the maximum amount of NIOX-14 substance (n = 5) was shown, IC ₅₀ = 0.004 ± 0.002 μg / ml; TC ₅₀ = 420 ± 76 μg / ml. This is consistent with our previous data on the study of the activity of the substance NIOX-14 against orthopoxviruses [see link for Example 1].

Example 4. The technology of obtaining the finished dosage form in capsules effective against VNO and other pathogenic for humans and animals orthopoxviruses,

The method of obtaining the substance NIOX-14, effective against VNO (RF patent No. 2543338), includes sequential dissolution and interaction of 4-trifluorobenzoic acid hydrazide and 3.3a, 4.4a, 5.5a, 6.6a-octahydro-1,3-dioxo 4,6-etheno-cycloprop [f] -furan in a 1: 1 molar ratio in a solvent according to scheme (I) below, followed by stirring the resulting suspension to obtain a precipitate that is removed from the solution, filtered and dried to give the final the product of the chemical compound 7- [N '- (4-trifluoromethylbenzoyl) -hydrazinocarbonyl] -tricyclo [3.2.2.0 ^2.4 ] non-8-en-6-carboxylic acid (NIOX-14). Ethyl or isopropyl alcohol is used as a solvent, the processes for preparing a suspension of reagents, separating and filtering the precipitate are carried out at a temperature of + 2-10 ° C, and the yield of dry product is 96.0%.

Scheme (I) for the preparation of NIOX-14 preparation:

There are no chemical processes in the production of the finished dosage form "NIOX-14 capsules 200 mg". At all technological stages, only physical processes are used (weighing, mixing, moisturizing, drying, dusting, filling capsules).

The process of obtaining "NIOC-14 capsules 200 mg" is divided into two main technological stages:

1. General support and preparatory work;

2. Obtaining "NIOC-14 capsules 200 mg".

Each of the main technological stages includes auxiliary stages and stages of the technological process.

The auxiliary stages include the following:

1. Incoming quality control of raw materials;

2. Staff training;

3. Obtaining purified water;

4. Preparation of washing and disinfecting solutions;

5. Preparation of premises and equipment;

6. Washing and sterilization of accessories for the preparation of a medicinal mixture.

The technological stages are the following processes:

1. Obtaining a granulate of the drug;

2. Filling capsules;

3. Packing in cans of polymer material;

4. Packaging and labeling.

Example 5. Determination of cytotoxicity and antiviral activity of GLF of the drug NIOX-14 in vitro

Materials Viruses. We used vaccinia viruses (WWII, Copenhagen strain), cow pox (EQA, Grishak strain), mouse pox (ectromelia, VE, strain K-1) and smallpox (VNO, strain India3a) obtained from the State collection of viral infections and rickettsioses, Federal State Budgetary Institution Scientific Center of the World Bank "Vector" of Rospotrebnadzor (Koltsovo, Novosibirsk Region). Viruses were cultured in a Vero cell culture in DMEM (FBUN SSC VB "Vector" Rospotrebnadzor, Koltsovo, Novosibirsk Region). The virus concentration in the culture fluid was determined by plaque by titration of samples in a Vero cell culture, calculated and expressed in lg of plaque forming units (PFU / ml) [Virology Methods Manual. Edited by: Brian W.J. Mahy and Hillar O. Kangro. San Diego: Academic Press; 1996; Sachs L. Statistical Evaluation. Translation from German. M .: Statistics; 1976]. The virus concentration in the samples used in the work ranged from 5.6 to 6.7 lg PFU / ml. The virus series obtained and used in the work with the indicated titer was stored at -70 ° C.

Cell culture. Viruses were cultured in a Vero cell culture, from the cell culture collection of the Federal State Institution of Biology Research Center “Vector” of Rospotrebnadzor (Koltsovo, Novosibirsk Region) in DMEM medium (Federal State Pedagogical Research Center, Vektor of Rospotrebnadzor, Koltsovo, Novosibirsk Region). Cells were grown in DMEM in the presence of 5% cattle fetal serum (cattle) supplemented with penicillin (100 IU / ml) and streptomycin (100 μg / ml). The same medium was used as a support medium for virus cultivation, but with the addition of 2% serum.

Method for determination of cytotoxicity and antiviral activity of GLF of the NIOX-14 preparation in vitro. Evaluation of the antiviral effectiveness of the substance and GLF of the drug was carried out according to our adapted and modified method [Selivanov B.A., Tikhonov A.Ya., Belanov E.F., Bormotov N.I. et al. Synthesis and antiviral activity of 1-aryl-3- {3,5-dioxo-4-azatetracyclo- [5.3.2.02,6.08,10] dodec-11-en-4-yl} urea. Chemical Pharmaceutical Journal. (2017) 51 (6), 13-17]. In wells of 96-well plates containing a monolayer of Vero cells in 100 μl of DMEM medium with 2% fetal serum, 50 μl of serial dilutions of NIOX-14 substance or GLF solutions with capsule were first added, and then 50 μl of vaccinia virus dilution in dose of 1000 PFU / well. The toxic activity of the drugs was determined by cell death under its influence in the wells of the tablet, in which the virus was not introduced. As controls, monolayers of cells in the wells of the plate into which the virus was added without drugs (virus control) and monolayers of cells in the wells into which neither the virus nor the compound were added (control of cell culture) were used.

The GLF capsule was opened, the contents (200 mg of substance + 200 mg excipients) were poured into one pen bottle, the gelatin capsule itself was placed in another pen bottle. 10 ml of DMSO was added to the contents of the capsule. 10 ml of serum-free DMEM medium was added to the gelatin capsule. The resulting solutions were mixed in equal volumes. To assess the cytotoxicity and antiviral activity of drugs against BOB and VNO, 2 series of serial dilutions with a 3-fold step were prepared. Eight dilutions were used to assess cytotoxicity, starting at a concentration of 1200 μg / ml. Received 1200; 400; 133; 44; 14.8; 4.9; 1.64; 0.54 mcg / ml. Eight dilutions were used to evaluate antiviral activity, starting at 4 μg / ml. Received 4.00; 1.33; 0.44; 0.148; 0.049; 0.016; 0.0054; 0.0018 mcg / ml. Dilutions of the preparations were prepared in DMEM growth medium with 2% serum, and each dilution was added in a volume of 50 μl to the wells of 96-well plates with cells containing 100 μl of culture medium. The concentration of the drug in the wells for determining cytotoxicity was 300; 100; 33.3; 11.11; 3.7; 1.23; 0.411; 0.137 μg / ml, and for determination of antiviral activity it was 1.00; 0.33; 0.11; 0.037; 0.012; 0.0041; 0.0013; 0,00045 mcg / ml.

When assessing the cytotoxicity and antiviral activity of the pure substance NIOX-14 without fillers and capsule components, the concentration of the drug in the wells of the plate with cells (without virus and with the virus) was the same as when studying the GLF of the NIOX-14 preparation.

After incubation for 4 days, the cell monolayer was stained with neutral red vital dye for 2 hours. After removing the dye and washing the wells from its unbound fraction, lysis buffer was added. The amount of dye adsorbed by the living cells of the monolayer was estimated by the optical density (OD), which is an indicator of the number of cells not destroyed under the influence of the virus in the monolayer. OD was measured on an Emax spectrophotometer (Molecular Devices, USA) at a wavelength of 490 nm. Results were calculated using an Emax plate spectrophotometer and SoftMax 4.0 software (Molecular Devices, USA), which automatically calculated a 50% cytotoxic concentration (TC ₅₀ in μg / ml) and a 50% virus inhibitory (effective) concentration (IC ₅₀ in mcg / ml) of the preparations. From the ratio of 50% cytotoxic and effective concentrations, the selectivity index (SI) was determined: SI = TC ₅₀ / IC ₅₀ .

Example 6. The study of the antiviral activity of GLF drug NIOX-14 against variola virus in vitro

At this stage of the study, we studied the activity of the substance and GLF of the NIOX-14 preparation (using DMSO to obtain a solution) against variola virus (VNO, strain India3a) in vitro (Figs. 1 and 2). Dilution and concentration of drugs in wells with cells with and without virus are given in the materials and methods section (example 5).

From the data shown in FIG. Figure 1 shows that the substance of the NIOX-14 preparation with respect to VNO was active at IC ₅₀ ≈ 0.004 μg / ml and cytotoxicity at TC ₅₀ ≈ 300 μg / ml. When statistical processing of the indicators of antiviral activity against VNO and cytotoxicity of the substance NIOX-14 (n = 5), it was shown that IC ₅₀ = 0.004 ± 0.001 μg / ml; TC ₅₀ = 410 ± 101 μg / ml. This is consistent with our previous data on the study of the activity of the substance of this drug against orthopoxviruses [see link for Example 1].

In FIG. 2 presents the results of testing the antiviral activity of GLF of the drug NIOX-14 against VNO in vitro. It was shown that the indicators of anti-VNO activity and GLF cytotoxicity of the NIOX-14 drug (IC ₅₀ ≈0.004 μg / ml at TS ₅₀ ≈300 μg / ml) are comparable with the efficacy and cytotoxicity of the NIOX-14 substance that we previously discovered in experiments with orthopoxvirus [cm. link for Example 1]. When statistical processing of indicators of cytotoxicity and antiviral activity against VNO GLF drug NIOX-14 (n = 5) showed that IC ₅₀ = 0.004 ± 0.001 μg / ml; TC ₅₀ = 370 ± 32 μg / ml.

In addition, it was shown that solutions of the substance and GLF (capsules with the substance and excipients) of the NIOX-14 preparation exhibit the same activity against BOB and VNO in cell culture (Table 1).

Example 7. The study of anti-arterial efficacy of the finished dosage form of the preparations NIOX-14 and ST-246 against variola virus (VNO) in vivo experiments

In experiments on outbred mice of an ICR population weighing 8-10 g infected with live VNO (Ind-3a strain at a dose of 30 ID ₅₀ ), a study was carried out of the specific activity of the finished dosage form (GLF, capsule) of the NIOX-14 drug when administered orally. The comparison groups used the substance NIOX-14, as well as the drug GLF and the substance ST-246, prepared for research purposes. In the control group of infected mice, the methylcellulose solution used to prepare the drug suspensions was orally administered.

Table 2 shows the anti-venipuncture efficacy of GLF of the NIOX-14 and ST-246 preparations against VNO in in vivo experiments.

It was shown that in the groups of animals treated with the preparations, the number of infected mice was 2 times less than in the control group. Moreover, the titers of the virus in the lungs in mice in the groups treated with the preparations were almost 1000 times less than in the control group.

In addition, with the introduction of NIOX-14 and ST-246 substances or finished dosage forms of NIOX-14 and ST-246, no differences were found between the number of infected mice and VNO titers in the lungs of mice.

Example 8. The study of anti-venous efficacy of the finished dosage form of the drug NIOX-14 against ectromelia virus (VE) in in vivo experiments.

In experiments on outbred mice of an ICR population weighing 12-14 g infected with ectromelia virus (VE, strain K-1, at a dose of 10 LD ₅₀ ), a study was carried out of the specific activity of the finished dosage form (GLF, capsule) of the NIOX-14 drug for oral administration the introduction of a dose of 50 μg / g of body weight of the mouse. GLF drug NIOX-14 began to be administered in 1 day. before infection, after 1 and 4 days. after infection and further for 9 days. after the first injection of the drug (a total of 10 times in each group). In the control group, mice infected with VE were orally injected with a solution of methylcellulose used to prepare drug suspensions 1 hour later and for 9 days after VE infection (10 times in total).

Table 3 shows the anti-venipuncture efficacy of the GLF of the NIOX-14 preparation at different times of administration relative to the time of infection of VE in in vivo experiments.

It was shown that in groups of animals treated with the drug 1 day before infection, 1 and 4 days after infection, and then within 9 days after the first injection of the drug, the number of dead mice was significantly less than in the control group. Moreover, the average life expectancy (LNG) of mice in the groups treated with the drug was significantly higher than in the control group of mice infected with VE at a dose of 10 LD ₅ about.

Example 9. The study of the anti-venous efficacy of the finished dosage form of the drug NIOX-14 against smallpox virus of cows (EQA) in experiments in vivo.

In experiments on outbred mice of an ICR population weighing 12-14 g infected with the smallpox virus of cows (EQA, Grishak strain, at a dose of 10 LD ₅₀ ), a study was carried out of the specific activity of the finished dosage form (GLF, capsules) of the NIOX-14 drug when administered orally at a dose of 50 μg / g of body weight of the mouse. The GLF of the NIOX-14 preparation and the NIOX-14 substance were administered 1 hour after infection and then for 9 days after infection of the wok (10 times in each group). In the control group, mice infected with wok were orally injected with the methylcellulose solution used to prepare the drug suspensions 1 hour later and for 9 days after the infection of VE (10 times in total).

Table 4 shows the anti-venipuncture efficacy of GLF of the NIOX-14 preparation and the NIOX-14 substance with respect to EQA in in vivo experiments.

It was shown that in the groups of animals treated with GLF of the NIOX-14 preparation and the NIOX-14 substance, no death of mice was observed, which significantly differed from the mortality rate of mice in the control group. At the same time, the average life expectancy (LSS) of mice in the groups treated with the preparations was significantly higher than in the control group of mice infected with wok at a dose of 10 LD ₅₀ .

Thus, it follows from the foregoing that a domestic dosage form of the drug was created that is effective against VNO and other orthopoxviruses pathogenic for humans and animals for use in medicine and veterinary medicine, which confirms the achievement of the claimed technical result of this invention, which consists in expanding the range of finished dosage forms for treatment and prevention of diseases caused by orthopoxviruses. In addition, in comparison with the prototype, the composition of the target GLF additives of the claimed preparation is improved by reducing their quantity and using harmless and proven components, such as potato starch and sodium chloride.

Claims (3)

1. The oral dosage form of the drug in capsules for the treatment and prevention of diseases caused by orthopoxviruses, containing a chemical compound having anti-orthopoxvirus activity, lactose monohydrate, silicon dioxide, magnesium stearate and microcrystalline cellulose (MCC), characterized in that it additionally contains potato starch, sodium chloride and purified water, as a chemical compound with anti-orthopoxvirus activity, it contains 7- [N- (4-trifluoromethylbenzoyl) -hydrazinocarbonyl ] -tricyclo- [3.2.2.0 ^2,4 ] non-8-ene-6-carboxylic acid (NIOX-14 substance), and anhydrous amorphous silicon dioxide (aerosil) as the silicon dioxide with the following quantitative content of components in one capsule with a total weight of 400 mg:

2. A dosage form according to claim 1, characterized in that the potato starch, sodium chloride and purified water are incorporated in the form of a paste.

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