RU2740660C1 - Antiviral composition - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.

SUBSTANCE: group of inventions relates to pharmaceutical industry, specifically to a composition for relieving clinical symptoms, clinical course and/or cure of a disease caused by exposure to a virus whose genome is encoded by a single-stranded RNA strand and which uses viral RNA-dependent RNA polymerase for its replication. Pharmaceutical composition for alleviating clinical symptoms, clinical course and/or cure of the disease caused by exposure to the virus, genome of which is encoded by a single-stranded RNA suture and which uses viral RNA-dependent RNA polymerase for its replication, containing an effective amount of favipiravir and an effective amount of a zinc compound selected from zinc sulphate, zinc acetate, zinc lactate, zinc-diethyl-bis(N-4-methylthiosemicarbazone), zinc dithiocarbamate, in weight ratio of favipiravir to zinc salt of 1:1–10:1, where the effective amount of favipiravir is 50–800 mg, the effective amount of the zinc salt is 15–250 mg. Method of treating and/or preventing a disease caused by exposure to a virus whose genome is encoded by a single-stranded RNA suture and which uses viral RNA-dependent-RNA polymerase for its replication, involving administering the above pharmaceutical composition to a subject in need thereof.

EFFECT: above described composition has pronounced action to alleviate clinical symptoms, clinical course and/or cure of the disease caused by exposure to the virus, genome of which is encoded by a single-stranded RNA suture and which uses viral RNA-dependent RNA polymerase for its replication.

10 cl, 5 dwg, 4 tbl, 4 ex

Description

The present invention relates to the field of pharmacy and medicine, more specifically to a pharmaceutical composition having excellent antiviral activity, intended to alleviate clinical symptoms, course and cure a wide range of diseases caused by exposure to viruses, the genome of which is encoded by a single-stranded RNA strand and which use viral RNA-dependent RNA polymerase for its replication, including influenza viruses, both high and low virulent strains, coronaviruses, picornaviruses, viruses of the arena, flavi, bunya, toga, calcium and noravirus families, causing severe and mostly lethal fever.

Single-stranded RNA viruses constitute a wide and varied group of pathogens that pose a serious threat to human health. Every year, they affect millions of people around the world and, depending on the family of the virus, cause diseases ranging from simple ARVI affecting the upper respiratory tract to pneumonia and acute distress of the lower respiratory tract syndrome such as SARS and MERS [70], and in the regions Asia and Africa - incurable tropical haemorrhagic fevers such as Dengue virus (DENV) and renewed outbreaks of Chikugunya (CHIKV) and Zika (ZIKV) viruses. For the majority of these viruses, regardless of the family, there is no specific antiviral therapy that leads to a complete cure of patients and does not lead to significant complications, as well as prevents death [68, 69]. Single-stranded viruses with a sense RNA genome use the replicative and translational apparatus of the cell itself, so any drugs acting on the virus will be a priori quite toxic for the cells of the person himself.

In particular, many therapeutic agents that suppress the synthesis of DNA and RNA and have been proven to block the replication of a and b - coronaviruses are cytostatic agents that cause serious toxic side effects [71, 76]. An additional health threat from these viruses is that many RNA viruses, in addition to their affinity for the tropic organ, exhibit high neuroinvasiveness, which leads to more significant damage to the body. These are, in particular, influenza viruses, most coronaviruses, flaviviruses, enteroviruses, Chikugunya [72]. They enter the central nervous system by various routes: through the olfactory neurons of the nasal cavity, sensory fibers of the vagus, innervating organs of the respiratory tract, peripheral neurons, and the facial nerve [73, 74]. Most RNA viruses have learned to "trick" the immune system, slipping imperceptibly past the immediate recognition system and interferon response of the body, causing acute disease, when viral titers are already colossal, and the damage to the tropic organ is extensive and the immune response is either absent or, on the contrary, off scale. Flavi, bunya, Dengue virus, coronaviruses have learned to bypass the immediate immune response by completely different ways of turning off both the expression of interferon genes and the ability of RNases to recognize foreign single-stranded RNA, as well as the NFκB pathway [75]. RNA viruses tend to mutate very quickly, thus adapting to a whole spectrum of zoonotic hosts, and then “jumping” to humans and causing life-threatening conditions requiring hospitalization, as well as periodically causing new unexpected outbreaks in different endemic regions [72].

It is clear that dangerous single-stranded RNA viruses require the development of new approaches and the use of new improved schemes with a multifactorial effect on diseases. And even well-studied influenza viruses that cause annual pandemics with complications, as well as the ubiquitous coronaviruses that lead to a variety of acute respiratory syndromes and viral rhinitis, pharyngitis, laryngitis, etc., are causative agents of much more dangerous lethal conditions.

One of the approaches to the development of highly specific antiviral drugs remains targeted targeting of virus-specific functional molecules, such as structural and non-structural proteins, viral proteases, and RNA-dependent RNA polymerase, which is absent in animal cells. The latter is therefore of particular interest as a target for inhibitors of viral replication.

The public need to create a composition with activity against the above viruses is currently very urgent.

For example, having erupted in Wuhan (China) in December 2019, a new virus from the coronavirus family (SARS-CoV-2), which causes SARS, turning into acute respiratory distress syndrome (ARDS) with systemic hypoxemia and inflammatory endovasculitis, has spread rapidly. to different parts of the planet, plunging it into a long-unprecedented pandemic. In less than 2 months, as of February 10, 2020, the virus reached 25 countries, 4 continents, and the number of confirmed cases exceeded 400,000. At that time, the mortality risk was about 2%. By April 15, 2020, the number of confirmed cases of the disease worldwide amounted to 1 991 562 people, which claimed 130 885 lives [1]. These indicators reduce the mortality rate to 0.066%, however, as the clinical practice of the last 3.5 months has shown, it varies in different groups of the population, depending on age, representing the greatest danger of its aggressive course for people aged 70 and over with concomitant morbid states.

Unfortunately, at the moment there are no drugs that counteract the infection, there are also no exact established clinical prescriptions, since the course of the virus is not uniform as the disease progresses in an individual and is different in different groups of the population, both age and ethnic. The mechanism of the onset of COVID-19 symptoms is being studied by many scientific and clinical laboratories in an emergency mode, directly in the process of a pandemic, providing doctors and scientists with only fragmentary knowledge about the nature of the virus and the physiology of the course of the disease, in particular the scenario leading to the development of acute respiratory distress syndrome, which does not always successfully removed by supporting the patient on a mechanical ventilation device (IVL).

However, the belonging of SARS-CoV-2 to a fairly well-studied family of b-coronaviruses, which means that understanding, in general, the life cycle of the virus in the human body, allows us to make some clinical assumptions and choose selective and effective therapeutic solutions from the arsenal of drugs used for subtypes of that the same family - coronaviruses, based on the experience of outbreaks of SARS and MERS in 2002 and 2012, respectively, proceeding with similar clinical symptoms and much higher mortality.

2019-nCoV or SARS-CoV-2 is a capsid coronavirus of the b-coronaviridae family that carries a single-stranded RNA sense strand. By analogy with SARS and MERS, the 2019-nCoV genome encodes non-structural proteins (3-chymotrypsin-like protease, papain-like protease, helicase, RNA-dependent RNA polymerase (RNAzRNApol / PsRn)), which ensure replication and assembly of viral particles, structural proteins, including S-glycoprotein, necessary for the recognition of the cell receptor and entry into the cell, M (membrane protein), E (envelope protein), N (nucleocapsid protein), and auxiliary proteins that play a role in maintaining the vital activity of the virus, its replication and genomic stability [2,3]. All 5 non-structural proteins and RNA-s-RNA-pol are direct targets for a potential drug against the 2019-nCoV coronavirus.

Analysis of the 2019-nCoV genome (Fig. 1) showed that catalytic proteases are rather conserved in the family of b-coronaviruses, being homologous to the corresponding enzymes of the SARS and MERS viruses [4]. Therefore, at the moment, multiple international studies are underway on the repositioning of successfully working drugs used from SARS and MERS.

Thus, there are reasonable assumptions about candidate substances for potential pharmaceutical use for the above purposes.

Potential candidates for repositioning for 2019-nCoV therapy.

Medicines that interact with the viral replication apparatus.

Approved nucleoside analogs, in particular favipiravir, have good clinical potential against viruses using RNA-dependent RNA polymerase in replication, and therefore, in particular, against 2019-nCoV. Nucleoside analogs, both adenine and guanine derivatives, are directly aimed at blocking the activity of RNA-dependent RNA polymerase and blocking the synthesis of the viral RNA chain for a wide range of RNA viruses, including the human coronavirus family [5]. Favipiravir (T-705), a guanine analogue approved in clinical practice for the treatment of influenza, has been proven to effectively block the RNA-dependent RNA polymerase of influenza viruses (various types), Ebola virus, yellow fever, Chikungunya, noroviruses, and enteroviruses [5]. A recent study showed its efficacy against 2019-nCoV (EC ₅₀ = 61.88 μM in Vero E6 cell culture) [6, 33]. The study tested the combined use of favipiravir with interferon-α (ChiCTR2000029600) and favipiravir with balokvavir marboxil (an approved cap-dependent endonuclease inhibitor for influenza) (ChiCTR2000029544) in patients with SARS-CoV-2. Favipiravir has a proven targeting effect against SARS-CoV-2. This is easily explained by its properties as an inhibitor of RNA-dependent-RNA polymerase, proven in many studies of RNA viruses of different families, all using this enzyme, listed below.

The mechanism of action of Favipiravir

Compound 1

Favipiravir (Compound 1) was initially discovered as a potent inhibitor of viral replication in studies on the influenza virus [7]. In in vitro studies on a model of Madin Darby Canine Kidney cell lines infected with the influenza virus, the effect of favipiravir was weakened in the presence of purine nucleosides or purine bases, indicating a selective competition of the substance with purine bases in the synthesis of new RNA strands during replication. Favipiravir was added to the MDCK cells, and the cell metabolites were then analyzed by high pressure liquid chromatography. As a result, favipiravir ribofuranosyl-5'-triphosphate (favipiravir - RTF), favipiravir-ribofuranose (favipiravir R) and favipiravir ribofuranosil 5'-monophosphate (favipiravir RMF) were identified, indicating that the action of favipiravir in PMF occurs, indicating that the action of favipiravir in PMF occurs. Chemically synthesized favipiravir - RTF inhibits viral RNA-dependent-RNA polymerase at nano- and micro-molar concentrations (Fig. 2, 3) [7]. These data showed that favipiravir as a substance is a pro-drug and, being phospho-ribosylated in the cell to favipiravir-RTF, inhibits viral RNA-dependent RNA polymerase.

Currently, it is believed that favipiravir integrates into the growing chain of viral RNA, interrupting it, and simultaneously structurally interacts with the domains of the viral RgRn, thus inhibiting both transcription and translation of the viral genome [8]. Also in studies on the influenza virus, it was shown that favipiravir leads to lethal mutagenesis of the viral genome, dramatically reducing viral titers in cell culture [9]. It is significant that for the entire time of many years of research on this substance, it was not possible to identify a single viral strain resistant to favipiravir. Based on the described mechanism of action, mutagenesis caused by favipiravir can also occur in other RNA viruses, as well as inhibition of RgRp, which makes it a powerful, effective and universal inhibitor of a whole group of epidemiologically significant viruses of various families that cause serious, often fatal diseases [10-12].

RNA synthesis is an integral part of human cell life. Unlike viruses, humans do not have RgRp, but there is DNA-dependent RNA polymerase (DsRp) and DNA-dependent DNA polymerase. Favipiravir-RTF is able to inhibit these two human enzymes at different concentrations of the active drug. It was shown that favipiravir-RTF blocks influenza PgRp at an IC _{50 of} 0.341 μmol / liter, but did not have any inhibitory effect on DNA polymerase α, β, γ at concentrations up to 1000 μmol / liter. Favipiravir insignificantly inhibited human RNA polymerase II, which belongs to DsRp, but at high concentrations - IC ₅₀ 905 μmol / liter [13]. At concentrations of 637 μmol / liter in vitro in MDCK cells, favipiravir blocked neither cellular DNA synthesis nor cellular RNA synthesis.

This universal mechanism of action of favipiravir is specific to the underlying enzyme of the viral replicative apparatus, suggesting a wide spectrum of antiviral activity of this substance, which has been demonstrated in many of the studies listed below.

Antiviral activity of favipiravir

Using favipiravir to treat COVID-19 patients.

According to the global report on pharmaceutical substances, favipiravir is currently included in clinical trials against COVID-19 disease, based on its proven mechanism of action against viral RhRp (https://www.clinicaltrialsarena.com/comment/influenza-favipiravir-covid- 19/). In a clinical study by the National Center for Clinical Research on Infectious Diseases in Shenzhen, favipiravir was administered to 340 patients (age groups and morbidity not specified) in two doses (2 doses) 1600 mg on the first day and two doses of 600 mg for the next 13 days in addition to inhalation aerosol of interferon-alpha (5 million units * 2 / day). This dosage led to a more rapid disappearance of the virus to values undetectable in the blood) than in the group of patients taking a combination of anti-HIV proteases - lopinavir / ritonavir, with a median removal of viral particles at 4 days, versus 11, respectively, and was assessed by CT control thoracic region. According to the current study, the use of favipiravir reduces the number of detectable viral particles in the blood, which means that it demonstrably inhibits viral replication, delays the development of an aggressive course of COVID-19 or prevents it altogether.

Taking into account the versatility of the mechanism of action of favipiravir, this drug can be positioned as a broad-spectrum RgRp inhibitor and can be successfully used to combat other diseases caused by RNA viruses equipped with a RgRp replicative apparatus, as described below. This is especially true in light of the increased incidence of zoonotic transmission of viruses around the world. Thus, the outbreak of the Ebola and Lassa virus in West Africa in 2014 caused a great wave of social concern about possible subsequent outbreaks of deadly viruses in Asia and Africa [15, 16].

Effect of using favipiravir against influenza virus

An influenza epidemic occurs around the world every year. The disease is caused by strains of the influenza virus of varying virulence. The highly pathogenic avian influenza A (H5N1) virus caused the first outbreak in Hong Kong in 1997 and continues to cause local outbreaks of this type of influenza every year. The 2013 avian influenza A (H7N9) epidemic in China and the 2009 influenza A (H1N1) pandemic resulted in 17,700 deaths, and influenza is still a major public health problem worldwide, not only in terms of morbidity, but also for critical health complications that it causes [14]. Epidemic A (H1N1) showed that this strain is resistant to ocetalmivir (tamiflu), a neuramidase inhibitor, and to amantadine, an inhibitor of the non-structural protein M2. In this regard, in medical practice, a drug of a different mechanism of action is urgently needed.

Favipiravir (T-705; 6-fluoro-3-hydroxy-2-pyrazinecarboxamide) is effective against a wide range of influenza strains, including A (H1N1) (pandemic 2009), A (H5N1) and A (H7N9), due to the fact that that viral PgRp mistakenly takes the metabolite favipyrovir-RTF for a purine nucleotide. Favipirovir has undergone Phase III studies in Japan and Poi in the United States for influenza treatment.

In addition to antiviral activity against influenza, favipiravir inhibits the replication of arenaviruses (Yunin, Machupo and Pichinde), phleboviruses (Rift-Valle fever, phlebotoma fever and Punta Toro fever virus), hantaviruses (Maporal, Dobillrava) and Prospectus flaviviruses (yellow fever and West Nile fever); enteroviruses (full and rhinophyiruses); Western encephalitis alphavirus, respiratory syncytia paramyxovirus and norovirus [8].

Use of favipiravir in in vitro influenza models

In in vitro studies, favipiravir showed high antiviral activity against all strains of influenza virus, A, B and C. Based on the calculation of plaque-forming units (PFU) in the MDCK cell culture, the effective concentration indicator (hereinafter EC ₅₀ ) ranged from 0.014 to 0.55 μg / ml [17]. The drug is not cytotoxic to MDCK cell culture. Table 1 shows the measured activities of favipiravir against 53 strains of influenza virus, including seasonal strains A (H1N1), A (H3N2), and strains of influenza type B; A (H1N1) pdm09 pandemic virus, highly pathogenic avian influenza A (H5N1) isolated from humans, strains A (H1N1) and A (H1N2) isolated from pigs, and A (H2N2), A (H4N2), A (H7N2) [78]. This study included a large number of strains resistant to existing anti-influenza drugs, such as amantadine, rimantadine, ocetalmivir, zanamivir [77].

The 50% cytotoxic concentration of favipiravir (CC ₅₀ ) in MDCK cells was over 2,000 μg / ml, demonstrating highly selective inhibition of influenza virus replication (selective index over 3000). The only limitation in the use of favipiravir is its embryo- and teratogenicity [18], thus preventing its use for pregnant and lactating women.

Use of favipiravir in in vivo influenza models

In vivo in mouse models of viral infection with lethal doses of H3N2 (A / Victoria / 3/75), H3N2 (A / Osaka / 5/70) or H5N1 (A / Duck / MN / 1525/81) strains, favipiravir was administered one hour after infection ... Survival of mice at doses from 30 mg / kg / day 2 or 4 times a day was significant, while all infected mice in the control group died (Fig. 4).

At 60 to 300 mg / kg / day, favipiravir has been shown to be effective in lowering viral load in the lungs of H1N1-infected mice (A / California / 04/09), as well as in delayed use, up to 96 hours after infection [19, twenty].

Favipiravir showed a significant therapeutic effect in comparison with ocetalmivir in mice, which were injected with a dose of the virus 100 times greater, and the treatment itself was delayed by 96 hours post-infection [79].

Efficacy of favipiravir for other families of RNA viruses in in vitro and in vivo studies.

Arenaviridae

Arenaviruses cause fatal human diseases [21], for which there are no antiviral drugs, except for ribavirin, which has a pronounced toxic effect.

Use of favipiravir in in vivo arenavirus models

In vitro, favipiravir has shown greater selectivity than ribavirin. When measuring the cytopathic effect in the cell culture, the EC ₅₀ values for the drug were 0.79-0.94 μg / ml for the Junin, Pichinde and Takaribe viruses. Also, the viral load when using favipiravir significantly decreased by the third day. In the study by counting the foci of hemolysis EC ₉₀ s against the highly pathogenic Romero strain of the Guanarito virus JUNV (Romero) and the Machupo virus it was 3.3-8.4 μg / ml (21-53 μM) (Table 2) [80].

When favipiravir was administered orally in a model line of hamsters infected with the Pichinda virus, the drug prevented death, reduced the number of viral titers in the blood and tissues, at doses of 60 mg / kg / day, it twice prevented liver destruction when used for 7 days, starting at 4 hours post-infection [22] The viral load was also significantly reduced at the beginning of treatment from 4, 5 and 6 days post-infection. When used in dosages of 100 mg / kg / day, the survival rate of animals was significantly increased [23].

In the model of infection of guinea pigs, favipiravir demonstrated its effectiveness even after the onset of acute symptoms of the disease [24].

When using favpiravir at a dose of 300 mg / kg / day, it showed significant effects on survival: 100% of the animals participating in the experiment survived, at a dose of 150 mg / kg / day, the indicators decreased to 50 and 25%, the animals retained body weight, in their temperature dropped to normal and all indicators were generally better than those for ribavirin at a dosage of 50 mg / kg / day. The concentration of aspartate aminotransferase (AST), a prognostic marker of the severity of Lassa fever in the blood, significantly decreased by the tenth day of illness during treatment with favipiravir. The viremia of the mean infectious dose per ml of eluate decreased to an average of 2.1, 1.3, and 1.6 log ₁₀ CCID ₅₀ / ml in the groups treated with high and medium doses of favipiravir and ribavirin, respectively.

The efficacy of oral administration of favipiravir has also been shown in models infected with the lethal Lassa virus in guinea pigs and mice [25]. The therapeutic effect was observed on the 2nd day after infection. Subcutaneous administration of favipiravir at a dosage of 300 mg / kg / day once a day reduced temperature, prevented weight loss, and increased animal survival. The effects of favipiravir were many times greater than the therapeutic effects of ribavirin at a dosage of 50 mg / kg / day. A significant improvement in the survival rate of guinea pigs infected with Lassa virus was observed even on days 5.7 and 9 of post-infection [25].

Bunyaviridae

Viruses of the Bunyavirida family, including La Croce virus (LACV), Rift-Vallee fever virus (RVFV), Crimean-Congo hemorrhagic fever virus (CCHFV), acute fever with thrombocytopenic syndrome (SFTSV) and hantavirus, cause severe concomitant hemorrhagic fever and renal complications.

In vitro studies have shown the superiority of favipiravir over other drugs in targeting, effectiveness and speed of action against a number of similar viruses (Table 3 [8, 26]). EC ₅₀ values in studies of plaque-forming units (PFU) in cell culture started at 0.9-30 μg / ml of the drug for Lassa, Punta Toro, Rifa-Valle viruses, acute fever (SFTSV), phlebotomic fever, and Dobrava, Maporal and Prospectus hantaviruses Hill [27].

Use of favipiravir in in vitro and in vivo models of bunyaviruses

Thrombocytopenia acute fever virus (SFTSV) emerged several years ago as a seasonal disease in China, Korea, and Japan [28, 29]. Favipiravir inhibited SFTSV replication in cell culture with EC50 values of 0.71-1.3 μg / ml.

The therapeutic effect of favipiravir has been demonstrated in interferon- a receptor knockout (IFNAR ^{- / -} ) murine models that do not elicit an immediate immune response.

With oral administration of favipiravir at a dose of 300 mg / kg / day for 5 days, starting from day 3 post-infection, all experimental mice survived (P <0.001), and starting from 4 and 5 days post-infection, the survival rates in the group significantly improved ... On the basis of data from in vivo studies in Japan, the drug entered clinical trials and successfully completed them by now [30]. It showed the efficacy of favipiravir even after the onset of the disease and the onset of clinical symptoms (Table 4) [8].

Flaviviridae

Favipiravir blocks the replication of viruses of the flaviviride family, including yellow fever virus (YFV) and West Nile virus (WNV) [31,32], however, at higher concentrations than are required to block influenza virus activity.

EC _{90 of} favipiravir against YFV is 51.8 μg / ml in in vitro studies to determine the release of active viral particles in Vero cell culture.

Use of favipiravir in in vivo flavivirus models

YFV-infected hamsters were treated orally with favipiravir at doses ranging from 200 to 400 mg / kg / day for 8 days, starting 4 hours prior to infection. This therapy significantly reduced the mortality rate of animals at the start of treatment [31].

Complete recovery was achieved with an injection of 400 mg / kg / day starting on day 2 post-infection.

In vitro and in vivo antiviral efficacy of favipiravir against West Nile virus was achieved at EC ₅₀ concentrations of 53 μg / ml in Vero cell culture.

Oral administration of favipiravir at doses of 400 mg / kg / day 2 times a day, 4 hours after infection, saved 90% of mice from death, and also significantly reduced the expression of viral proteins and viral RNA in brain tissues. The same efficacy was shown in lines infected with WNV, hamsters at the same doses. The envelope proteins of the WNV virus were not detected in the brains of the treated animals.

Zika virus (ZIKV) is a newly emerged arbovirus of the Flaviviridae family, transmitted mainly by mosquito bites. The data indicate a link between infection in the developing fetus and microcephaly. Favipiravir blocked ZIKV replication in Vero cell culture at EC ₅₀ from 3.5-3.8 μg / ml [34].

Togaviridae

Favipiravir exhibits antiviral activity against Western Equine Encephalitis Virus (WEEV) in Vero cell culture, reaching EC ₉₀ at 49 μg / ml [35].

In WEEV-infected mice, oral administration of favipiravir significantly increased the survival and lifespan of the infected animals when twice administered at a dose of 400 mg / kg / day for 7 days, starting at the 4th hour post-infection. Viral titers in brain tissues were reduced on the 4th day of post-infection, however, a complete cure of infection with the use of favipiravir did not occur.

Favipiravir showed antiviral activity against Chikungunya virus (CHIKV) in Vero cell culture, reaching EC50 at 0.3-9.4 μg / ml. In CHIKV-infected mice, oral administration of favipiravir improved survival rates at two-fold doses from 300 mg / kg / day, starting one day before or 4 hours after infection [36].

Picornaviridae

Use of favipiravir in in vitro models of picornaviruses

Replication of vesicular stomatitis enterovirus was inhibited by favipirovir in in vitro studies with an EC50 of 14 μg / ml [37, 38].

Favipiravir also blocked the replication of poliomyelitis virus in Vero cell culture and rhinovirus in HeLa cell culture at EC ₅₀ s at 4.8 and 23 μg / ml, and with a selectivity index of 29 and> 43, respectively [39]. Favipiravir inhibited Enterovirus replication at an EC ₅₀ of 23 μg / ml [40].

Caliciviridae

Favipiravir is active against murine noravirus with EC ₅₀ values from 39 μg / ml in studies of viral plaque counting in the RAW 264.7 murine macrophage cell line. Real-time PCR revealed the blocking of RNA synthesis using favipiravir with an EC ₅₀ of 19 μg / ml [41].

Use of favipiravir in in vivo models of the noravirus genus from the Calicivirus family

In a mouse model of persistent infection, oral administration of favpiravir at a dose of 600 mg / kg / day twice for 8 weeks, after 4 weeks post-infection, resulted in a significant decrease in viral titers in the feces of mice and in the number of norovirus-positive mice. Scientific evidence also confirms that favipiravir-RTF inhibits the RNA polymerase activity of human Noraviruses [42].

Filoviridae

Favipiravir showed antiviral activity against the Zaire Ebola virus (Mayinga 1976 strain) in a Vero E6 cell culture with EC ₅₀ at 10.5 cg / ml. In a strain of mice infected with the Mayinga 1976 strain lacking the interferon-alpha receptor (IFNAR ^{- / -} C57BL / 6), oral administration of favipiravir avoided death and reduced viral titers in the blood when twice applied from 300 mg / kg / day for 8 days from day 6 post-infection, whereas all mice in the placebo group died [43]. Similarly, in the IFNAR ^{- / -} IFNAR ^{- / -} interferon receptor knockout A129 strain infected with Ebola E718 shitamm, oral favipiravir treatment completely saved all infected mice from death when the drug was twice administered at a dose of 300 mg / kg / day for 14 days, starting from 1 hour after infection [44].

In the 2014 Ebola outbreak in West Africa, the French Institute for Health and Medical Research (INSERM) and the Government of Guinea conducted a clinical trial of favipiravir in patients [45]. Favipiravir was well tolerated by patients and reduced deaths in patients with low viral titers [46]. A group of Chinese researchers have also noted an increase in the survival of Ebola patients with favipiravir in Sierra Leone [47].

Rhabdoviridae

The activity of favipiravir against rabies virus (RABV) was detected on the Neuro-2a murine neuroblastoma cell line with EC ₅₀ s at 5.1–7.0 μg / ml [48]. Favipiravir significantly reduced morbidity and mortality in RABV-infected mice when taken orally at doses of 300 mg / kg / day, twice for 7 days, starting at 1 hour post-infection.

All the generalized data described above on the indisputable efficacy of favipiravir against a very wide range of RNA viruses show that this drug perfectly “closes” the still effectively uncovered niche of incurable, acute and fatal viral diseases, including a wide range of tropical fevers. Prior to studies on favipiravir, ribavirin and / or alpha-interferon were used against the aforementioned RNA infections, but the former has significantly less antiviral activity and effectiveness against RNA viruses, and both drugs, with long-term use, lead to debilitating side effects. Favipiravir, unlike other drugs that block RNA polymerase, is well tolerated in clinical trials and deserves widespread use due to the knowledge and universality of the mechanism.

It is clear that dangerous single-stranded "sense" -RNA viruses require the development of new approaches and the use of new improved schemes with a multifactorial effect on diseases. And even well-studied influenza viruses that cause annual pandemics with complications, as well as the ubiquitous coronaviruses that lead to a variety of acute respiratory syndromes and viral rhinitis, pharyngitis, laryngitis, etc., are causative agents of much more dangerous lethal conditions.

One of the most effective approaches to the development of highly specific antiviral drugs remains targeted targeting of virus-specific functional molecules, such as structural and non-structural proteins, viral proteases, and RNA-dependent RNA polymerase, which is absent in animal cells. The latter is therefore of particular interest as a target for inhibitors of viral replication.

Favipiravir, relying on the universal and proven mechanism of its action in relation to RgRp, can also facilitate the course of viral diseases, with the timely start of admission, and also significantly reduce the viral load in the course of diseases with complications.

However, given the multicomponent and complexity of the clinical picture of viral diseases, in particular, the COVID-19 demonstrated today, the ability of viruses to mutate, as well as the restructuring of the physiological mechanisms of fighting the virus inside the body, there is a need to create effective antiviral combinations of favipiravir with other drugs, relying on, both on the cell cycle of the virus in the body, and on the physiological response for an effective fight.

Previously, it has been suggested that clinically approved retroviral protease inhibitors, ritonavir and lopinavir, are active against SARS and MERS viruses, hypothetically by acting on the chymotrypsin-like proteases of both coronaviruses. Clinical trials have achieved an improvement in the clinical performance of SARS patients treated with the combination of drugs [2], which was the beginning of studies of the combination of lopinavir with ritonavir (ChiCTR2000029539) for patients with 2019-nCoV. Within the framework of the current pandemic, several attempts have been made to influence the replication of the virus by repositioning the anti-HIV proteases: ritonavir, enhanced by the action of lopinavir, however, it is obvious that these attempts could not give significant clinical results, since the site of recognition and cleavage of the polypeptide chain by HIV proteases does not coincide with papain- and chymotrypsin-like proteases, and, accordingly, the structure of their inhibitors does not coincide, and ritonavir did not show an inhibitory effect on other proteases.

Cai et al conducted an open-label, controlled study comparing the effects of favipiravir (1600 mg twice daily on day 1 followed by 600 mg twice daily from days 2 to 14) versus lopinavir and ritonavir (400 mg / 100 mg twice) in combination with interferon-a1b at a dosage of 60 mg 2 times a day by inhalation for the therapy of COVID-19 [66]. Preliminary results show significant clinical differences between favipiravir (35 patients) versus lopinavir / ritonavir (45 patients), with a median viral load clearance over time of 4 days versus 11 days assessed by improvement in chest CT (91.43% vs. 62.22%) ... The poor clinical results of this study regarding the use of antiretroviral proteases clearly indicate the need for the use of highly specific inhibitors of chymotrypsin-like and papain-like proteases.

Lopinavir, which inhibits chymotrypsin-like proteases of the 3CL ^pro type, in theory, could act as a successful therapeutic agent, however, it has been shown that the proteases of the SARS-CoV-2 coronavirus contain a C2-symmetric interaction site where lopinavir cannot enter. Computer modeling in silico for the search for specific structural blockers of chymotrypsin-like proteases based on available approved drugs has shown that among retroviral proteases, darunavir can interact with the 3CL ^pro pocket specific for SARS-CoV-2 proteases [67].

However, the introduction of a large number of additional active components cannot be a solution to the problem, since it is often associated with the occurrence of complications in patients due to the side effects of each of the components of the combination. In addition, in this case, it is necessary to check the safety and effectiveness of the mutual action of each of the active components, which is objectively difficult for multicomponent compositions.

The objective of the present invention is to provide a composition that exhibits prophylactic and / or therapeutic efficacy against a wide range of diseases caused by exposure to viruses, the genome of which is encoded by a single-stranded RNA strand and which use viral RNA-dependent RNA polymerase for its replication, which includes an effective the amount of favipiravir in combination with an effective amount of another agent. Moreover, the new composition should act more effectively than favipiravir alone.

Based on the above data, the authors propose the use of a combination of favipiravir with at least one zinc compound for therapeutic approaches to treating viruses using Pgrp for replication and chymotrypsin-like proteases for translation, confirming their effectiveness in in vitro and in vivo studies.

It has been found that the combined use of at least one zinc compound and favipiravir in effective amounts leads to a significant enhancement of the mutual effect against viruses whose genome is encoded by a single-stranded RNA strand and which use viral RNA-dependent RNA polymerase for their replication. in the absence of additional side effects. The therapeutic effect of the combination may be due to the multidirectional effect of the components of the combination on the virus. This multidirectional action allows maintaining the necessary high activity even in cases of mutation of the target virus, which is especially important with viruses that have relatively recently overcome the interspecies barrier. The conclusion about the enhancement of the antiviral therapeutic effect of introducing an effective amount of zinc and favipiravir into the composition is made on the basis of the described examples and observations. An accurate explanation of the mechanisms of such unexpectedly effective joint action requires further in-depth research.

The above properties can be regarded as a technical result manifested when using the present invention - a pharmaceutical composition for alleviating clinical symptoms, course and cure of a disease caused by exposure to a virus whose genome is encoded by a single-stranded RNA strand and which uses viral RNA-dependent RNA polymerase for its replication containing an effective amount of favipiravir and at least one zinc compound.

It has previously been shown that the combined use of Zn2 ⁺ and ionophores Zn2 ^+, such as pyrithione (PT) in the concentrations (2 uM Zn2 + and 2 Fr .mu.M) is effective in reducing virus replication rate and also influences the protsessirovanie viral polyprotein chain RNA viruses carrying a sense RNA strand in the genome and using PsRn [50].

It is reliably known that zinc plays one of the key roles in the homeostasis of the immune system [83]. Zinc deficiency is associated with limitation and deterioration of the effectiveness of cellular immunity, accompanied by an imbalance between Th1 and Th2 [84, 85]. Zinc deficiency also leads to decreased synthesis of interleukins and interferon gamma [86].

At the level of cellular mechanisms, zinc is required to maintain the catalytic activity of many cellular enzymes, and also binds to proteins containing zinc-finger sequences and playing important roles in maintaining metabolic and immune homeostasis [87]. Therefore, it is not surprising that zinc salts have the ability to block the proliferation of a wide range of viruses, including HIV, gastroenteritis virus, herpes simplex virus, vaccine virus, acute respiratory syndrome virus (SARS-CoV), equine encephalitis, rhinovirus, respiratory syncytia virus [88-94 ]. The antiviral effect is achieved through direct inhibition of viral RNA-dependent RNA-polymerase and indirect blocking of catalytic processing of polypeptide viral chains [94].

Direct inhibition of PsRp corona and arteri-viruses by Zn2 + ions in an in vitro model.

The addition of ZnOAc ₂ to the active RNA polymerase of equine encephalitis viruses (EAV) and SARS-CoV led to a strong dose-dependent inhibition of the enzymatic activity of the enzyme of both viruses, similar to that observed in studies on the rate of RNA synthesis for these viruses. In comparison with the activity of other divalent atoms, in particular, Co ²⁺ and Ca ²⁺ , which usually bind polypeptide chains at oxygen atoms rather than at sulfur-containing radicals, Zn ²⁺ was the most effective inhibitor of the SARS-CoV nsp12 protein P3Pn. At a concentration from 2 mM to 6 mM Zn ^{2+, the} effect of complete blocking of the synthesis of RNA strands, as well as the production of sgRNA in vitro, was achieved [92].

Inhibition of PsRp by Zn2 + ions of coronavirus and respiratory syncytium virus (RSV) in an in vitro model.

USP-grade zinc acetate (AMEND Chemical, Irvington, N.J.) or zinc lactate or sulfate (Sigma Chemical, St. Louis, Mo.) were used to prepare zinc solutions. Stock solutions were used at a concentration of 250 mM salts in deionized water, sterilized by filtration through a 0.22 µm pore syringe filter.

(a) Yield of viral particles. When zinc was added during the absorption of the virus by cells, penetration and release of viral particles, a significant decrease in the yield of viriomas was observed, even in the presence of the lowest concentrations of 10 μM solution, in comparison with the "empty" - salt-free control and control with salts of other divalent metals - Mg ²⁺ and Co ²⁺ . The average viral titers were 5.3 log ₁₀ PFU / ml (standard mean error [SEM] = 0.7 log ₁₀ PFU / ml) versus 2.8 log ₁₀ PFU / ml (SEM = 0.7 log ₁₀ PFU / ml) for any zinc salt ( P <0.01). A decrease in viral titers of 10,000 or more times was achieved at a zinc salt concentration of 100 μM. In contrast, calcium acetate, magnesium sulfate and manganese sulfate did not show similar inhibitory properties even at the highest concentration of 10 mM.

(b) Complete inhibition of RSV viral plaque formation was observed at 10 mM zinc salt concentrations, while 88% inhibition was already achieved at 1 mM concentration (SEM, 4%; P <0.001) [94].

(c) Effect of zinc on viral penetration.

A significant blocking effect of zinc salts on viral entry into the cell was found at concentrations of 100 μM, 1 mM, and 10 mM compared to the effect of control salts and a control culture, where the average viral titer was 6.6 log ₁₀ PFU / ml (SEM, 0.2 log ₁₀ PFU / ml).

Zinc compounds also block other RNA viruses, in particular HIV, due to inhibition of RNA transcription on the DNA template [95].

Inhibition by Zn2 + ions of RsRp RNA of vesicular-invasive foot and mouth disease virus.

Zinc at a concentration of 0.5 mM was added to the culture of FMD-infected cells over time at intervals. It blocked the production of new viral particles almost instantly, provided it was added to the cells no later than 90 minutes after infection, and significantly reduced the number and yield of viral particles when added after 120 and 150 minutes post-infection [96]. The 0.5 mM Zn2 + concentration shown to be sufficient to completely inhibit the replication of vesicular vesicular foot and mouth disease is comparable to the results in blocking the replication of human rhinovirus, encephalomyocarditis, and poliomyelitis [97]. As well as for these viruses, with an increase in the intracellular concentration of zinc ions in the shunts, the amount of unprocessed polypeptide chains of viral protein precursors increased. The main structural proteins of the FMD capsid, weighing 30,000 Daltons, were no longer produced in the required quantities at a zinc concentration of 0.5 mM, apparently due to the effect of zinc on the proteolytic processing of precursor proteins [96].

However, when zinc enters the body, there may be a problem of its excessive accumulation locally in a certain organ, as well as the problem of a lack in the intracellular system where it is needed.

In this regard, it is recommended to use it together with "ionophores" that improve its transmembrane delivery, such as dithiocarbamates in equimolar concentrations and zinc - diethyl bis (N4-methylthiosemicarbazone) - Zn-DTSM. Zn-DTSM significantly increased the intracellular zinc content (p <0.0001; + 287% in comparison with the control), and at the same time did not significantly change the content of other metals (Fe and Cu).

The effects disclosed above can be explained by the fact that zinc ions are involved in a large number of different cellular processes and are absolutely necessary for the correct folding of cellular enzymes and transcription factors. Hence, it is possible to assume that Zn2 ⁺ can also act as a co-factor for viral proteins. The intracellular concentration of Zn2 ⁺ ions is normally maintained at low concentrations using metalloteoneins, most likely because Zn2 ⁺ can act as a secondary messenger as a trigger of apoptosis and a substance that reduces an increased level of protein synthesis [51, 52]. In many studies in cell culture, it was shown that increased concentrations of Zn2 ⁺ ions, together with other components that stimulate an increase in its intracellular concentration, in particular with chinokitol, pyrrolidine dithiocarbamate and pyrithione, inhibit the replication of RNA viruses, including influenza virus [53] , respiratory syncytia virus [54] and several species of picornaviruses [55-58]. Despite the fact that these earlier studies do not reflect the specific mechanism of action of Zn2 ⁺ , nevertheless, they conclusively describe that zinc ions are involved in viral replication [50].

For corona- and arterivirus infections, a characteristic feature of the replication cycle is the transcription of the 5'- and 3'-terminal small fragments of subgenomic messenger RNA (sg mRNA), from which the translation of structural and non-structural auxiliary viral proteins occurs [59, 60]. In vitro cell culture studies, as in the case of the picornavirus family, it was shown that zinc ions inhibit certain sites of proteolytic processing of polyproteins of the coronavirus replicase complex in infected cells and cell-free systems [61, 62]. In particular, zinc pyrithione ionophore in combination with zinc ions has been shown to be a potent inhibitor of the replication of SARS coronavirus (SARS-CoV) and equine arteritis virus (EAV). In addition, this effect was observed in studies on the recombinant RgRp SARS-CoV and EAV [63].

In a study on coronaviruses, in particular on the SARS-CoV virus, which is similar in structure to SARS-CoV-2, it was shown that increasing the concentration of Zn2 ⁺ to micromolar concentrations in the culture of infected cells dramatically reduces the efficiency of PgRp binding to the matrix of genomic and messenger viral RNA [50 ] (Fig. 5).

Most families of RNA viruses express their genome in the form of long polypeptide chains - precursors of functional proteins that are processed by viral and cellular proteases. This strategy of combined transcription and translation allows simultaneously and with minimal energy and biochemical costs to activate a large number of diverse proteins from one precursor. Viral proteases, although different in structure, are highly specific for their polypeptide substrates and the targeted recognition of individual cleavage sites. The processing cascade is a tightly regulated process, which in some cases involves the participation of co-factor proteins - modifiers of the action of individual proteinases [64]. Many families of single-stranded sens-RNA viruses have similar chymotrypsin-like and papine-like proteases, in particular, Picorna-, Flavi-, and Coronaviridae [65].

We hypothesized that inhibition of these, as well as other types of proteases specific for RNA (+) - viruses, is a successful antiviral therapeutic strategy, in parallel with the blocking of viral replication described above. Based on studies of the inhibition of polypeptide processing sites of coronaviruses using zinc, as well as on the need to perform polypeptide processing for the vital activity of the virus, inhibitors of papain-like and chymotrypsin-like proteases, in particular, encoded in the SARS-CoV genome, should be effective in the therapy of RNA viruses. -2. And their combined use with favipiravir will lead to an even more effective and rapid inhibition of the virus, not only of the coronavirus, but also of most of the RNA (+) viruses discussed above.

Thus, it has been found that the addition of at least one zinc compound to favipiravir within the stated limits enhances the required pharmaceutical activity, while not causing side effects during antiviral therapy.

The invention also relates to a method of treatment which comprises administering to a patient in need of such antiviral treatment, for example, an oral or injectable effective amount of the composition according to the invention.

Images explaining the essence of the invention:

FIG. 1: (a) genomic structure of 2019-nCoV showing proteases and RNA-dependent RNA polymerase, (b) nucleoside blockers of RNA-dependent RNA polymerase.

FIG. 2: Effect of favipiravir-RTF, favipiravir and favipiravir-MTF on the activity of RNA-dependent RNA polymerase (RgRn) of the influenza virus [7]. The PgRp activity was measured by the incorporation of ³² P-GTP into the native RNA strand. P <0.01; the results differ significantly from the control, according to the Tukey test [8].

FIG. 3: Schematic representation of the mechanism of action of favipyrovir and its transformations upon entering the cell [8].

FIG. 4: Therapeutic effect of favipiravir in a mouse influenza infection model [8]. 10 females were infected with 100% lethal dose of influenza virus of one of the strains: A / Victoria / 3/75 (H3N2), A / Osaka / 5/70 (H3N2) or A / Duck / MN / 1525/81 (H5N1) and monitored survival for 21 days post-infection. Favipiravir was administered orally, 1 hour after infection, 2 times a day in the Osaka and Duck groups or 4 times in the Victoria group for 5 days. **, p <0.001 compared to control (Chi-square test). ++, p <0.001 compared with control (Kaplan-Meier method).

FIG. 5: Effect of the presence of increasing concentrations of zinc ions on the binding of SARS-CoV nsp12 PgRp virus on binding to the substrate.

Implementation of the invention

The pharmaceutical composition according to the invention contains an effective amount of favipiravir.

The effective amount of favipiravir is 50 mg to 800 mg. Preferably, the effective amount of favipiravir is 150 mg to 600 mg. Preferably, the effective amount of favipiravir is 200 mg to 400 mg. Preferably, the effective amount of favipiravir is 100 mg to 800 mg.

The pharmaceutical composition according to the invention contains an effective amount of at least one zinc compound.

However, the zinc compound can be selected from, but not limited to, zinc acetate, zinc lactate, zinc aspartate, zinc sulfate, zinc undecylenate.

In this case, the zinc compound can be represented as a salt or an ionophoric form, for example, zinc pyrithione, dithiocarbamates, zinc-diethyl bis (N4 methylthiosemicarbazone) zinc-DTSM, quinokitol, pyrrolidine, zinc dithiocarbamate, but is not limited to them.

The effective amount of zinc salts, if used, is 15 to 250 mg. The maximum concentration is up to 900 mg. Ionophores, if used, are used in equimolar amounts. Preferably, said zinc salts can be used in the composition in an amount of 15 to 250 mg, and also preferably 100 to 150 mg.

The mass ratio of components favipiravir: zinc / zinc-ionophore (if used) can be 1: 1 to 10: 1, more preferably 1: 1; 1.5: 1; 2: 1; 2.5: 1.

The pharmaceutical composition according to the invention may further comprise pharmaceutically acceptable substances.

The pharmaceutical composition according to the invention may contain one or more excipients selected from solvents, dispersants, fillers, surfactants, solubilizers, emulsifiers, stabilizers, lubricants, preservatives, antioxidants, buffers.

The pharmaceutical composition according to the invention may contain one or more solvents selected from water, for example, water for injection, distilled water, pyrogen-free water, buffer solution, but is not limited to them.

The pharmaceutical composition according to the invention may contain one or more dispersants selected from, but not limited to, starch, agar, alginic acid or salts thereof.

The pharmaceutical composition according to the invention may contain one or more surfactants selected from, but not limited to, polyethylene glycol, glycerol, organic acid glyceride, polysorbate, Tween-80.

The pharmaceutical composition according to the invention may contain one or more solubilizers selected from Tween-80, PEG, benzene, glycerol, sorbitol and its derivatives, xylitol, alginic acid, but is not limited to them.

The pharmaceutical composition according to the invention may contain one or more stabilizers selected from citric acid, ascorbic acid, sodium bicarbonate, sodium sulfite, unithiol, tocopherol, but is not limited to them.

The pharmaceutical composition according to the invention may contain one or more preservatives selected from sorbic acid, benzoic acid, sodium metabisulfite, benzyl alcohol, but is not limited to them.

The pharmaceutical composition according to the invention may contain one or more lubricants selected from, but not limited to, stearic acid and its derivatives.

The pharmaceutical composition according to the invention may contain one or more fillers selected from sucrose, magnesium carbonate, silicon dioxide, starch, mannitol, polyvinylpyrrolidone, povidone, lactose monohydrate, various cellulose derivatives, for example, microcrystalline cellulose, croscamellose, but is not limited to them.

The pharmaceutical compositions can be prepared by any means known in the pharmaceutical art, for example, by mixing the active ingredients with pharmaceutically acceptable substances under sterile conditions.

The pharmaceutical compositions can be in the form of a tablet or capsule, in lyophilized form, or in a liquid dosage form as an injection.

The tablet can be film-coated.

Compositions of the present invention can be made by a variety of methods including, but not limited to, extrusion, spray drying, and solvent evaporation.

The pharmaceutical compositions of the present invention have excellent antiviral activity and are directed to the prevention and / or therapy of diseases caused by exposure to viruses, the genome of which is encoded by a single-stranded RNA strand and which use viral RNA-dependent RNA polymerase for their replication.

The pharmaceutical compositions of the present invention are aimed at significantly alleviating the clinical symptoms, course and cure of a wide range of diseases caused by exposure to viruses, the genome of which is encoded by a single-stranded sense (+) - strand, as well as an antisense (-) - RNA strand and which use viral RNA-dependent -RNA polymerase for its replication.

Viruses against which the compositions of the present invention are active are influenza viruses of both high and low virulence strains, viruses of the Coronaviridae, Picornaviridae, Arenaviridae, Flaviviridae, Bunyaviridae, Togaviridae, Orthomyxoviridae families.

The administration of the composition, namely, the relief of symptoms, up to recovery, in a patient in need of treatment may be associated with infection of the patient with a virus, which is a calicivirus or noravirus.

The pharmaceutical compositions of the present invention are used in treatment or prophylaxis by a course.

The duration of the course and the daily dose is determined by the task, the complexity of the disease and the individual characteristics of the patient and can be adjusted during therapy and / or prevention.

The duration of the prophylactic course can be from one to four weeks and include taking from one to thirty dosage forms per day.

The duration of a therapeutic course can be from one to four weeks and include taking from one to thirty dosage forms per day.

The course for the treatment of diseases may include taking from 3 doses of the composition per day in the first 2 days, then 2 doses.

The course for the treatment of diseases may include taking from 2 doses per day.

Preferably, one dose comprises 200 to 6000 mg favipiravir, 10 to 100 mg Zn2 + compound (in terms of zinc).

Preferably, one dose comprises 200 to 1600 mg favipiravir, 10 to 60 mg Zn2 + compound (in terms of zinc).

Examples of

The present invention and methods for its implementation will be clear to the specialist from the following examples.

These examples, relating to the compositions of favipiravir, at least one zinc compound, are not limiting of the invention and relate only to its particular variants of implementation.

Example 1. Manufacturing of variants of finished medicinal products.

1. Tablet, film-coated

Structure:

Lactose monohydrate, favipiravir and croscarmellose sodium are loaded into a high shear mixer, mixed for 5 minutes at a speed of 100 rpm on the lower mixer. A humidifier, 10% povidone KZO solution is added at a uniform speed, with constant stirring of a mixer 150 rpm and a chopper 500 rpm, moisten until the humidifier is completely added. The moistened mass is granulated with stirring stirrer 200 rpm and chopper 650 rpm.

The granules obtained in this way are passed through a mesh with a mesh size of 1 mm and dried in a fluidized bed installation at an inlet air temperature of 50-60 ° C and a product temperature of 30-45 ° C to a residual moisture content of not more than 2%.

The resulting granules of favipiravir, microcrystalline cellulose, potato starch, colloidal silicon dioxide mixed with zinc sulfate are loaded into the bin of the gravimetric mixer, mixed for 30 minutes (15 minutes on each side).

Load the magnesium stearate and dust for 2 minutes (1 minute on each side).

The resulting mass is tableted on a rotary tablet press with a force of 120 -150 N, and an average core mass of 5 80 mg.

The tablets are coated with a finished film coating in a coater with a perforated drum until the weight gain is 5%.

2. Hard gelatin capsule

Structure:

Lactose monohydrate is loaded into a mixer with a high shear force, favipiravir is half of the potato starch composition, mixed for 5 minutes at a speed of 100 rpm on the lower mixer. Add a humidifier, 10% povidone K30 solution, with constant stirring, a stirrer 150 rpm and a chopper 500 rpm, and moisten until the humidifier is completely added. The moistened mass is granulated with stirring stirrer 200 rpm and chopper 650 rpm.

The granules obtained in this way are passed through a mesh with a mesh size of 1 mm and dried in a fluidized bed installation at an inlet air temperature of 50-60 ° C and a product temperature of 30-45 ° C to a residual moisture content of not more than 2%.

The obtained granules of favipiravir, microcrystalline cellulose, the rest of potato starch, colloidal silicon dioxide mixed with zinc sulfate are loaded into the bin of the gravimetric mixer, mixed for 30 minutes (15 minutes on each side).

3. Injection solution

Composition of the composition per 100 ml of an aqueous solution:

The claimed solution was prepared in the following way: measured amounts of favipirafir, propylene glycol, sorbitol and povidone K15 are placed in a glass or stainless steel apparatus for preparing solutions equipped with an mixing device and a thermostatic jacket and heated with continuous stirring to a temperature of 40-45 ° C. The resulting mixture is stirred at this temperature until the components are completely dissolved. Next, water for injection, pre-measured and heated to a temperature of 40 ° C, is added to the solution and stirred until a homogeneous solution is obtained.

Then the solution is cooled to room temperature, the calculated amount of zinc sulfate is added and stirred until it is completely dissolved. The resulting solution is filtered through membrane plates of the "Millipore" type with a pore diameter of 0.22 μm and poured into ampoules or vials.

The presented examples demonstrate variants of finished dosage forms according to the invention and possible methods of their manufacture.

Example 2. Measurement of inhibition of influenza virus replication in the lungs of infected mice with favipiravir in combination with zinc salts.

Example 3. Therapeutic effect of favipiravir and favipiravir in combination with zinc salts in a mouse model infected with highly pathogenic avian influenza virus.

Example 4. Therapeutic effect on influenza-infected immunodeficient mice

The examples carried out allow us to conclude that the claimed combinations have increased therapeutic activity against various types of pathogens in comparison with individual components.

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Claims (10)

1. Pharmaceutical composition for alleviating clinical symptoms, course and / or cure of a disease caused by exposure to a virus, the genome of which is encoded by a single-stranded RNA strand and which uses viral RNA-dependent RNA polymerase for its replication, containing an effective amount of favipiravir and an effective amount of a zinc compound , selected from zinc sulfate, zinc acetate, zinc lactate, zinc-diethyl bis (N-4-methylthiosemicarbazone), zinc dithiocarbamate, in a weight ratio of favipiravir to zinc salt 1: 1-10: 1, where the effective amount of favipiravir is 50- 800 mg, effective amount of zinc salt is 15-250 mg. 2. A pharmaceutical composition according to claim 1, wherein the virus is an influenza virus, coronavirus, picornavirus, arenavirus, flavivirus, bunyavirus, togavirus, calicivirus, or noravirus. 3. The pharmaceutical composition of claim 2, wherein the influenza virus is a highly virulent virus or a low virulent virus. 4. The pharmaceutical composition according to PP. 1-3, in which the amount of favipiravir is from 100 to 800 mg. 5. The pharmaceutical composition according to PP. 1-3, in which the amount of favipiravir is 200 to 400 mg. 6. A pharmaceutical composition according to claim 1, wherein the amount of the zinc compound is 50 to 250 mg. 7. A pharmaceutical composition according to claim 1, wherein the amount of the zinc compound is 100 to 150 mg. 8. A pharmaceutical composition according to claim 1, in solid or liquid form. 9. The pharmaceutical composition of claim 8, wherein the solid form is a film-coated tablet or a compression tablet. 10. A method of treating and / or preventing a disease caused by exposure to a virus, the genome of which is encoded by a single-stranded RNA strand and which uses viral RNA-dependent RNA polymerase for its replication, comprising administering to a subject in need of a pharmaceutical composition according to claims. 1-9.

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