RU2794315C1 - Method for prevention or treatment of coronavirus and other acute respiratory viral infections - Google Patents

Abstract

FIELD: medicine; therapy; infectology.

SUBSTANCE: group of inventions relates to the field of medicine, namely to therapy and infectology, and is intended for the prevention or treatment of respiratory viral infections. The combined drug for the prevention or treatment of RNA viral infections is made in the form of a solution for inhalation and contains recombinant interferon α2b, lopinavir, remdesivir, amantadine, sodium chloride, ethyl alcohol and tween 80. The components are used in the stated amounts. In a different the embodiment presents a method for the prevention or treatment of RNA viral infection, includes the use of the specified drug in the form of inhalations, which carry out 5 ml of the solution for 10 minutes from 2 to 6 times a day for 1-14 days.

EFFECT: use of the group of inventions provides effective treatment or prevention of respiratory viral infections while reducing the drug load on the patient's body.

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Description

1. Technical field to which the invention belongs

The invention relates to medicine and biology and is intended for the prevention or treatment of respiratory viral infections, including coronavirus infections, in particular, Сovid-19, by using the drug "TRIAVIR OVID®".

2. State of the art

At the end of 2019, an outbreak of a new coronavirus infection occurred in the People's Republic of China (PRC), with its epicenter in the city of Wuhan (Hubei Province). On February 11, 2020, the World Health Organization (WHO) officially named the infection caused by the novel coronavirus COVID 19 (“Coronavirus disease 2019”). On February 11, 2020, the International Committee on Taxonomy of Viruses assigned the official name of the infectious agent - SARS CoV 2.

The emergence of COVID 19 has presented healthcare professionals with the challenges of rapidly diagnosing and providing medical care to patients. Currently, information on the epidemiology, clinical features, prevention and treatment of this disease is limited.

Coronaviruses (Coronaviridae) are a large family of RNA viruses that can infect both animals (their natural hosts) and humans.

Currently, four coronaviruses (HCoV 229E, OC43, NL63 and HKU1) are known to circulate among the population, which are present all year round in the ARVI structure and, as a rule, cause mild to moderate upper respiratory tract damage.

Prior to 2002, coronaviruses were considered as agents causing mild upper respiratory infections (with extremely rare fatalities). Between 2002 and 2004 coronavirus SARS-CoV from the genus Betacoronavirus (reservoir - bats, intermediate hosts - camels) for the first time caused the development of an epidemic of the so-called SARS - severe acute respiratory syndrome (SARS) and a confirmed cause of death of 774 people in 37 countries of the world. Another epidemic caused by the coronavirus MERS-CoV (reservoir - one-humped camels), also from the genus Betacoronavirus Middle East coronavirus syndrome, began in 2012 in the Arabian Peninsula (82% of cases in Saudi Arabia). Until 2020, 866 deaths from MERS have been registered. At present, MERS-CoV continues to circulate and cause new cases.

SARS-CoV-2 is a single-stranded RNA virus belonging to the Coronaviridae family, Betacoronavirus genus. Considering the high pathogenicity of SARS-CoV, SARS-CoV-2 and MERS-CoV viruses are assigned to pathogenicity group II. The genetic sequence of SARS-CoV 2 is similar to that of SARS-CoV by at least 79%.

The main entry gate of the SARS-CoV-2 virus is the epithelium of various parts of the upper respiratory tract. At the same time, the main and quickly achievable target of SARS-CoV-2 is alveolar cells of type II (AT2) of the lungs, which determines the development of diffuse alveolar damage.

It was found that during the manifestation of the disease there is a significant and rapidly increasing viremia with dissemination of SARS-CoV-2.

Based on the analysis of clinical symptoms, we can assume the tropism of the virus to the epithelium of the larynx, ciliated epithelium of the respiratory tract throughout, alveolocytes I and II types. Apparently, viral lesions in such patients do not lead to the development of pronounced exudative inflammation and, accordingly, catarrhal phenomena.

The SARS-CoV-2 virus is detected by immunohistochemical, PCR, ultrastructural studies in the ciliated cells of the bronchi, the epithelium of bronchioles, in alveolocytes and macrophages, as well as in the vascular endothelium. In terms of observations, changes were also detected in other organs that can be presumably associated with the generalization of coronavirus infection: in the lymph nodes, intestines, pia mater, heart, pancreas, kidneys, and spleen. Severe lesions of the microvasculature with the development of disseminated intravascular coagulation (DIC) syndrome were noted, which were most pronounced in the lungs and kidneys. Skin manifestations typical of COVID-19 are described, ranging from hemorrhagic syndrome to rashes of various types, the pathogenesis of which is unclear.

Thus, as with other coronavirus infections, as well as influenza A / H1N1, the main morphological substrate of COVID-19 is diffuse alveolar damage. The term viral interstitial pneumonia, widely used in the clinic, essentially reflects its development.

Since the beginning of 2020, an undulating course has been noted, which proceeded against the background of mass vaccination of the population and the emergence of new mutations of the SARS-CoV2 virus, namely: “Delta strain” (B.1.617), “Delta Plus” (B.1.617.2.1), “ Omicron (B.1.1.529) and its new variant Omicron Stealth (BA.2), which has an even faster propagation speed.

The emergence of these strains has led to a significant change in the epidemiological properties and clinical course of COVID-19, namely:

A significant decrease in the effectiveness of vaccination;

A significant 5-10 times increase in the infectiousness of the virus.

A shorter incubation period of the disease and a longer period of development of viral pneumonia.

In addition, a new strain of coronavirus recently discovered in South Africa (NEOCoV) will obviously have a significantly higher lethality characteristic of the MERS virus (the causative agent of the Middle East respiratory syndrome, characterized by a rapid course of the disease and a very high lethality of more than 30%), with more a higher spread rate with even greater resistance to the preventive action of vector vaccines.

In connection with all these factors, it became objectively necessary to find other additional ways to prevent or treat coronavirus infection.

In addition, there is an urgent unmet need for optimal treatment of both coronavirus infection and generic antiviral agents, including specific antiviral therapy. More than 160 randomized and non-randomized trials have been registered so far. Various agents have been used to treat COVID-19, including corticosteroids, various combinations of ribavirin, lopinavir/ritonavir, chloroquine, hydroxychloroquine, interferons, and other agents. An RCT is underway using remdesivir for severe COVID-19 (NCT04257656). [15].

The prior art describes the following approaches to the treatment of coronavirus infection.

Stopping the copying of the viral genome.

If an RNA-synthesizing enzyme is added to ordinary ribonucleotides with some analogue molecule similar to a ribonucleotide, then RNA synthesis will deteriorate: the enzyme will stop, will not finish the RNA thread it has started, or will continue to synthesize it, but now incorrect letters will often come across in RNA, which will enter into different chemical reactions and thereby provoke numerous mutations.

Coronaviruses and other RNA viruses use a special enzyme for synthesis - RNA-dependent RNA polymerase. There is no such enzyme in the cell itself, the cell does not synthesize RNA molecules on the RNA template, so you can try to act on the viral polymerase without interfering with the cell. Molecules-analogues of ribonucleotides have been synthesized for a long time, and some of them were created just to fight viruses. For example, the drug remdesivir was developed against Ebola viruses, but later it turned out that remdesivir suppresses the reproduction of MERS-CoV and SARS-CoV. Remdesivir is known to block SARS-CoV-2 infection in human and monkey cell culture. Clinical trials with sick people are underway in China and the United States, and at least one patient who took remdesivir has already recovered - although it is not yet clear how much he owes his recovery to this particular drug.

Another ribonucleotide analogue called EIDD-2801 has been developed against the influenza virus and, according to a recent article in Science Translational Medicine, also inhibits SARS-CoV and MERS-CoV infection in mice and the replication of the new SARS-CoV-2 in mice. human lung cells. EIDD-2801 provokes many mutations in viral RNA, while cellular RNA is not damaged. It remains to wait for the results of clinical trials.

You can try turning off the viral RNA polymerase itself. Chloroquine is used against the malaria parasite, sometimes against pathogenic amoebas. As it turned out, it also inhibits the activity of viral polymerase: chloroquine greatly increases the level of zinc ions in the cell, which just interfere with the work of the enzyme. In addition, chloroquine prevents the invading virus from releasing its genome into the cell cytoplasm. However, chloroquine has a serious disadvantage - because of it, abnormalities in the heart rhythm occur, and the higher the dose of the drug, the higher the risk of cardiac abnormalities. In the meantime, it cannot be said whether small doses of chloroquine can stop SARS-CoV-2 infection.

Another way is to prevent the virus from entering the cell. Outside the cell, the virus cannot multiply, and those viral particles that remain in the intercellular space and in the blood will be destroyed by the cells of the immune system. The S-protein plays the role of a master key that breaks the cell: in order for the virus to enter the cell, its S-protein interacts with the ACE2 protein, which sits on the surface of the cell membrane.

The viral S-protein can be provided with an ACE2-like decoy to distract it from the cells. For example, ACE2 itself, only freely floating around the virus, and not sitting on the cell. So, in the April article in the journal Cell, it is said about a human modified ACE2 grown in bacterial cells. Its action was tested on organoids, tiny models of the kidney and blood vessels grown from human stem cells, and such a protein did not allow the virus to penetrate into human cells [19].

Amantadine was synthesized in 1967 as an antiviral agent. Amantadine is a tricyclic amine belonging to the class of aminoadamantanes. Amantadine has a unique dual pharmacological profile (dopaminergic and non-dopaminergic). To date, we can talk about the following proven mechanisms of action of amantadines:

- stimulation of dopamine release from presynaptic endings;

- an increase in the synthesis of dopamine;

- decrease in dopamine reuptake; - increased sensitivity of dopamine receptors to it;

- blockade of glutamate NMDA receptors;

- mild anticholinergic effect (due to the blockade of the release of acetylcholine mediated by NMDA receptors;

- antiviral action associated with the ability to block the penetration of influenza A virus into cells).

Amantadine is used as an antiviral drug in the treatment of influenza type A, as it is believed to block the replication of the virus in the early stages. In 1976, the FDA approved the use of amantadine for the symptomatic and/or prophylactic treatment of influenza type A in adults.

To date, several observations have been published suggesting a possible protective effect of amantadine against COVID-19.

Such observations allowed a number of authors to recommend amantadine for the prevention of COVID-19 at a dose of 100 mg 2 times a day for 14 days after contact with the patient or receiving a positive test for COVID-19. Of course, clinical studies are required to confirm the protective effect of amantadines. A retrospective generalization of the results on a larger cohort of PD patients will also be useful [2].

Remdesivir (also known as GS-5734 in clinical studies) belongs to a class of antiviral drugs that inhibit RNA-dependent RNA polymerase, an enzyme required for the replication of a number of RNA viruses.

Since inhibition of the enzyme prevents the virus from replicating in infected cells, it exhibits antiviral activity against a number of single-stranded RNA viruses such as Ebola virus, Marburg virus, human respiratory syncytial virus, Lassa fever virus, and coronaviruses (including MERS and SARS viruses). Its use for the treatment of people with COVID-19 has been justified, and, based on the previously accepted treatment regimen for the disease caused by the Ebola virus, a 10-day regimen of remdesivir treatment has been proposed: on day 1, 200 mg, and then maintenance doses of 100 mg 1 r /d for 9 days.

On May 1, 2020, the US Food and Drug Administration (FDA) issued an "Emergency Use Authorization" for remdesivir for the treatment of COVID-19 in the US. Preliminary results from NCT04280705 showed that patients who received remdesivir recovered 31% faster than patients who received placebo. Specifically, the median time to recovery was 11 days in patients treated with remdesivir (95% confidence interval [CI] 9 to 12) compared to 15 days (95% CI 13 to 19) in patients treated with placebo (p< 0.001). Kaplan-Meier scores for mortality at 14 days were not significantly different for remdesivir compared with placebo (7.1 vs 11.9%; hazard ratio: 0.70; 95% CI 0.47 to 1.04). Mortality rates at day 28 are not shown because a significant proportion of patients have not yet completed the study. Viral load indicators were not measured. Serious side effects were reported in 21.1% in the remdesivir group and 27.0% in the placebo group. However, some researchers criticize the preliminary findings of this CT, in particular, the absence of objective indicators (mortality, survival) of patients as a primary endpoint. A phase III randomized open-label study (NCT04292899) compared 14-day clinical improvement in 397 patients with severe COVID-19. The first group of patients received treatment with remdesivir for 5 days, the second - 10 days. The study was open, randomized, but without a placebo control group. Patients in the 10-day treatment group had a more severe clinical condition at baseline than patients in the 5-day treatment group. After 14 days, the 10-day remdesivir group showed a 64% improvement in clinical status compared to 54% in the 5-day remdesivir group. The results after adjusting for baseline clinical status do not suggest a difference between the two groups. Furthermore, in the absence of a placebo group, it is not clear whether remdesivir changed the natural course of the disease and outcomes. As of 07/09/2020, 2 studies of remdesivir have been registered in the register of permits for conducting clinical trials in the Russian Federation. The first (WA42511) evaluated the efficacy and safety of remdesivir plus tocilizumab versus remdesivir plus placebo in hospitalized patients with severe COVID-19 pneumonia. The second (No. REMVR-2020/I) was to study the safety and efficacy of the drug Remdeform (INN - remdesivir), a lyophilisate for the preparation of a solution for intravenous administration of 100 mg.

In vitro, remdesivir is a substrate of CYP3A4, CYP2C8 isoenzymes, which indicates a possible interaction with inhibitors and inducers of these isoenzymes. In a number of protocols, the drug is not recommended for GFR 5 upper norms. In a study by Wang M et al. the proportion of patients who experienced any adverse event was similar in the remdesivir and placebo groups, but the proportion of patients with serious adverse events was higher in the placebo group than in the remdesivir group (26% vs. 18%, respectively). The proportion of patients who experienced adverse events leading to treatment discontinuation was higher with remdesivir than with placebo (12% vs. 5%, respectively). The most common adverse events with remdesivir were: constipation (14%), hypoalbuminemia (13%), anemia (13%), hypokalemia (12%), anemia (12%), thrombocytopenia (10%) and hyperbilirubinemia (10%) . The most common serious adverse events were respiratory failure or acute respiratory distress syndrome (10% on remdesivir and 8% on placebo) and cardiopulmonary failure (5% on remdesivir and 9% on placebo). At least one case of liver failure has been described in a 64-year-old patient five days after starting remdesivir. After discontinuation of the drug, liver function parameters returned to normal.

WHO does not recommend the use of remdesivir for the treatment or prevention of COVID-19 outside of clinical trials. Restrictions on the use of remdesivir as outlined in the guidance issued by the FDA should be taken into account, namely remdesivir: should not be used prophylactically; should not be used on an outpatient basis; is indicated only for patients with oxygen saturation less than 94% or requiring some form of respiratory support.

Preliminary results from several randomized clinical trials of the efficacy of remdesivir are inconsistent [3].

Lopinavir is a fixed-dose combination antiretroviral drug for the treatment and prevention of AIDS. The active ingredients are the protease inhibitors lopinavir and (at a low dose) ritonavir. It can be used for prophylaxis after a needle stick or other potential source of HIV infection. It is taken orally in the form of tablets, capsules or solution. Common side effects include diarrhea, vomiting, feeling tired, headaches, and muscle pain. Serious side effects may include pancreatitis, liver problems, and high blood sugar. Can be used during pregnancy. Both components of the drug are HIV protease inhibitors. Ritonavir slows down the breakdown of lopinavir. In 2020, a non-blind, randomized trial showed that lopinavir/ritonavir is not effective for the treatment of severe COVID-19 [4].

The guidelines for the treatment of coronavirus say: "Lopinavir / ritonavir. Used in adult patients with COVID-19 in China, Iran, USA. There is no evidence base for efficacy and safety in children. Currently, the drug is not recommended for either children or adults."

However, there are also studies indicating the safety of remdesivir, lopinavir/ritonavir, in the absence of a reduction in hospital mortality in patients with COVID-19 [5].

Thus, there is no data in the prior art indicating that any of the drugs: amantadine, remdesivir and lopinavir are really effective and safe in the treatment of coronavirus infection. different studies show contradictory results on the effectiveness of the use of these drugs [6].

Recombinant interferon alpha-2b is a water-soluble protein containing 165 amino acids, with a molecular weight of approximately 19300 Da, obtained using the Escherichia coli SG-20050/pIF 16 strain containing a genetically engineered plasmid hybrid encoding the human leukocyte interferon alpha-2b gene. Identical in characteristics and properties to human leukocyte interferon alfa-2b.

In 2020, a study was conducted to evaluate the preventive efficacy of recombinant interferon α-2b (Viferon) in the context of the COVID-19 pandemic in healthcare workers.

A pronounced prophylactic efficacy of interferon α-2b in various schemes was revealed, both for topical application in the form of a gel (3 out of 15 fell ill), and in a combination of rectal and topical forms of interferon α-2b (1 out of 60) compared with a group of medical workers without preventive therapy (11 out of 34). Such results are expected, they coincide with the previously obtained data on the effectiveness of interferon α-2b in ARVI. It is assumed that prophylactic intranasal administration or inhalation of recombinant interferons can limit viral replication in the upper respiratory tract. Thus, the article [7] concludes that the use of recombinant interferon for the prevention of COVID-19 in a pandemic is a promising area for further research.

Since theoretical data indicate a possible positive effect of the use of the above drugs for the treatment of coronavirus infection, researchers have made attempts to use these drugs in combination therapy.

Thus, in [8], a method for treating, preventing, improving the condition, slowing down the course, reducing the severity or preventing a coronavirus infection, or a COVID-19 infection, or an infection caused by a virus of the Orthocoronavirinae subfamily or a virus of the Coronaviridae family or a virus of the order Nidovirales, is disclosed, including the introduction to a person in need of to that person a therapeutic combination of drugs or a drug, pharmaceutical dosage form, drug delivery device, or a product of manufacture containing: (a) chloroquine, chloroquine phosphate, chloroquine diphosphate or hydroxychloroquine; (b) a macrolide antibiotic or macrolide preparation; (c) a drug selected from the group consisting of remdesivir, oseltamivir, zanamivir, and combinations thereof; and d) zinc, as well as a combination of lopinavir and ritonavir and interferon-beta-1b or interferon alpha-2b; or amantadine, type II interferon and/or type III interferon), remdesivir, lopinavir.

[9] disclosed a method for preventing or treating virus-induced organ damage or failure in an individual, comprising administering to the individual an effective amount of an IL-22 dimer and additionally a combination of remdesivir, lopinavir, IFN-α, amantadine. In this case, virus-induced organ damage or failure is induced by SARS-CoV-2.

In the prior art, the use of a combination of lopinavir, remdesivir and amantadine as an additional component of the composition for the treatment of coronavirus infection [10-15] is widely presented, including in the form of inhalation.

The disadvantages of the above methods is that interferon, lopinavir, remdesivir and / or amantadine, are used as part of combination therapy, and not as part of a single agent in the form of monotherapy.

As you know, the disadvantage of combination therapy is that sometimes patients have to take a medicine that is not necessary. In addition, such therapy leads to a significant increase in the cost of treatment. It is the economic aspect, which today acquires significant and sometimes decisive importance, that in many cases was the reason for refusing combination therapy. At the same time, admission schemes are often complex, and the need to monitor the implementation of such schemes leads to an additional burden on the staff of medical institutions or places an additional burden on the patient. In this case, errors and violations of the optimal reception scheme are not ruled out.

Thus, the use of a single combination drug is preferred over multiple single drug combination therapies.

Interest in the creation of combined drugs (fixed combinations) containing two or more active substances in one dosage form has been growing in recent years. However, the development of such drugs is difficult, including from a methodological point of view, since there is no unified program for pre-registration clinical trials of combined drugs. On the other hand, for each combination medicinal product, the pre-registration clinical study program should be developed individually. The choice of the optimal strategy for clinical trials allows obtaining sufficient information about the efficacy and safety of the combined drug for its subsequent registration. Just determining the optimal strategy for clinical trials every time is a challenge for developers of a combination drug.

Combined drugs (fixed combinations) - medicines contain two or more active substances in one dosage form. The expected advantages of combination drugs compared to monocomponent ones are greater efficacy, accelerated onset of effect, greater safety and better tolerability with comparable efficacy, which is possible due to the use of lower doses of one or more components of the combination, as well as ease of use: a decrease in the number of tablets taken simultaneously, simplification of the scheme of reception. In addition, the use of a combination drug in some cases can reduce the incidence of adverse reactions to one of the components of the combination or to treat simultaneously existing (comorbid) diseases with different pathogenesis [17].

The following publication can be taken as the closest analogue. From [18], a therapeutic combination of drugs or a drug, a pharmaceutical dosage form, a drug delivery device or a production product is known, including: a combination of type II interferon, lopinavir, amantadine and remdesivir. In this case, the specified tool is used for the treatment of Covid-19. The delivery device may be an inhaler.

In the closest analogue, these drugs are used as part of combination therapy, not as a single drug for monotherapy, which makes all the disadvantages inherent in this type of treatment relevant. In this source, the task of obtaining one drug based on interferon, lopinavir, remdesivir and amantadine was not set. Thus, the question of doses and administration regimens remains open. In addition, in the closest analogue, a type II interferer was used, and not type I, as proposed in this solution. The use of type 2 INF for the treatment or prevention of viral diseases, and in particular coronavirus infections, is inferior to the use of type 1 INF, which will be demonstrated later.

The technical result of the proposed technical solution is to simplify the regimen for the treatment or prevention of viral infections, reduce the drug load on the patient's body, while obtaining an effective result of prevention or treatment. In addition, another effect is the ease of use of the drug in its application. Efficiency is expressed in inhibition of virus replication, with a decrease in the infectious titer of the virus by 5.25 lgTCID50. The technical result achieved by implementing the proposed solution is also effective prevention or treatment of patients with a viral infection, including coronavirus infection.

Innovative solutions in the development of the drug was the use of the inhalation route of administration of a combination of antiviral drugs with different mechanisms of antiviral action aimed at various cellular and molecular targets.

At the same time, two components of the composition, interferon alfa and amantadine, have a pronounced antiviral activity against a wide range of acute respiratory infections, including influenza, which is confirmed by literature data [1,16,20].

3. The essence of the invention

The drug "TRIAVIR OVID®" is a combined inhalation drug for the prevention or treatment as part of the complex therapy of "COVID-19 coronavirus infection" and other acute respiratory infections, including influenza.

Recipe of the drug "TRIAVIR OVID®".

Component name Quantity for 1 vial 100 ml Active substances Interferon α2b recombinant 200,000 IU Lopinavir 10 mg Remdesivir 10 mg Amantadine 10.0 mg Excipients Sodium chloride 900 mg Ethanol 2.0 ml Twin 80 200 mg

The doses used in the formulation can be selected from the following range: interferon alfa 50,000 - 1,000,000 IU / 100 ml, lopinavir 0.1-100 mg / 100 ml, remdesivir - 0.1-200 mg / 100 ml, amantadine 0.1- 200 mg/100 ml.

The expediency of combining four components in one preparation, confirming the specific activity of the composition, is based on the following considerations.

The combination of four different pharmacological agents, possibly effective against the SARS-CoV-2 virus, with different molecular targets in one dosage form should provide a summed effect of the composition on the virus replication and its penetration into the epithelial cells of the respiratory epithelium.

Application using the inhalation method of administration should ensure rapid access of drugs to the pathological focus and blockade of virus replication in all parts of the upper respiratory tract with high exposure safety. This is important, especially with COVID-19, where the lower respiratory system is predominantly affected.

As our studies have shown, in situ inhalation exposure leads to a significant potentiation of the antiviral effect, presumably due to a significant increase in the active concentration of the substance when it is directly exposed to the virus. A very important argument in favor of this is the significantly higher safety of this route of administration compared to oral administration. This is due to the fact that the maximum dosage that is used for oral administration of lopinavir leaves 100 mg twice a day, while the average maximum plasma concentration (Cmax) is 9.8 ± 3.7 μg / ml and is reached after about 4 hours after taking. It is obvious that the inhalation administration of the drug at a dose of 100 μg/5 ml, i.e., 2000-4000 times less, is practically safe (in terms of systemic and local irritating effects) and at the same time affects the SARS 3C-like protease of the virus on average 10-20 times more effective dose in terms of its blocking effect on virus infection.

The prior art has already studied the effect of lopinavir/ritonavir on novel coronavirus infection, but the results of lopinavir/ritonavir have been unsatisfactory. Typically, one skilled in the art would expect to enhance the effect of a drug by changing the route of administration to intravenous or at least intramuscular. Therefore, the specialist could not assume that by changing the route of administration, the desired level of therapeutic effect will be achieved. Therefore, one of ordinary skill in the art would not assume that inhalation administration would be effective in comparison with oral administration.

Effective therapeutic doses of IFN by inhalation route of administration are thousands of times lower than possible toxic ones, and for an adult with dosed inhalation administration are 125-500 pg / ml (31250-125000 IU / ml). At the same time, therapeutic doses of interferons, due to their low concentration, practically do not have any local irritating effect and can be used several times a day for a long time (up to several months).

Indications for use are: contact with a viral infection, especially an infection caused by RNA viruses, in particular coronavirus infections, including COVID-19, for the prevention of infection, during the incubation period of the disease, and also as therapy at all stages of the course of the disease.

Method of application and dose. From a 100 ml vial, 5.0 ml of inhalation solution is taken, which are placed in the inhaler cuvette. After that, for 10 minutes or until the entire solution is used, continuous inhalation is carried out using the mask supplied with the device. The solution for inhalation can be used using any modern inhalation equipment, such as a nebulizer or any other cold, compression inhaler. The use of ultrasonic and hot steam inhalers is prohibited, as this leads to the destruction of interferons during inhalation.

The drug can be used to prevent infection with an RNA virus during the entire period of the epidemic. The duration of the drug is not limited.

For prevention after visiting crowded places, it is enough to do 2 inhalations a day. In this case, there is no need to strictly observe the intervals between inhalations. It is advisable to carry out inhalation before visiting a possible focus of infection and after.

In case of established contact with persons possibly infected with RNA viruses, or when establishing a diagnosis, it is necessary to take the drug 4-6 inhalations per day at regular intervals. Such a course should be carried out within 1 to 14 days. If symptoms appear, this regimen should also be used until the symptoms disappear completely.

The action of one inhalation lasts 3-4 hours, after which the risk of infection resumes.

Despite the fact that a specific example of the use of the drug "TRIAVIR OVID®" on the SARS-CoV-2 virus will be given below, it is clear to the specialist that the effect of the drug will be equally effective against any representative of the coronaviridae family, since they are all RNA- viruses, and in addition, the drug is effective for the prevention and early treatment of any respiratory tract infection caused by an RNA virus. This is understandable to a specialist, since on the one hand the drug contains interferon, which is a factor of non-specific protection against any viral infections. At the same time, interferon of the first type (IFN-α) was used in the preparation, which has antiviral and antiproliferative effects, and to a lesser extent - immunomodulatory. It is produced immediately after a meeting with a pathogen, its action is aimed at localizing the pathogen and preventing its spread in the body. This interferon provides early and non-specific protection of the body from an infectious agent. On the other hand, the drug contains lopinavir, which is an inhibitor of the SARS 3C-like protease, and has been extensively studied in vitro in SARS and MERS pathogens, showing some results in terms of inhibiting the replication of these viruses. At the same time, comparative genetic analysis showed almost complete identity of the structure of the SARS 3C-like protease in the SARS-CoV-2 virus, compared with those in SARS and MERS. Thus, if lopinavir has an effect on SARS and MERS, then there is no reason to believe that it will not work on other types of coronaviruses. The next component of the drug is remdesivir, which is an adenosine nucleotide prodrug that is metabolized in the cells of the body to form a pharmacologically active metabolite of nucleoside triphosphate. Remdesivir triphosphate acts as an analog of adenosine triphosphate (ATP) and competes with the natural ATP substrate for incorporation into nascent RNA chains by the virus's RNA-dependent RNA polymirase, resulting in delayed chain termination during viral RNA replication. Just as remdesivir showed in vitro antiviral activity against a clinical isolate of the SARS-CoV-2 virus in primary human respiratory tract epithelial cells with a half maximum effective concentration (EC50) of 9.9 nM 48 hours after the start of treatment, so it shows antiviral activity. activity against any RNA virus, since the structure of the RNA molecule is the same in all organisms. Remdesivir EC50 values against SARS-CoV-2 in Vero cells were 137 nM at 24 hours and 750 nM at 48 hours from the start of treatment.

Another component of the drug is amantadine, which also acts directly on the virus in low concentrations, which determines the feasibility of its inhalation use. The main mechanism of its action is the inhibition of the replication of respiratory viruses, primarily influenza A, by interacting with the M2 protein, which is responsible for the formation of ion channels in the lipid membrane, presumably ensuring the transport of protons into the virion and its undressing. In addition, the drug can also act at the stage of virus assembly.

Examples

Dosage and administration

From a 100 ml vial, 4.0 ml of the test solution for inhalation, the preparation "Triavir Ovid®", are taken, which are placed in the inhaler cuvette. After that, for 10 minutes (until the entire solution is used up), continuous inhalation is carried out using the mask supplied with the device. After that, the removable parts are thoroughly washed with hot water with the addition of safe antiseptic solutions.

The solution for inhalation can be used using any modern inhalation equipment, such as a nebulizer or any other cold, compression inhaler. The use of ultrasonic and hot steam inhalers is prohibited, as this leads to the destruction of interferons during inhalation.

Inhalations are carried out 3-6 times a day at regular intervals.

Example 1

Obtaining the drug "TRIAVIR OVID®".

To prepare a solution for inhalation, Tween 80 and sodium chloride are dissolved in ethyl alcohol, at the rate of 2.0 ml of ethyl alcohol, 900 mg of sodium chloride and 200 mg of Tween 80.

Next, recombinant interferon α2b, lopinavir, remdesivir and amantadine are added to the resulting mixture, at the rate of interferon 50,000 - 1,000,000 IU / 100 ml, lopinavir 0.1-100 mg / 100 ml, remdesivir - 0.1-200 mg / 100 ml, amantadine 0.1-200 mg/100 ml.

Mixing is carried out by standard methods used in the pharmaceutical industry on standard equipment, observing the standard specifications for the preparation of drugs.

Example 2

Analysis of cell viability with the preparation without virus was carried out according to the “G” row, in which dilutions of the preparations were made, but no dilutions of the viral suspension were made. Cell viability was assessed visually by the integrity of the cell monolayer, as well as by the morphological structure of the cells.

Determination of the antiviral efficacy of the tested drug "TRIAVIR OVID®" according to the treatment and prophylactic scheme (drug administration 1 hour before infection).

The assessment of the antiviral activity of the drug was taken into account by reducing the infectious titer of the virus in the Vero E6 cell culture according to the cytopathic effect. The results obtained are presented in Table 1.

As can be seen, amantodine had practically no antiviral effect, and there was practically no decrease in lgTCID ₅₀ .

As a result of the study of the antiviral activity of the above substances in in vitro experiments against the SARS-CoV-2 coronavirus strain according to the therapeutic and prophylactic scheme of adding the drug to the Vero E6 cell culture, it was found that the test drug "TRIAVIR OVID®" inhibited the replication of the SARS-CoV- 2 in Vero E6 cell culture, reducing the infectious titer of the virus by 5.25 lgTCID50. What is the highest indicator for the investigated substances. At the same time, this indicator is significantly higher than the indicator for the combined use of INF and remdesivir, which is unexpected, since, based on the state of the art, it was for each of these drugs that the greatest therapeutic effect was revealed. In addition, it was unexpectedly found that the use of IFN α2b in comparison with IFN γ significantly increases the effectiveness of the combination of IFN + LDL + RMDR + AMN, which the specialist could not expect, in particular, up to 5.25 lgTCID50 from 4.25.

To assess the antiviral efficacy of the combination in relation to each of the incoming components, the formula for the ratio of ECAV and EΣ (A + [B + C]) was used, where:

Antiviral effect of the combination (EC):

A- Antiviral effect of Interferon α2b

[B+C] - antiviral effect of lopinavir + remdesivir

A + [B + C] \u003d 5.0 lgTCID50. ECAB \u003d 5.25 lgTCID50

EKAV> EΣ (A + B), that is, the composition has a potentiating effect. [Potentiating effect - EC is greater than the total effect of the combinants: ECAV> EΣ (A + B)].

Thus, according to the criteria for the scope of this composition, as a drug for the treatment of COVID-19, it has a potentiating antiviral effect in relation to the constituent combinations and can be used to prevent and treat this disease.

Example 3

In accordance with Decree of the Government of the Russian Federation of April 3, 2020 No. 441 "On the peculiarities of the circulation of medicinal products for medical use, which are intended for use in conditions of a threat of the emergence and liquidation of an emergency", as well as the Order of the Ministry of Health and Social Development of the Russian Federation of August 9 2005 N 494 "On the procedure for the use of drugs in patients for health reasons" we conducted low-intervention clinical trials.

In a clinical trial involving 286 patients (198 patients) received Triavir Ovid®, and 88 patients were included in the comparison group, which received treatment in accordance with the Interim Guidelines for the Prevention, Diagnosis, and Treatment of Novel Coronavirus Infection (COVID-19 )" of the Ministry of Health of the Russian Federation, Versions 7-9). All patients were diagnosed with: “A novel coronavirus infection with COVID-19 of moderate course, which were observed on an outpatient basis, it was found that Triavir Ovid® can reduce the median time to clinical improvement (according to the World Health Organization (WHO) Ordinal Scale of Clinical Improvement) In the Triavir Ovid® group, clinical improvement occurred on day 4 (±1.1), and in the standard therapy group on day 7.4 (±5.0) (p<0.05). Triavir® "allows on average to reduce the period until the onset of clinical improvement by 3.4 days. It is important to note that on the 10th day in the Triavir Ovid® group, almost complete recovery occurred in all patients, while in the comparison group, in which used standard therapy, it occurred only on day 14 (figure 1) .

A low-intervention study was also conducted in 70 patients with moderate COVID-19 who are being treated in hospital. The study was conducted with the following design: “Pilot, randomized, controlled in parallel groups, low-intervention, single-center therapeutic study of the combined inhalation mixture of Triavir Ovid® for the prevention and treatment of COVID-19: in patients 18-65 years old.” As a result of a clinical study, it was found that improvement (according to the Ordinal Scale of Clinical Improvement of the World Health Organization (WHO), corresponding to positive dynamics or resolution of clinical symptoms, in the Triavir Ovid® group occurred on day 6.2 (± 1.4), and in the standard therapy group (according to the outpatient treatment regimen in accordance with the Interim Guidelines for the Prevention, Diagnosis, and Treatment of Novel Coronavirus Infection (COVID-19) of the Ministry of Health of the Russian Federation, Versions 7-9) by 12.4 (±5, 0) day (p<0.005) Thus, the use of "Triavir®" allows to reduce the period until the onset of clinical improvement by 6.2 days on average (Fig. 2).

It should be noted that while taking Triavir Ovid®, in the course of clinical trials, not a single case was registered in which transfer to the ICU was recommended, while in the comparison group that did not receive appropriate drug therapy, 3 patients (8.6%) were transferred to the ICU due to the increase in the severity of the course of the disease.

An equally important result was the confirmation of the favorable safety profile of Triavir Ovid®. Thus, the overall frequency of adverse events in the group of patients who received the therapy being tested is comparable to similar indicators against the background of standard therapy. No specific side effects associated with the use of the drug "Triavir Ovid®" were noted.

A low-intervention clinical study was conducted in 298 volunteers who were in contact with persons infected with COVID-19, who were observed on an outpatient basis.

Table 1 shows the optimal potentiating effect of the IFN+LPN+RMDR+AMN composition. This is consistent with the following dosages of Triavir Ovid:

IFN 500 IU / ml corresponds to an inhalation dose - 4 ml of a solution with a concentration of 200,000 IU / 100 ml

LDL - 2 mg / 100 ml

RMDR - 2 mg / 100 ml

AMN - 1.5 mg / 100 ml

Ratio 100,000/1/1/0.75

In the indicated dosage - 100,000/5/5/5.

In this case, the ratio of components in itself does not matter, only the optimal concentrations of active substances are important.

Doses can be adjusted if necessary.

Table 2 in vitro comparison of the solution of the present invention with the prior art solution.

A drug Drug concentration TCID ₅₀
lg Virus control Δlg _max is the maximum decrease in the value of the infecting viral dose in the experiment compared to the control, expressed in decimal logarithms IFNα2b+ IFNγ+LPN 500 IU/ml+50 IU/ml +5 mcg/ml 1.25 6.0 4.75 IFN+LPN+RMDR+AMN 500 IU/ml + 5 µg/ml + 5 µg/ml + 3.75 µg/ml 0.75 6.0 5.25

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Claims (5)

1. Combined preparation for the prevention or treatment of RNA viral infections, made in the form of a solution for inhalation, containing the following components per 100 ml: interferon α2b recombinant 200,000 IU lopinavir 10 mg remdesivir 10 mg amantadine 10 mg sodium chloride 900 mg ethanol 2 ml twin 80 200 mg 2. A method for the prevention or treatment of an RNA viral infection, including the use of the drug according to claim 1 in the form of inhalations, where inhalations are carried out with 5 ml of the solution for 10 minutes from 2 to 6 times a day for 1-14 days. 3. The method of claim 2, wherein the RNA viral infection is a coronavirus infection, including COVID-19. 4. The method according to claim 2, where inhalations are carried out through a nebulizer.

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