CN118176014A - Methods for preventing or treating coronavirus infection - Google Patents

Abstract

The present invention relates to medicine and biology, aimed at using

Description

Methods for preventing or treating coronavirus infection

Technical Field

The present invention relates to medicine and biology, and aims at preventing or treating coronavirus infection by taking a 'TRIAVIR' formulation.

Background

Coronaviruses (CoV) are a large family of viruses that cause diseases including more severe diseases from the common cold to the middle east respiratory syndrome (MERS-CoV), severe acute respiratory syndrome (SARS-CoV), and the like. The novel coronaviruses (nCoV, COVID-19, SARS-CoV-2) are novel strains of coronaviruses that have never been found in humans before [10].

Avian Infectious Bronchitis Virus (IBV) is the first coronavirus known to humans and has heretofore caused a number of complications for poultry farming. This virus was found in 1937. There are 39 coronaviruses known to date, each of which may comprise tens or hundreds of strains, and in addition 10 coronaviruses under investigation [16].

Several new coronaviruses (covs) have emerged in the 21 st century. The novel viruses are characterized by their extremely potent virulence and pathogenicity, and the lesions they cause are often fatal [1,2]. Severe acute respiratory syndrome coronavirus (SARS-CoV) was first discovered in 2003 [3]. Ten years later, a second novel coronavirus, middle east respiratory syndrome (MERS-CoV), was developed, which was first discovered in sauter arabia [1]. Since the first discovery, SARS-CoV has been infected by about 8000 people, and it has been reported that about 10% of the infected people die accordingly [4]. MERS-CoV is not as widely spread as SARS-CoV, but is more active and therefore more pathogenic and toxic, with mortality rates as high as 35-50% in all cases of established infection [1,2].

It is alarming that other covs are also found in animal populations, which increases the likelihood of re-outbreaks of similar epidemic [4]. Since both SARS-CoV and MERS-CoV are zoonotic, they cause more severe disease than the disease normally caused by CoV. SARS-CoV and MERS-CoV infection can lead to severe lung injury, which manifests as Acute Lung Injury (ALI) with concomitant pulmonary edema and respiratory failure. In this case, ALI may dynamically develop into Acute Respiratory Distress Syndrome (ARDS) [5].

The above-mentioned syndromes also occur in the case of novel coronavirus infection caused by SARS-CoV-2. It was found that immune dysfunction plays a key role in its pathogenesis [6,7,8,9]. Note the presence or absence of lymphopenia and neutropenia. Peripheral blood lymphocytes mainly belong to HLA-DR and CD38 marker cells. They belong to the perforin and/or granulocyte positive cd8+ T lymphocyte subpopulations or inflammatory Th17 cells. Since the latter has a pronounced tissue damaging effect and immunosuppressive activity, such changes can cause severe damage to lung tissue, manifest as diffuse damage to alveoli, and exacerbate ARDS conditions. Such changes are consistent with high expression of SARS-CoV-2 receptor on lung cells. Recent studies have shown that antibody-dependent enhancement (ADE) is a biological mechanism of SARS-CoV-2 lung injury [8,9]. The antibody-dependent increase is induced not only by neutralizing antibodies but also by non-neutralizing antibodies. For SARS-CoV-2, ADE is induced by CD32 expressing cells, including monocytes, macrophages and lung macrophages.

2019-NCoV is a virus that has a serious impact on public health, economic and political areas. COVID-19 control should become urgent for all countries [13].

Since this is a novel infectious disease, there is currently no registered vaccine against the novel coronavirus [14]. However, due to the risk of ADE effect, vaccines that mainly elicit humoral immunity in the body require very careful safety tests in simulation systems and clinical trials that reproduce the true infection as closely as possible. At the same time, vaccines that elicit cellular immunity from conserved antigenic determinants do not elicit ADE effects.

There is also a need in the art for optimal therapeutic methods to meet, including specific antiviral therapies, studies of immune system modulation, and how to provide optimal support for multiple organ failure. Currently, 160 more random trials and non-random trials have been registered. Various medications are being used to treat COVID-19, including corticosteroids, various ribavirin formulations, lopinavir/ritonavir, chloroquine, hydroxychloroquine, interferons, and other drugs. A randomized controlled trial study using ramitevir to treat severe COVID-19 (NCT 04257656) is underway. [15] Traditional Chinese medicine is also used in the treatment.

In addition to the above viruses, there are four coronaviruses that infect humans, two of which (HCoV-229E and HCoV-OC 43) have been found prior to SARS-CoV, and the other two (HCoV-NL 63 and HCoV-HKU 1) were found in 2004 and 2005. Although coronavirus colds are common, accounting for 15-30% of the total number of colds worldwide, these four coronaries do not have more severe symptoms than the mild ones [16].

However, all coronaviruses have the following characteristics.

The round virus particles had protrusions on their surface, which formed corona-like envelopes around the virus, as observed under electron microscopy. These protrusions are the dentate molecules of the S protein through which the virus formally needs to penetrate the cell. (notably, another class of viruses, coronavirus' relatives, carina viruses (usually infecting animals, rarely humans) — also have "coronal" S proteins.) some coronaviruses have "sub-coronal" projections, i.e., an additional layer of smaller S proteins protruding from the envelope. This smaller protein is known as HE, hemagglutinin esterase. The interaction between cells also requires HE, which is present in addition to coronaviruses in the carina virus and some influenza viruses.

Both the S protein and HE protein are located in the lipid envelope of the virus, which is obtained from the host cell when the virus is released outside the host cell. In addition to the S and HE proteins, there are a large number of M proteins and a small number of E proteins in the lipid envelope that support and structure the membrane. The protein lipid envelope surrounding the viral genome is a chain of RNA molecules synthesized from N proteins: it coils the viral RNA into a compact helical structure. When RNA infects cells, proteins can be synthesized immediately, and this RNA in the virus is marked with a plus sign. Based on these characteristics, coronaviruses are classified as RNA viruses, whose genomes are linear single-strand positive strand RNA viruses.

Coronaviruses approach cells and come into contact with the cells via "coronal" spike protein S. There are many proteins on the cell surface, including proteases, i.e. enzymes capable of cleaving other protein molecules. Cells first phagocytose the virus, a vesicle in which the virus enters the cytoplasm, by surrounding an envelope that interacts with the virus. The protease binds to and cleaves the S protein, eventually changing the spatial structure of the remainder of the viral S protein. The deformed S protein contributes to the fusion of the viral membrane with the intracellular vesicle membrane, fusion of the two membranes, disruption of the viral envelope, and release of viral RNA into the cytoplasm [16].

The following methods for treating coronavirus infections are described in the prior art.

Preventing viral genome replication.

If, in addition to the usual ribonucleotides, a similar molecule of ribonucleotides is added to the enzyme that synthesizes DNA, the synthesis of RNA is destroyed: the enzyme will stop, not complete the RNA strand that has been synthesized, or continue to synthesize, but now often contains wrong letter proteins in the RNA that will participate in different chemical reactions, thereby causing a large number of mutations.

Coronaviruses and other RNA viruses are synthesized by a specific enzyme, the RNA-dependent RNA polymerase. The cells themselves do not have this enzyme and the cells do not participate in the synthesis of RNA molecules on RNA templates, so we can try to exert an effect on viral polymerase without interfering with the cells. The synthesis of ribonucleotide analogue molecules has long been established, some of which have been developed in order to combat viruses. For example, dimension Lei Midi is a drug developed for ebola virus, but dimension Lei Midi was later found to be able to inhibit MERS-CoV and SARS-CoV proliferation. Lei Midi d was known to prevent SARS-CoV-2 infection in human and monkey cell cultures. Clinical trials are being conducted on patients in China and the United states, and at least one patient taking Lei Midi dimensions has recovered, but it is currently unclear how much effect this drug has on the recovery of that patient.

Another ribonucleotide analog named EIDD-2801 has been developed to combat influenza virus, and it is reported in a paper recently published under Science Translational Medicine (science of transformation), that this analog also inhibits SARS-CoV and MERS-CoV infection in mice, and the proliferation of novel SARS-CoV-2 in human lung cells. EIDD-2801 are capable of inducing multiple mutations in viral RNA without damaging the cellular RNA. Clinical trial results remain to be observed.

Attempts may be made to block the viral RNA polymerase itself. Chloroquine is used to inhibit plasmodium and sometimes also pathogenic amoeba. As a result, it was found that it also inhibits the activity of viral polymerase: chloroquine can greatly improve the content of zinc ions in cells, and the zinc ions can just block the enzyme from working. In addition, chloroquine also prevents invasive viruses from releasing their genome into the cytoplasm. However, chloroquine has a serious disadvantage in that it causes heart rhythm abnormalities, and the larger the dosage of the drug, the higher the risk of causing heart abnormalities. There is no current certainty as to whether a small dose of chloroquine can prevent infection with SARS-CoV-2.

Another approach is to block the entry of viruses into cells. Outside the cell, the virus cannot propagate, and virus particles left in the cell gap and blood are destroyed by the immune system cells. Protein S this acts as a "key" to open the cell gate: to enter a cell, the S protein must interact with ACE2 protein on the surface of the cell membrane.

The viral S protein can be provided with a bait similar to ACE2, keeping it away from the cell. For example, ACE2 itself is only free floating around the virus, rather than resting on cells. For example, CELL (a paper in the journal of CELL, april et al mentions that human recombinant ACE2 cultured in bacterial CELLs has been tested for its effects on organoids (micro-models of kidneys and blood vessels cultured from human stem CELLs), a protein capable of blocking viral invasion into human CELLs [17].

However, general solutions for preventing and treating coronavirus infections have not been found, and there is still an urgent need for effective means and methods for preventing or treating new coronavirus infections and any known and future reoccurring coronavirus infections.

Disclosure of Invention

"TRIAVIR" formulation recipe.

Component name 100ml per bottle

Active ingredient

Recombinant human interferon alpha 2b 100000IU

Recombinant interferon gamma 10000IU

Lopinavir 2.0mg

Auxiliary component

Sodium chloride 900mg

Ethanol 2.0

The above formulation versions are not intended to limit the scope of the claims, but rather to demonstrate specific examples of embodiments.

The dosage used in the formulation may be selected from the following ranges: lopinavir 0.1-32mg/100ml, alpha interferon 50000-1000000 IU/100ml, gamma interferon 5000-500000IU/100ml.

The characteristics of the components of the inhalation mixture formulation "Triavir" are combined.

Interferon (IFN) is a family of polygene-induced cytokines with multiple functions of antiviral, cell proliferation inhibition, antitumor, and immunity regulation.

IFNs can be divided into three types:

type i interferon: known as viral interferons, including IFN- α (leukocyte type, synthesized by activated monocytes and B lymphocytes), IFN-B (fibroblast type, synthesized by fibroblasts and epithelial cells, macrophages), IFN-w and IFN-k;

type ii interferon: known as immunointerferons, including IFN- γ (produced by activated T cells and NK cells);

type iii interferon: the time was found to be later than for type I and type II, and information about this suggests that type III IFN is important in certain types of viral infections;

The first type (IFN-. Alpha.) has mainly antiviral and cytostatic effects, and secondly has an immunity-regulating effect. They are produced directly after meeting the pathogen, which acts to locate the pathogen and prevent it from spreading in the body. These interferons provide the body with early and nonspecific defenses against pathogen attack.

The second type of interferon (IFN-. Gamma.) has a major role in participating in immune responses. In the latter stages of the infection process, interferons start to be produced by already sensitized T lymphocytes and actively participate in the cascade of specific immune responses.

Induced during infection by antiviral interferon virus, mitogen or antigen stimulation activates the synthesis of type II interferon (IFN-gamma). Most virus-infected cell types are capable of synthesizing IFN a/b in cell culture. In contrast, IFN-gamma can only be synthesized by certain cells of the immune system, including Natural Killer (NK) cells, CD 4T cells and CDS cytotoxicity-inhibiting cells [18].

According to the russian federal health department temporary method recommendation [19], "recombinant human interferon alpha 2b (IFN-alpha 2 b) can be used as intranasal administration solution with immunoregulatory, anti-inflammatory and antiviral effects. The mechanism of action is to inhibit replication of viruses entering the human body through the respiratory tract.

The efficacy of inhaled IFN-. Alpha.2b on COVID-19 has also been demonstrated to have very high confidence [20-23,29]. As such, inhalation of IFN- α2b interferon is recommended in all COVlD-19 treatment regimens, according to the current clinical guidelines of the people's republic of China. At the same time, IFN-alpha 2b inhalation therapy is incorporated into treatment regimens of different periods and different severity of the condition, and is applicable to children over 5 years [25].

Thus, IFN-. Alpha.2b inhaled administration is significantly better than intranasal administration, a phenomenon which is particularly pronounced when treating COVID-19 where the respiratory system is primarily affected [25,20-23,29]. Notably, this method of administration is extremely safe. Thus, when using a metered dose nebulizer, an adult single inhalation IFN-. Alpha.2b dose is 100 tens of thousands to 1.2 million IU. At an inhaled dose of 1-1800 ten thousand IU, IFN-. Alpha.2b was not detected in the plasma, but at a dose of 6000 ten thousand IU, a trace amount of IFN-. Alpha.2b was detected in the plasma. In patients with an inhaled IFN-. Alpha.2b dose of 1.2 million IU, a blood concentration of IFN-. Alpha.2b of 11 to 35IU was detected. Systemic or local side effects are not observed when doses of 100 to 1800 ten thousand IU of IFN- α2b are inhaled, but patients may develop hyperthermia, headache and systemic discomfort when doses of 6000 to 1.2 million IU are administered. These symptoms begin to appear 3-6 hours after inhalation, with the most severe symptoms after 8-10 hours and the disappearance of symptoms after 12-24 hours [26]. Thus, administration of IFN- α2b is a traditional therapy for treatment COVID-19, whose effectiveness and safety have been demonstrated. In addition to intramuscular and subcutaneous injections, interferon gamma (IFN-gamma) is also suitable for intranasal aerosol inhalation, including children [29-31,32,28,27]. Unlike other interferons, IFN-gamma enhances the expression of Major Histocompatibility Complex (MHC) class I and class II antigens on different cells, even by inducing those cells that do not express these molecules. This increases the efficiency of antigen presentation and the ability of T lymphocytes to recognize antigens. IFN-gamma blocks replication of DNA and RNA viruses, viral protein synthesis and assembly of mature virions. Gamma interferon exerts a cytotoxic effect on virus-infected cells. IFN-gamma is used in new coronavirus infections because only this type of IFN blocks the synthesis of beta-TGF, which leads to pulmonary fibrosis, a common long-term complication of COVID-19 [32,33]. Currently, the clinical pulmonary sciences are increasingly interested in the potential role of IFN-gamma in the treatment of respiratory diseases, and delivering drugs directly to target organs by inhaled administration is considered the most effective, safest therapy [34,35]. A comparative experiment of IFN-gamma inhalation administration and subcutaneous administration in healthy volunteers showed that parenteral administration, unlike inhalation administration, can significantly increase IFN-gamma blood concentration, but does not increase IFN-gamma blood concentration in lung lavage [32]. A large number of clinical data indicate significantly higher efficacy and safety of treatment of respiratory viral infections by aerosol inhalation of IFNα [32,34,35]. By inhalation of IFN alpha, the effective therapeutic dose is thousands of times lower than the possible toxic dose, and the effective therapeutic dose for adults is 125-500pg/ml (31250-125000 IU/ml). Meanwhile, because of low interferon concentration of therapeutic dose, hardly any stimulation is generated locally, and the composition can be used for a long term (up to several months) multiple times per day [34]. Thus, the safety of therapeutic doses of IFN-gamma by aerosol inhalation is clearly demonstrated by reliable clinical trial results. Furthermore, an important argument supporting this statement is that IFN-. Gamma.and IFN-. Alpha.2b molecules are functionally and structurally similar, which determines that there are neither intermolecular interactions nor similar toxicological properties between them.

Lopinavir is an inhibitor of SARS 3C-like protease and has been extensively studied in vitro for SARS and MERS pathogens while exhibiting some effect in inhibiting replication of these viruses [29-30,35]. Meanwhile, gene comparison analysis shows that the structure of SARS 3C-like protease in SARS-CoV-2 virus is almost completely consistent as compared with SARS and MERS virus [36].

A recent systematic review of the clinical efficacy of oral lopinavir/ritonavir treatment COVID-19 [20,38,24] shows that the clinical efficacy of lopinavir/ritonavir is not apparent.

Therefore, since the prior art has studied the efficacy of lopinavir/ritonavir in the treatment of novel coronavirus infections, the administration of lopinavir/ritonavir is not satisfactory and the skilled person would not expect to change the route of administration to achieve the desired therapeutic effect.

Since inhaled administration has been shown to be effective in treatment COVID-19, the efficacy of prevention and treatment of COVID-19 can be generalized to any coronavirus infection given the genetic affinity of coronaviruses to 3C-like proteases.

Our studies indicate that inhalation exposure in situ can significantly enhance antiviral effects, probably because the active concentration of the substance is significantly increased upon direct exposure to the virus. Another very important argument supporting this view is that the safety of this method of administration is significantly higher than that of oral administration. This is because lopinavir is orally administered at a maximum dose of 400ml twice daily reaching a maximum blood concentration (Cmax) of 9.8±3.7 μg/ml about 4 hours after administration. It is apparent that the inhaled dose is 400 μg/ml, i.e. 1000 times lower, and is practically safe (in terms of systemic and local stimulation) while inhibiting the viral SARS 3C-like protease at a dose 40-50 times higher than the average effective dose to block viral infection.

However, similar considerations are not included in the prior art. Thus, it is not possible for the average expert to assume that inhaled administration is more effective than oral administration. Often, specialists will wish to enhance the therapeutic effect of the drug by changing the route of administration to intravenous or at least intramuscular injection.

Comprehensive safety tests were performed on the oral drug kalelta (lopinavir/ritonavir) according to the Food and Drug Administration (FDA) regulations and russian federal drug registration preclinical test regulations, and existing preclinical toxicological study data indicate that the active substance belongs to a low-toxicity drug (half-lethal dose (LD 50) of lopinavir for single administration is more than 2500mg/kg, low toxicity (GRAS state).

There is little risk of inhalation poisoning when administered at therapeutic doses [39,40].

The technical result achieved by implementing the proposed solution is an effective prevention or treatment of patients suffering from coronavirus infections.

We developed a randomized controlled, parallel group, prospective, single-center treatment trial with subjects 25 to 90 years of age and with the combined inhalation cocktail formulation of prophylaxis and treatment COVID-19The result of this test was the development of an effective/>The mixture is inhaled to aid in the treatment and prevention COVID-19 of patients 25-90 years old.

Example

The total duration of the trial for each patient did not exceed 14 days, divided into 3 phases:

screening period (day 1) -study day 1;

test drug treatment period (14 days (+ -2 days) -study days 1-10 (+ -2 days);

end of trial (day 1) -study day 14 (+ -2 days).

Screening period:

v0 th visit (day 1),

Collecting epidemiological history, demographic data, main complaints and medical history, developing physical examination, evaluating clinical manifestations of the disease, performing pulmonary computed tomography (MSCT), electrocardiogram, measuring pulse oxygen saturation and SpO2, and detecting whether nasopharyngeal and oropharyngeal swabs contain SARS-CoV-2RNA by PCR; routine (clinical) blood analysis was performed to determine leukocyte levels, leukocyte counts, lymphocyte absolute values and percentages; performing biochemical analysis of blood; performing routine urine analysis; the level of C-reactive protein (CRP) in serum was determined.

Based on the obtained results, an inclusion/non-inclusion criterion was evaluated.

Treatment period of test drug:

visit V1 (day 1).

After final evaluation of the inclusion/non-inclusion criteria based on the observations,

Collecting complaints, registering concomitant therapy, developing physical examination, assessing clinical manifestations of the disease, then deciding whether to enroll patients in the trial, and randomly assigning them to one of four groups:

patients in the first group (control group) received standard treatment according to the "temporary guidelines for prevention, diagnosis and treatment of novel coronavirus (covid-19) infections issued by russian ministry of health" (5 th edition (2020, 4/8).

-A second group: in addition to receiving standard treatment according to the novel guidelines for prevention, diagnosis and treatment of coronavirus (covid-19) infections issued by russian health ministry (5 th edition (4 th, 8 th year of 2020)), patients inhale the combination 1 inhalation mixture by nebulizer 4 times a day for 10 minutes each for a total of 14 days.

-A third group: in addition to receiving standard treatment according to the novel guidelines for prevention, diagnosis and treatment of coronavirus (covid-19) infections issued by russian health ministry (5 th edition (4 th, 8 th year of 2020)), patients inhale the combination No. 2 inhalation mixture by nebulizer 4 times per day for 10 minutes for a total of 14 days.

-A fourth group: in addition to receiving standard treatment according to the novel guidelines for prevention, diagnosis and treatment of coronavirus (covid-19) infections issued by russian health ministry (release 5 (day 4, 8 of 2020)), patients inhale the combination 3 inhalation mixture via a nebulizer 4 times a day for 10 minutes each for a total of 14 days.

-A fifth group: in addition to receiving standard treatment according to the New coronavirus (covid-19) infection prevention, diagnosis and treatment temporary guidelines issued by Russian health sector (5 th edition (4/8/2020)), patients were inhaled by nebulizerThe preparation was administered 4 times daily for 10 minutes for a total of 10 days.

For this purpose, a random number was assigned to each test participant by means of random number generation using SPSS STATISTICS 19.0.0 statistical software. The number random assignment schedule determines the type of treatment (T1-T5) used. Throughout the trial, the number was attributed to the trial participant.

Patients received hospitalization throughout the trial period until complete recovery or other outcomes occurred.

Pulse oximetry, symptom and complaint recordings, physical examination, and body temperature measurements were performed for each study day starting with random assignment to observe disease progression.

Adverse reactions were subsequently assessed.

Visit V2 (day 4 after visit V1).

This visit included collecting complaints (changes since the last visit), recording concomitant therapy, performing physical examination, assessing clinical manifestations of the disease, measuring pulse oxygen saturation and SpO2, detecting whether nasopharyngeal and oropharyngeal swabs contained SARS-CoV-2RNA by PCR.

Visit V3 (day 8 after visit V1).

Collecting complaints from this visit (changes since the last visit), recording concomitant treatments, performing physical examination, assessing clinical manifestations of the disease, measuring pulse oxygen saturation and SpO2, detecting whether nasopharyngeal and oropharyngeal swabs contain SARS-CoV-2RNA by PCR, performing routine (clinical) blood analysis, determining leukocyte levels, leukocyte counts, lymphocyte absolute values and percentages; performing biochemical analysis of blood; performing routine urine analysis; the level of C-reactive protein (CRP) in serum was determined.

Visit V4 (day 14 (±2) after visit V1).

Collecting complaints from this visit (changes since the last visit), recording concomitant treatments, performing physical examination, assessing clinical manifestations of the disease, performing pulmonary computed tomography (MSCT), electrocardiogram, measuring pulse oxygen saturation and SpO2, detecting whether nasopharyngeal and oropharyngeal swabs contain SARS-CoV-2RNA by PCR, performing routine (clinical) blood analysis, determining leukocyte levels, leukocyte counts, lymphocyte absolute values and percentages, performing blood biochemical analysis; performing routine urine analysis; the level of C-reactive protein (CRP) in serum was determined.

The trial included 70 adult male and female patients with a moderate to severe weight COVID-19 who were 25-90 years old.

During the study period, all patients were randomized into 5 groups: control group 36 patients; 9 patients per study group.

The test subjects included: patients with "novel coronavirus COVID-19 infection, moderately severe, nosocomial infection bilateral pneumonia, no respiratory failure" were diagnosed based on nasopharyngeal swab and pulmonary computed tomography (MSCT) results. No lopinavir-containing drug was taken 7 days prior to the trial. The interval between onset of symptoms (hyperthermia, cough) and random distribution does not exceed 7 days. Female patients were pregnancy test negative or confirmed no fertility.

Patients with any of the following conditions cannot be the subject:

1. Is allergic to any component of the medicament.

2. Type i or type ii Respiratory Failure (RF), acute respiratory distress syndrome, occurs.

3. Bacterial sepsis exists and has led to lung lesions (pathogen is not detailed)

4. There is a contraindication of lopinavir administration

5. There are contraindications to the use of interferon

6. There are risk factors that lead to severe disease progression and death in COVID-19 patients;

type 7.1 or type 2 diabetes;

8. Bronchial asthma;

9. Chronic obstructive pulmonary disease;

10. Heart and lung function insufficiency;

11. The presence of transplanted organs and tissues;

12. any part suffers from tumor diseases;

13. Obesity;

14. HIV, hepatitis and other acute infections (excluding COVID-19);

15. Other diseases and conditions identified as risk factors in the test;

16. Pregnancy and lactation;

17. patients need to take treatment forbidden drugs;

18. Active tuberculosis, cystic fibrosis, systemic connective tissue disease;

19. Serious, decompensated or unstable somatic diseases (any disease or condition that endangers the life of the patient or leads to a poor prognosis or failure to conduct a clinical trial);

20. Suffering from mental disorders (including claustrophobia) that interfere with the evaluation of treatment;

21. There is a history of alcoholism and drug abuse at present or once;

22. The patient lacks a willingness to cooperate;

23. patients underwent any other clinical trial over the past 30 days.

The time for taking the test drug was 14 days (+ -2 days).

Patient follow-up time was 14 days (+ -2 days).

The safety and tolerability of the drug was observed throughout the course of the drug administration and follow-up.

Observations include assessment of frequency, nature and intensity of adverse and severe adverse reactions.

Composition of the inhalation mixture:

inhalation mixture No. 1-IFN alpha 2b-2000IU/ml

Inhalation mixture No. 2-IFN gamma-200 IU/ml

Inhalation mixture No. 3-IFN alpha 2b,2000IU/ml+IFN gamma, 200IU/ml

Inhalation mixture No. 4(IFN alpha 2b,1000IU/ml + IFN gamma, 100IU/ml + lopinavir 80 u g/ml)

Therapeutic effects

TABLE 1 characterization of test population

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T1-standard therapy

T 2-inhalation combination No. 1 combination inhalation mixture except standard therapy

T 3-inhalation combination No. 2 combination inhalation mixture except standard therapy

T4-inhalation combination No.3 inhalation mixture except standard therapy

T5-inhalation in addition to standard therapyFormulations

Inhalation mixture No. 1-IFN alpha 2b-2000IU/ml

Inhalation mixture No. 2-IFN gamma-200 IU/ml

Inhalation mixture No. 3-IFN alpha 2b,2000IU/ml+IFN gamma, 200IU/ml

"TRIAVIR" formulation (IFN alpha 2b,1000IU/ml + IFN gamma, 100IU/ml + lopinavir 80. Mu.g/ml).

Table 2.

Statistical data

Table 3.

Age of

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Fig. 1 also reflects age characteristics.

TABLE 4 weight of body

Figure 2 also reflects the body weight characteristics.

TABLE 5 height

Figure 3 also reflects height characteristics.

TABLE 6 BMI

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Fig. 4 also reflects the characteristics of the BMI.

Thus, the patient samples of the test group were homogeneous.

The results of the experimental and control groups were not significantly different.

The treatment effect was evaluated using the following sections:

composition of the inhalation mixture:

inhalation mixture No. 1-IFN alpha 2b-2000IU/ml

Inhalation mixture No. 2-IFN gamma-200 IU/ml

Inhalation mixture No. 3-IFN alpha 2b,2000IU/ml+IFN gamma, 200IU/ml

Inhalation mixture number 4-preparation of "TRIAVIR" (IFN. Alpha. 2b,1000IU/ml+IFNγ,100 IU/ml+lopinavir 80. Mu.g/ml),

The results were as follows according to the following criteria.

Main conclusion:

1. Compared to the screening results, the lung specificity changes of patients infected with the novel coronaviruses COVID-19 were confirmed by the results of the pulmonary computed tomography (MSCT) to be in good condition.

2. Pulse oximetry measurements are well-liked with SpO2 measurements compared to visit 1.

3. The relative numbers of lymphocytes in conventional clinical blood analysis normalize well.

4. Good potential (normalization of C-reactive protein levels in blood tests).

Secondary conclusion:

in the detection of whether the nasopharyngeal swab and the oropharyngeal swab contain SARS-CoV-2RNA by PCR, the number of patients whose result is negative was determined.

Frequency of exacerbations (exacerbations of pneumonia).

Table 7.

Results of a computed tomography (MSCT) study of the lung

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Table 8.

Conclusion of test results:

1. all patients, including the control group, showed a positive trend.

2. The positive trend was more pronounced in the inhaled mixture treatment group (see fig. 5-10).

3. The expression of the forward trend was highest in the group receiving "TRIAVIR" treatment (see figures 5-10).

TABLE 9 categorization of CT results based on detected degree of variation

TABLE 10 development stage of COVID 19 viral pulmonary lesions

All researchers emphasized the importance of "changes in ground glass, nodules, reticulation, and lesion area, which are consistent with the belief that these changes reflect the severity of the disease and can predict further progression of the disease. However, a combination of these variations is widely controversial, resulting in a large number of classification approaches. Clearly, there is a reliable correlation between the severity of lung lesions, CT method and clinical course of the disease, which is also a realistic concern for quantitative assessment of the extent of lung parenchyma changes.

TABLE 11 pulse oximetry and SpO2 measurement analysis

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Statistics of the 1 st visit

TABLE 12 summary of observations report

TABLE 13 descriptive statistics

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Stem and leaf map of 1 st visit

Group = 1.00

Stem and leaf map of 1 st visit

Group = 2.00

Fig. 11 also depicts the case of visit 1.

TABLE 14 summary of observations report

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TABLE 15 Normal distribution Standard

A. Li Erye Focus significance correction

If the error probability p of the result is less than 0.05, it is interpreted that the distribution is significantly different from the normal distribution. In this example, the distribution may be regarded as a normal distribution.

By plotting a normal distribution map, i.e., a Q-Q map (see fig. 11), it can be intuitively determined whether a given distribution is sufficiently close to a normal distribution. Here, each observed value is compared with an expected value under normal distribution. Assuming that the normal distribution is completely met, all points lie on a straight line. The observed values lie on the X-axis and the expected values lie on the Y-axis, all values being normalized (z-transformed). In this example, the observations are very close to a straight line.

In the excluded trend graph, the deviation of the observed value from the expected value under normal distribution is a function of the observed value. In the case of normal distribution, all points lie on a horizontal straight line passing through the zero point. If the deviation from the straight line is significant, it is indicated that the distribution is different from the normal distribution. In this figure, all values are also normalized (z-transformed) (see fig. 11).

Statistics of the 4 th visit

TABLE 16 summary of observations report

TABLE 17 descriptive statistics

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Stem and leaf panel for the 4 th visit = 1.00

Stem and leaf panel for the 4 th visit = 2.00

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Fig. 12 also depicts the case of the 4 th visit.

TABLE 18 Normal distribution Standard

A. Li Erye Focus significance correction

Visit 1

TABLE 19 group statistics

TABLE 20 independent sample criteria

Visit 4

TABLE 21 group statistics

TABLE 22 independent sample criteria

The analysis results were as follows: since the p value is less than or equal to 0.05, it can be concluded that there is a significant statistical difference in the expression levels of hemoglobin oxygen saturation before and after treatment; considering that the t standard value is negative= -4.171, it can be concluded that a significant statistical increase in SpO2 value occurs after treatment; that is, the treatment performed is effective.

The results obtained include:

observation number, average value, standard deviation of two groups, standard error of average value,

And (3) checking the result of the alignment standard of the column Wen Fangcha.

In general, if the p-value from the column test is less than 0.05 (variance heterogeneity), then the assumption of equal variances (homogeneity) is not accepted. For both homogeneous (equal) and heterogeneous (unequal), the following characteristics can be derived:

t test results: distribution value T, degree of freedom value, error probability p (labeled "significance (bilateral)"), mean difference, standard error thereof, and confidence interval. In this example, there was a significant difference in the effect of the treatment regimen on the oxygen content in the patient's blood (p=0.000).

TABLE 23 statistics of paired samples

TABLE 24 correlation of paired samples

TABLE 25 paired sample criteria

The oxygen content varied significantly throughout the treatment period (p=0.001).

TABLE 26 statistics of paired samples

TABLE 27 correlation of paired samples

TABLE 28 paired sample criteria

And differences within the control group.

Conclusion: since the p value is less than or equal to 0.05, it is concluded that there is a significant statistical difference in the expression levels of hemoglobin oxygen saturation before and after treatment; considering that the t standard values are negative = -10.073 (all patients) and-11.441 (control group), we can say that there is a significant statistical increase in SpO2 values after treatment; that is, the treatment performed, particularly the treatment performed using the inhalation mixture, is effective.

TABLE 29 descriptive statistics SpO2

TABLE 30 variance alignment criteria

Table 31.

ANOVA

SpO2

In this example, analysis of variance gave significant results (p=0.003).

SpO2 data is also shown in fig. 13 a-summary of SpO2 results (calculated panel mean) and panel comparison fig. 13 b.

Table 32.

Detection of SARS-CoV-2RNA by PCR

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FIG. 14 shows the results of the PCR tests for each group as negative and positive samples after the end of the treatment.

FIG. 15 shows a comparison of PCR results for each set of 1-3 visits.

The differences given by the results of the PCR standard are not reliable, which is obviously related to the unreliability of the validity determination standard itself (the level of false negative results corresponds to 45%).

Routine (clinical) blood analysis, determination of leukocyte levels, leukocyte counts

TABLE 33 lymphocyte percentage

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TABLE 34 descriptive statistics

Table 35 ANOVA

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The lymphocyte content is also reflected in figure 16.

The data fully demonstrate that the control group had better therapeutic effect than the control group. Meanwhile, in the control group,The group has the best curative effect.

Table 36 serum C-reactive protein (CRP) levels.

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TABLE 37 descriptive statistics

TABLE 38 ANOVA

The data is also shown in fig. 17.

The above table and fig. 17 fully demonstrate that the control group had better therapeutic effect than the control group. In this case, in the control group,The group has the best curative effect. Meanwhile, in the control group,/>The group has the best curative effect. The above examples clearly demonstrate the industrial applicability of the claimed invention, as these examples show the reproducibility and realisation of the use. At the same time, they also fully demonstrate the broad validity of the claims, since, according to the national law, in order to demonstrate the general degree of validity used by the applicant in disclosing the essential features of the invention, it is necessary to provide information about the specific form in which the essential features of the invention are implemented. The foregoing presents a sufficient number of embodiments of the invention to demonstrate the potential for achieving the technical results described by the applicant when using a private implementation of the essential features of the invention. As mentioned above, all coronaviruses share substantial similarities in structure, permeation mechanism and vital activity. According to national legislation, one example can be given with reference to graphic materials. The examples given illustrate the possibilities of realising the technical result of the invention and should not limit the scope of the applicant's rights. In summary, the basic feature of the achievement of the present invention is to take the "TRIAVIR" formulation in the form of aerosol inhalation. In this case, the expert may choose the effective dosage, the time of use and the frequency according to the weight and condition of the patient. As described above, the above information is obtained in patients who are general and do not have excessive weight. Thus, for other patient populations, the frequency and dosage of use and the time of treatment may be modified according to methods commonly used in medicine.

As can be seen from the above information, "TRIAVIR" can be used for both combination prophylaxis or therapy, as well as for both primary and mild infection or single agent prophylaxis. The mode and dosage of administration of the combination and the individual will be selected by the specialist in accordance with the condition of the patient.

Drawings

FIG. 1 illustrates age characteristics of a subject;

FIG. 2-body weight characteristics of subjects;

FIG. 3-height characteristics of the subject;

FIG. 4-BMI feature;

FIG. 5 a-CT results for control group at visit 1;

FIG. 5 b-CT results for the control group at visit 1;

FIG. 6 a-CT results for control group at visit 4;

FIG. 6 b-CT results for the control group at visit 4;

FIG. 7 a-group 2 CT results at visit 1;

FIG. 7 b-CT results for group 2 at visit 4;

FIG. 8 a-group 3 CT results at visit 1;

FIG. 8 b-CT results for group 3 at visit 1;

FIG. 9 a-group 4 CT results at visit 1;

FIG. 9 b-CT results for group 4 at visit 4;

FIG. 10 a-group 5 CT results at visit 1;

FIG. 10 b-CT results at group 5 at visit 4;

FIGS. 11 a-1.00 and 2.00 stem and leaf diagrams;

FIG. 11 b-histogram of group 1.00;

FIG. 11 c-histogram of group 2.00;

FIG. 11 d-normal Q-Q plot for group 1.00 visit;

FIG. 11 e-normal Q-Q plot for group 2.00, visit 1;

FIG. 11 f-normal Q-Q plot for group 1.00 with remote trend;

FIG. 11 g-2.00 sets of normal Q-Q and remote trend graphs for the 1 st visit;

fig. 12 a-1.00 and group 2.00, 4 th visit stem and leaf map;

FIG. 12 b-histogram of group 1.00;

FIG. 12 c-histogram of group 2.00;

FIG. 12 d-normal Q-Q plot of group 1.00 with remote trend for visit 4;

FIG. 12 e-normal Q-Q plot of group 2.00 with remote trend for the 4 th visit;

FIG. 12 f-normal Q-Q plot of group 1.00 with remote trend for visit 4;

FIG. 12 g-normal Q-Q plot of group 2.00 for visit 4 with remote trend for visit 4;

FIG. 13 a-SPO 2 summary calculated from the mean values of the groups;

FIG. 13 b-pulse oximetry and oximetry SpO2 measurement

Fig. 14 a-control group visit 1;

fig. 14 b-control group visit 2;

Fig. 14 c-control group visit 3;

fig. 14 d-control group 4 th visit;

fig. 15 a-control group visit 1;

Fig. 15 b-control group visit 2;

Fig. 15 c-control group visit 3;

fig. 15 d-control group visit 3;

FIG. 16 a-average number of lymphocytes at visit 1;

FIG. 16 b-average number of lymphocytes at visit 2;

FIG. 16 c-average number of lymphocytes at visit 3;

FIG. 16 d-average number of lymphocytes at visit 4;

fig. 17 a-average CRP at visit 1;

Figure 17 b-average CRP at visit 2.

Claims (6)

1. A method of preventing or treating a coronavirus infection comprising inhalation of an effective dose of a "TRIAVIR" formulation by a nebulizer.

2. The method of claim 1, wherein the coronavirus infection is designated COVID-19.

3. The method of claim 1, inhaled by the nebulizer for 10 minutes, 4 times per day, for 10 days.

4. The method of claim 1, wherein "TRIAVIR" is used as monotherapy or in combination therapy.

Use of a formulation of "TRIAVIR" for the prevention or treatment of coronavirus infection.

6. The use according to claim 4, wherein the coronavirus infection is designated COVID-19.