CN114159442A - Application of favipiravir in preparation of drugs for preventing or treating coronavirus infectious diseases - Google Patents

Abstract

The invention discloses application of Favipiravir in treatment of coronavirus infectious diseases. The technical scheme is innovative by verifying the new application and the effective dosage range of the Favipiravir in a mouse test infected with the human coronavirus HCoV-229E, and a curative effect test of the Favipiravir in treating pneumonia caused by HCoV-229E virus infection is carried out, and the result shows that the Favipiravir shows obvious drug effect in the pulmonary index and the inhibition rate of a mouse infected with the HCoV-229E virus pneumonia.

Description

Application of favipiravir in preparation of drugs for preventing or treating coronavirus infectious diseases

Technical Field

The invention relates to the field of drug treatment, in particular to application of favipiravir in preparation of a drug for preventing or treating coronavirus infection diseases.

Background

Favipiravir was first reported in 1999. In 3 months 2014, favipiravir (favipiravir) is approved to be marketed in japan as a novel broad-spectrum anti-RNA virus drug for antiviral treatment of influenza a and b.

Coronaviruses belong to the order of the nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae), and the genus coronaviruses (Coronavirus), are important pathogens of diseases of many domestic animals and pets, including humans, and cause a variety of acute and chronic diseases.

Common human coronaviruses include four groups of HCoV-229E, HCoV-OC43, HCoV-NL63 and HCoV-HKU1, and 10% to 30% of upper respiratory tract infections worldwide are caused by these four groups of coronaviruses, which are second-ranked in the etiology of common cold, and are second only to rhinoviruses. The infection is seasonal, the disease is high in incidence in spring and winter every year, the latent period is 2-5 days, and the population is generally susceptible.

The human coronavirus HCoV-229E is a positive strand RNA coronavirus, and clinically causes infection symptoms of upper respiratory diseases such as rhinorrhea, headache, cough, sore throat, fever and the like; the medicine can also cause lower respiratory tract diseases such as pneumonia or bronchitis, gastrointestinal tract diseases and nervous system symptoms, and is particularly common to patients with cardiopulmonary diseases, people with low immunity, infants and the elderly, and research shows that when HCoV-229E infects the lung, proinflammatory cytokines can be released to cause pneumonia. However, there is no report on the use of Favipiravir for treating diseases caused by human coronavirus HCoV-229E infection, and there is no specific administration mode for infants and children without the ability of spontaneous inhalation and patients with active dyspnea.

Disclosure of Invention

The invention aims to provide a new application of Favipiravir in treating coronavirus, and simultaneously provides a safe and effective drug application with convenient administration route, clear dosage range and low dosage.

In order to achieve the purpose, the invention adopts the following technical scheme:

an application of Favipiravir in preparing a medicament for treating coronavirus infectious diseases;

the coronavirus infectious diseases are diseases caused by human coronavirus HCoV-229E infection;

the disease caused by the infection of the human coronavirus HCoV-229E is constructed by a BALB/c mouse model infected by the HCoV-229E virus;

the diseases caused by the infection of the human coronavirus HCoV-229E comprise: upper respiratory tract disease infection diseases, lower respiratory tract disease infection diseases, gastrointestinal tract diseases, nervous system diseases, nephritis and hepatitis; the upper respiratory tract infection diseases comprise: common cold, viral pharyngitis and laryngitis, herpetic pharyngolaryngitis, pharyngoconjunctivitis and tonsillitis; the lower respiratory tract infectious diseases include: bronchitis and pneumonia;

the medicament for preventing or treating the coronavirus infectious diseases is a medicinal preparation containing Favipiravir, and specifically is an inhalation preparation, an injection, an oral preparation or a nasal spray;

the daily administration dose of the inhalation preparation comprises Favipiravir of 27.3mg/60kg-272.7mg/60 kg;

the inhalation preparation is a Favipiravir inhalation solution;

the Favipiravir inhalation solution comprises active ingredients Favipiravir, meglumine, an osmotic pressure regulator and a solvent, and the pH value of the solution is 6-8;

preferably, the content of the favipiravir is 1-50 mg/ml;

preferably, the content of the meglumine is 3-110 mg/ml;

preferably, the osmolality adjusting agent is selected from sodium chloride in an amount such that the osmolality of the inhalation solution is 270-330 mOsmol/kg;

preferably, the solvent is water for injection.

The preparation method of the Favipiravir inhalation solution comprises the following steps:

adding a solvent accounting for 80% of the total amount of the required preparation solution into a liquid dispenser, adding Favipiravir accounting for the amount of the prescription, stirring until the Favipiravir is fully dispersed in the solution, gradually adding meglumine and sodium chloride, stirring until the meglumine and the sodium chloride are fully dissolved, adjusting the pH value to 6-8 by using the meglumine, adding the solvent to a constant volume to the total amount of the prescription, filtering, subpackaging and filling.

In the test of the application of Favipiravir in a BALB/c mouse model infected by human coronavirus HCoV-229E, the Favipiravir inhalation solution is administrated for 10min at the concentrations of 0.5mg/ml, 1mg/ml, 2mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 40mg/ml, 50mg/ml and 60 mg/ml; the atomization mode is adopted for mouse administration, the human coronavirus HCoV-229E is adopted to infect a mouse pneumonia model, and the effectiveness of the medicine method plavir in the mouse lung index and the inhibition rate is detected.

Advantageous effects

The new application of the Favipiravir is proved through a mouse treatment test of the human coronavirus HCoV-229E, and meanwhile, a curative effect test of the Favipiravir inhalation solution in treating pneumonia caused by HCoV-229E virus infection is carried out, and test results show that the Favipiravir inhalation solution shows good drug effect in lung index and inhibition rate of mice infected with the HCoV-229E virus pneumonia, and can reduce the lung index of the mice.

Detailed Description

The treatment effect of the P.vera inhalation solution on the mouse pneumonia model of the human coronavirus HCoV-229E is evaluated from 2 aspects of lung index and inhibition rate by adopting the mouse pneumonia model infected with the human coronavirus HCoV-229E.

1 test Material

1.1 test drugs

Table 1: favipiravir inhalation solution specification and batch number

Table 2: favipiravir inhalation solution prescription composition

The preparation method comprises the following steps:

adding a solvent accounting for 80% of the total amount of the required preparation solution into a liquid dispenser, adding Favipiravir of a prescription amount, stirring until the Favipiravir is fully dispersed in the solution, gradually adding meglumine and sodium chloride, stirring until the solution is fully dissolved, adjusting the pH value to 6-8 by using the meglumine, and adding the solvent to fix the volume to the total amount of the prescription to respectively prepare Favipiravir inhalation solutions with the concentrations of 0.5mg/ml, 1mg/ml, 2mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 40mg/ml, 50mg/ml and 60 mg/ml.

The stability of the faplatavine inhalation solutions prepared in batches 200701, 200702, 200703, 200704, 200705, 200706, 200707, 200708 and 200709 was measured and refrigerated (2-8 liquid), and the measurement result after 10 days was found to be 3.

Table 3: favipiravir inhalation solution stability investigation

As can be seen from table 3, after the prepared favipiravir inhalation solution is refrigerated and left for 10 days, the properties, pH value and related substances have no significant change, i.e., the stability of the prepared favipiravir inhalation solution is better.

1.2 Positive drugs

Recombinant human interferon alpha 2b injection, Tianjin is not famous for production of biological medicine Limited company. Batch number: r0191201, production date 2019.11.26, expiration date to 2021.11.25. The specification is 300 ten thousand IU/1ml, and the liquid is colorless and transparent. The usage and dosage are as follows: referring to the seventh edition of the diagnosis and treatment plan for the novel coronavirus pneumonia, the clinical usage is recommended, 2ml of sterile water for injection is added for 500 ten thousand IU, and the mixture is atomized and inhaled for 2 times per day.

1.3 Experimental animals

Table 4: laboratory animal information

1.4 strains and cells

1.4.1 virus strains: human coronavirus 229E (HCoV-229E), supplied by institute of medical and biotechnology, national academy of sciences, was passed through the laboratory and stored in a freezer at-80 ℃ for further use.

1.4.2 cell lines: mouse mononuclear macrophage RAW264.7 purchased from Beijing Beinanna institute of Biotechnology, passaged in this laboratory, and stored in liquid nitrogen for further use.

1.5 test reagents

The test reagent is diethyl ether, the batch number is 20150417, and the test reagent is produced by national drug group chemical reagent limited company and is used for mouse anesthesia.

1.6 test apparatus

Table 5: test instrument information

2. Test method

2.1 drug dosage design and formulation

2.1.1 recombinant human interferon alpha 2b injection

The dosage of the usage is recommended according to the seventh edition of diagnosis and treatment plan for novel coronavirus pneumonia, 2ml of sterile water for injection is added into 500 ten thousand IU, and 2 times per day, and the medicine is atomized and inhaled. I.e. 20 ten thousand IU/kg/d. The mice used 440 ten thousand IU/kg/d, which is 2 times the human dose, during the test.

Preparing a liquid medicine: the test shows that the inhalation amount of each mouse is 0.25ml in 10min, so the medicine is prepared into liquid medicine with the concentration of 26.4 ten thousand IU/ml by using normal saline before the test, and the time is 1 time/day and 4 days are continued when 15g of mice inhale for 10min each time.

2.1.2 Favipiravir inhalation solution specifications

The daily dosage of the Favipiravir inhalation solution is 5.45mg/60kg-27.27mg/60kg, and the dosage is 9 specifications of batch numbers 200701, 200702, 200703, 200704, 200705, 200706, 200707, 200708 and 200709 according to the mouse coefficient of 11 and the human body weight of 60 kg. The preparation is administered by aerosol inhalation for 10min, once daily, 5ml once, and continuously for 4 days according to the above 9 specifications.

2.2 passage of Virus

Taking 25cm of RAW264.7 cells which have grown into a monolayer²Removing culture medium, washing cell surface with cell maintenance solution for 3 times, adding cell maintenance solution 5ml, and addingAdding 200 mul of HCoV-229E virus solution, culturing in an 3705% CO2 incubator for 72-96 h, observing the cytopathic condition under an inverted microscope every day until 80% of cells have obvious lesions (CPE), placing a cell culture bottle in a low-temperature refrigerator at minus 80 ℃ for freezing and storing, and repeatedly freezing and thawing the virus solution for 3 times for measuring the virus titer.

2.3 Virus titre assay

Collecting 96-well plate grown into single layer of RAW264.7 cells, discarding culture solution, washing cells for 3 times with cell maintenance solution, and mixing at 10 times ratio (10 times of cell maintenance solution)^-1～10^-8) HCoV-229E virus solution with different titers is inoculated by dilution, 8 dilutions are carried out, 100 mul/well, each dilution is carried out for 4 multiple wells, and a normal cell control is set. 96-well plate device 37 and 5% CO₂Culturing for 72-96 h in an incubator, observing the cytopathic condition under an inverted microscope every day, and recording the cytopathic condition of each hole. 50% cytopathic concentration (TCID) calculated by Reed-Muench method₅₀)。

2.4 construction and administration of human coronavirus pneumonia mouse model

96 ICR mice, randomly divided by body weight: a normal control group, a model control group, an interferon alpha 2b positive drug group, a Favipiravir 0.5mg/ml group, a Favipiravir 1mg/ml group, a Favipiravir 2mg/ml group, a Favipiravir 5mg/ml group, a Favipiravir 10mg/ml group, a Favipiravir 20mg/ml group, a Favipiravir 40mg/ml group, a Favipiravir 50mg/ml group and a Favipiravir 60mg/ml group, and 12 groups in total, wherein each group comprises 8 male and female halves. After mild ether anesthesia of mice in each group except the normal control group, 100TCID was used₅₀HCoV-229E was administered to nose in drops of 50. mu.l/case. On the day of infection, each administration group was inhaled by nebulization 10min each time, 1 time daily for 4 consecutive days. After weighing the body weight one day after the last administration, the lung was dissected and weighed, and the lung index inhibition rate were calculated.

2.5 mouse Lung index and inhibition calculation

After weighing the weight of the mouse, dissecting and taking lung tissues and weighing the lung weight, and calculating the lung index and the inhibition rate of the mouse by using the specific calculation formula as follows:

lung index ═ lung wet weight (g) × 100/body weight (g)

2.6 statistical analysis

Data are expressed as (MEAN ± SD), differences between groups are measured by T-test, and P <0.05 is statistically significant.

3 results of the experiment

TABLE 6 Effect of the tested drugs on the human coronavirus pneumonia mouse model (pulmonary index and inhibition Rate)

Note: comparing with normal control group^##P<0.01; comparison with model control group^*P<0.05，^**P<0.01。

Table 1 the results show that: the lung index of the mice in the model control group is obviously increased, and the mice have obvious difference compared with the normal control group (P < 0.01); the 7 tested drugs were administered by aerosol inhalation 10min each time, 1 time per day for 4 consecutive days, with the following results:

favipiravir inhalation solution: the lung index of seven specification groups of mice, namely 0.5mg/ml, 1mg/ml, 2mg/ml, 5mg/ml, 10mg/ml, 20mg/ml, 40mg/ml, 50mg/ml and 60mg/ml, is reduced, compared with a model control group, the lung index is obviously different except 0.5mg/ml (P <0.05 and P <0.01), and the lung index inhibition rate is obviously reduced at 60mg/ml in combination with the comprehensive consideration of the lung index inhibition rate. Therefore, the preferred administration range of the Favipiravir inhalation solution is 1mg/ml to 50 mg/ml.

Claims (10)

1. Application of favipiravir in preparation of drugs for preventing or treating coronavirus infectious diseases.

2. The use according to claim 1, wherein the coronavirus infectious disease is a disease caused by infection with human coronavirus HCoV-229E.

3. The use according to claim 2, wherein the disease caused by infection with human coronavirus HCoV-229E comprises: upper respiratory tract infection diseases, lower respiratory tract infection diseases, gastrointestinal tract diseases, nervous system diseases, nephritis and hepatitis, wherein the upper respiratory tract infection diseases comprise: common cold, viral pharyngitis and laryngitis, herpetic pharyngolaryngitis, pharyngoconjunctivitis and tonsillitis; the lower respiratory tract infectious diseases include: bronchitis and pneumonia.

4. The use of claim 1, wherein the disease caused by HCoV-229E infection with human coronavirus is constructed by infecting a BALB/c mouse model with HCoV-229E virus.

5. The use according to claim 1, wherein the medicament for preventing or treating coronavirus infectious diseases is a pharmaceutical preparation containing Favipiravir, and is specifically an inhalation preparation, an injection, an oral preparation or a nasal spray.

6. The use according to claim 5, wherein the inhaled formulation comprises fapirovir in an amount from 27.3mg/60kg to 272.7mg/60kg per daily dose for human administration.

7. The use according to claim 5, wherein the inhalation formulation is a Favipiravir inhalation solution.

8. The use according to claim 7, wherein the inhalation solution comprises the active ingredient Favipiravir 1-50mg/ml, meglumine, an osmotic pressure regulator and a solvent, and the pH of the solution is 6-8.

9. The use as claimed in claim 7, wherein the meglumine content is from 3 to 110mg/ml, the tonicity modifier is selected from sodium chloride in an amount such that the osmolality of the inhalation solution is 270-330mOsmol/kg, and the solvent is selected from water for injection.

10. Use according to claim 7 or 8, wherein the inhalation solution is prepared by:

adding a solvent accounting for 80% of the total amount of the required preparation solution into a liquid dispenser, adding Favipiravir accounting for the amount of the prescription, stirring until the Favipiravir is fully dispersed in the solution, gradually adding meglumine and sodium chloride, stirring until the meglumine and the sodium chloride are fully dissolved, adjusting the pH value to 6-8 by using the meglumine, adding the solvent to a constant volume to the total amount of the prescription, filtering, subpackaging and filling.