Use of Favipiravir in the Treatment of Coronavirus Infection

Abstract

To provide a drug with antiviral activity against coronavirus, which can be used for the treatment of a related disease caused by infection with a coronavirus, especially SARS coronavirus (SARS-CoV) and SARS-CoV-2.SOLUTION: The present application relates to a Favipiravir compound represented by Formula I, a geometric isomer, a pharmaceutically acceptable salt, a solvate and / or a hydrate thereof, and a pharmaceutical composition comprising the compound for treating a coronavirus infection.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] This application relates to the use of favipiravir represented by the following formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates, and pharmaceutical compositions containing said compounds for the purpose of treating infections caused by coronaviruses, particularly SARS coronavirus (SARS-CoV) and SARS-CoV-2.

[0002]

Chemical Formula

Background Art

[0003] This application is based on Chinese Patent Application No. 202010070142.0 filed on January 21, 2020, and claims the benefit of priority therefrom. The entire disclosure of that application is incorporated herein by reference in its entirety.

[0004]

[0005] Favipiravir (compound represented by formula I, T-705), a nucleoside analog drug with the chemical name 6-fluoro-3-hydroxypyrazine-2-carboxamide, is a viral RNA polymerase inhibitor. This drug is a broad-spectrum antiviral drug approved as an anti-influenza drug in Japan.Favipiravir has excellent inhibitory effects in vitro and in vivo against members of infectious RNA virus families such as the Filoviridae, Bunyaviridae, Arenaviridae, and Togaviridae, as well as members of other virus families such as the Orthomyxoviridae, Paramyxoviridae, Picornaviridae, and Flaviviridae, but its activity against coronaviruses has not been reported. After being taken up by cells, favipiravir is converted into a triphosphorylated active form, incorporated into the RNA strand during RNA transcription and replication, and specifically blocks the elongation of the viral RNA strand, thereby exerting an antiviral effect.

[0006] Favipiravir can effectively inhibit the viral titer in the supernatant of the Vero E6 cell line after viral infection, and has a 50% inhibitory concentration (IC 50) have been reported. The results of in vivo pharmacokinetic studies using the type I interferon receptor-deficient IFNAR- / - C57BL / 6 mouse model showed that at a challenge dose of 100% lethality in the placebo group administered at a dose of 300 mg / kg / day from day 6 to day 13, favipiravir showed a 100% protection rate, and at the same time, parameters such as body weight, glutamate-pyruvate transaminase, glutamate-oxaloacetate transaminase, and viral load were also significantly improved. The relevant research results of the UK Defence Science and Technology Laboratory (DSTL) showed that favipiravir was not cytotoxic to Vero C1008 cells even at a high concentration of 1.95 g / L, and that the cytopathic effect (CPE) of EBOV-Zaire could not be completely inhibited at a drug concentration of 62.5 mg / L or higher. When the A129 mouse model with type I and type II interferon deficiency was challenged with a 100% lethal prescription dose in the placebo group and a dose of 300 mg / kg / day (150 mg / kg per administration, twice a day) of favipiravir was administered intragastrically for 14 consecutive days starting 1 hour after the challenge, 100% of the mice could be protected from death, and the body weight of the mice in the treatment group was significantly improved.

[0007] The novel coronavirus 2019 (2019-nCoV) is a novel strain of coronavirus that has never been found in humans before. On February 11, 2020, the International Committee on Taxonomy of Viruses (ICTV) announced that the official name of the novel coronavirus 2019 (2019-nCoV) is Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). On the same day, the World Health Organization (WHO) announced that the official name of the disease caused by this virus is COVID. The symptoms of SARS-CoV-2 virus infection are mainly pneumonia, but according to the severity of the disease, it is classified into simple infection, mild pneumonia, severe pneumonia, acute respiratory distress syndrome, sepsis, septic shock, etc. Simple infection patients present with non-septic symptoms such as fever, cough, sore throat, nasal congestion, fatigue, headache, muscle pain or discomfort, and the elderly and immunosuppressed patients may have atypical symptoms. Mild pneumonia patients mainly present with cough, dyspnea, and tachypnea. Severe pneumonia is seen in adolescents, adults or children, and its main symptoms include increased respiratory rate, severe respiratory failure or dyspnea, central cyanosis, lethargy, unconsciousness or convulsions, shortness of breath, etc. The lung imaging of acute respiratory distress syndrome is bilateral ground-glass opacities that cannot be fully explained by infiltration, lobar exudation or atelectasis, or it is a pulmonary tumor image, and its main symptom is pulmonary edema. Septic patients often have life-threatening multiple organ dysfunction, and most critically ill patients present with septic shock symptoms and have a high mortality rate.

[0008] At present, the novel coronavirus infection is mainly treated with supportive therapy in the hospital, and specific antiviral drugs are not available.

Summary of the Invention

[0009] The concept of the invention of this application The object of the present invention is to discover a drug having antiviral activity against coronaviruses that can be used for the treatment of related diseases caused by infection with coronaviruses, particularly SARS coronavirus (SARS-CoV) and SARS-CoV-2. Such related diseases include simple infections (such as fever, cough, and sore throat), pneumonia, acute or severe acute respiratory infections, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis, and septic shock. Through creative research, it has been found by the present invention that Favipiravir represented by Formula I has the function of inhibiting the replication of SARS coronavirus (SARS-CoV) and SARS-CoV-2, and potentially exhibits excellent therapeutic effects in the treatment of diseases caused by SARS coronavirus (SARS-CoV) or SARS-CoV-2.

[0010] The present invention relates to a compound represented by Formula I, its geometric isomers, pharmaceutically acceptable salts, solvates, and / or hydrates:

[0011]

Chemical formula

[0012] In some embodiments, the pharmaceutically acceptable salts of the compound represented by Formula I described herein include inorganic or organic acid salts and inorganic or organic base salts. The present invention relates to all forms of the above salts, and examples thereof include, but are not limited to, sodium salts, potassium salts, calcium salts, lithium salts, meglumine salts, hydrochloride salts, hydrobromide salts, hydroiodide salts, nitrate salts, sulfate salts, hydrogen sulfide salts, phosphate salts, hydrogen phosphate salts, acetate salts, propionate salts, butyrate salts, oxalate salts, pivalate salts, adipate salts, alginate salts, lactate salts, citrate salts, tartrate salts, succinate salts, maleate salts, fumarate salts, picrate salts, aspartate salts, gluconate salts, benzoate salts, methanesulfonate salts, ethanesulfonate salts, benzenesulfonate salts, p-toluenesulfonate salts, embonate salts, etc.

[0013] The compound represented by formula I can inhibit the replication of coronaviruses in cells and can reduce the nucleic acid load of coronaviruses in cell cultures.

[0014] After creative invention research, the inventors of the present application discovered novel characteristics of the compound represented by formula I in cells: First, the compound represented by formula I can reduce the viral nucleic acid load in cells infected with SARS-CoV-2 at the micromolar concentration level; Second, the compound represented by formula I has a significant protective effect on mice infected with SARS coronavirus (SARS-CoV).

[0015] The present invention also relates to the use of the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates in the manufacture of a medicament for treating diseases or infections caused by coronaviruses, particularly SARS coronavirus (SARS-CoV) and / or SARS-CoV-2 [for example, but not limited to, respiratory diseases such as simple infections (such as fever, cough and sore throat), pneumonia, acute respiratory infections, severe acute respiratory syndrome (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.].

[0016]

Chemical formula

[0017] The present invention also relates to the use of the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates in the manufacture of a medicament as a coronavirus inhibitor.

[0018] The present invention also relates to the use of a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salt salts, solvates and / or hydrates in the manufacture of a medicament for inhibiting the replication or propagation of coronaviruses in cells (e.g., mammalian cells).

[0019] The present invention also relates to a pharmaceutical composition comprising a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salt salts, solvates and / or hydrates.

[0020] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable salt carrier or excipient. Specifically, the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a composite preparation.

[0021] In some embodiments, the pharmaceutical composition comprises an effective amount of a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salt salts, solvates and / or hydrates.

[0022] The present invention also relates to the use of a pharmaceutical composition comprising a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salt salts, solvates and / or hydrates in the manufacture of a medicament for treating respiratory diseases, including but not limited to, simple infections (e.g., fever, cough and sore throat), pneumonia, acute respiratory infections, severe acute respiratory infections (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis and septic shock, severe acute respiratory syndrome (SARS), etc.

[0023] The present invention also relates to a method for treating and / or preventing a disease in a mammal in need of treatment, or a method for inhibiting the replication or propagation of a coronavirus in a mammal in need of treatment, the method comprising administering to the mammal in need of treatment a pharmaceutical composition comprising a compound represented by formula I, a geometric isomer thereof, a pharmaceutically acceptable salt, solvate and / or hydrate thereof, or a therapeutically and / or prophylactically effective amount of a compound represented by formula I, a geometric isomer thereof, a pharmaceutically acceptable salt, solvate and / or hydrate thereof, wherein the disease includes diseases caused by coronaviruses.

[0024] In some embodiments, diseases caused by coronaviruses, particularly SARS coronavirus (SARS-CoV) and SARS-CoV-2, include, but are not limited to, respiratory diseases [e.g., simple infections (e.g., fever, cough, sore throat, etc.), pneumonia, acute respiratory infections, severe acute respiratory infections (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, severe acute respiratory syndrome (SARS), etc.].

[0025] The present invention also relates to the use of a pharmaceutical composition in the manufacture of a medicament for treating a disease or infection caused by a coronavirus, particularly SARS coronavirus (SARS-CoV) and SARS-CoV-2 [including, for example, respiratory diseases (such as simple infections like fever, cough, and sore throat), pneumonia, acute respiratory infections, severe acute respiratory infections (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, severe acute respiratory syndrome (SARS), etc.], wherein the pharmaceutical composition comprises a compound represented by formula I, a geometric isomer thereof, a pharmaceutically acceptable salt, solvate and / or hydrate thereof,

[0026]

Chemical formula

[0027] The present invention also relates to the use of a pharmaceutical composition in the manufacture of a medicament as a coronavirus inhibitor, wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates,

[0028]

Chemical formula

[0029] The present invention also relates to the use of a pharmaceutical composition in the manufacture of a medicament for inhibiting the replication or propagation of coronavirus (e.g., mammalian cells), wherein the pharmaceutical composition comprises a compound represented by formula I, its pharmaceutically acceptable salts, solvates and / or hydrates,

[0030]

Chemical formula

[0031] The present invention also relates to a pharmaceutical composition comprising a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates, which is used for treating diseases or infections caused by coronavirus, particularly SARS coronavirus (SARS-CoV) and SARS-CoV-2, such as, but not limited to, respiratory diseases [e.g., simple infections (e.g., fever, cough, sore throat, etc.), pneumonia, acute respiratory infections, severe acute respiratory infections (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, severe acute respiratory syndrome (SARS), etc.].

[0032] The present invention also relates to a pharmaceutical composition comprising a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates, which is used as a coronavirus inhibitor.

[0033] The present invention also relates to a pharmaceutical composition comprising a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates, which is used to inhibit the replication or propagation of coronaviruses in cells (e.g., mammalian cells).

[0034] The present invention also relates to a pharmaceutical composition used for treating diseases or infections caused by coronaviruses, particularly SARS coronavirus (SARS-CoV) and SARS-CoV-2, such as respiratory diseases [e.g., simple infections (e.g., fever, cough, and sore throat, etc.), pneumonia, acute respiratory tract infection, severe acute respiratory tract infection (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, severe acute respiratory syndrome (SARS), etc.], wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates,

[0035]

Chemical formula

[0036] The present invention also relates to a pharmaceutical composition used as a coronavirus inhibitor, wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates,

[0037]

Chemical formula

[0038] The present invention also relates to a pharmaceutical composition used for inhibiting the replication or propagation of coronavirus in cells (e.g., mammalian cells), wherein the pharmaceutical composition comprises a compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates,

[0039] [Chemical formula] Preferably, the pharmaceutical composition further comprises a pharmaceutically acceptable salt, carrier or excipient. Specifically, the pharmaceutical composition is a solid preparation, an injection, an external preparation, a spray, a liquid preparation, or a combined preparation.

[0040] In some embodiments, the coronavirus in the present application is SARS coronavirus (SARS-CoV) or SARS-CoV-2.

[0041] In some embodiments, the coronavirus in the present application is SARS-CoV-2.

[0042] In some embodiments, the disease caused by the coronavirus in the present application is the disease caused by SARS-CoV-2, i.e., COVID-19.

[0043] In some embodiments, the disease caused by the coronavirus in the present application is atypical pneumonia caused by SARS coronavirus (SARS-CoV).

[0044] In some embodiments, the mammals in the present application include Bovinae, Equidae, Caprinae, Suidae, Canidae, Felidae, rodents, primates, such as humans, cats, dogs or pigs.

[0045] In the present application, the official name of the term "2019 novel coronavirus (2019-nCoV)" is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0046] In some embodiments, the official name of the term "disease caused by 2019 novel coronavirus (2019-nCoV)" is COVID-19.

[0047] In the present application, the terms "therapeutically effective amount" or "prophylactically effective amount" refer to an amount that is sufficient to treat or prevent a patient's disease, but is low enough to avoid serious side effects within reasonable medical judgment (appropriate benefit / risk ratio). The therapeutically effective amount of a compound will vary depending on various factors such as the specific compound selected (e.g., selected considering the efficacy, effectiveness, and half-life of the compound), the administration route selected, the disease being treated, the severity of the disease being treated, the patient's age, size, weight, and health status, medical history, treatment duration, the nature of the current therapy, the desired therapeutic effect, etc., but it can be routinely determined by those skilled in the art.

[0048] Moreover, the specific dosages and methods of use of the compounds represented by Formula I, their geometric isomers, pharmaceutically acceptable salts, solvates, and / or hydrates depend on a number of factors for each different patient, such as the patient's age, weight, gender, natural health status, nutritional status, activity intensity of the drug, administration time, metabolic rate, disease severity, and the subjective judgment of the physician. Here, it is preferred to use a dosage of 0.001 to 1000 mg / kg body weight / day.

[0049] The pharmaceutical compositions described in the specification of the present application can be prepared in various dosage forms according to various administration routes.

[0050] According to the present application, the pharmaceutical composition can be administered by any one of the following routes: oral administration, aerosol inhalation, rectal administration, nasal administration, buccal administration, intravaginal administration, topical administration, parenteral administration, for example, subcutaneous, intravenous, intramuscular, intraperitoneal, subarachnoid, intracerebroventricular, intrasternal and intracranial injection or infusion, or administration using an implant reservoir, wherein the preferred administration routes are oral, intraperitoneal or intravenous administration.

[0051] For oral administration, the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be prepared in any oral dosage form, for example, but not limited to, tablets, capsules, aqueous solutions or aqueous suspensions. Carriers used in tablets generally include lactose and corn starch, and lubricants such as magnesium stearate can also be added. Diluents used in capsules usually include lactose and dried corn starch. Aqueous suspensions are usually used by mixing the active ingredient with a suitable emulsifier and a suitable suspending agent. If necessary, sweeteners, flavoring agents or coloring agents can also be added to the oral preparations.

[0052] For rectal administration, the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be formulated in the form of suppositories prepared by mixing the drug with a suitable non-irritating excipient. The excipient is in a solid state at room temperature but melts at rectal temperature to release the drug. Such excipients include cocoa butter, beeswax and polyethylene glycol.

[0053] For topical administration, particularly when administered topically for the treatment of neurologic diseases of an easily accessible affected surface or organ, such as the eye, skin or lower intestinal tract, the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be prepared in various topical dosage forms according to the different affected surfaces or organs. Specific instructions are as follows:

[0054] When administered locally to the eye, the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be formulated into dosage forms such as micronized suspensions or solutions, and the carrier used in that case is isotonic sterile saline having a certain pH, and a preservative such as benzyl alcohol chloride may or may not be added. In addition, when administered to the eye, the compound can also be formulated in the form of an ointment such as petrolatum ointment. <tmp <tmp

[0055] <tmp When administered locally to the skin, the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be formulated into suitable dosage forms such as ointments, lotions or creams, in which case the active ingredient is suspended or dissolved in one or more carriers. Carriers for ointments include, but are not limited to, the following: mineral oil, light oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax, and water. Carriers for lotions and creams include, but are not limited to, the following: mineral oil, sorbitan monostearate, Tween® 60, cetyl ester wax, hexadecenyl aryl alcohol, 2-octyldodecanol, benzyl alcohol and water. <tmp <tmp

[0056] <tmp When administered locally to the lower intestinal tract, the compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be formulated into dosage forms such as the rectal suppositories or suitable enemas described above, and in addition, a transdermal patch can also be used. <tmp <tmp

[0057] <tmp The compound represented by formula I, its geometric isomers, pharmaceutically acceptable salts, solvates and / or hydrates can be administered in the form of a sterile injection, such as a sterile aqueous solution for injection or an oily suspension, or in the dosage form of a sterile injection, and the carriers and solvents that can be used here include water, Ringer's solution, and isotonic saline. In addition, a sterile non-volatile oil such as monoglyceride or diglyceride can also be used as a solvent or suspension medium. <tmp

[0058] The drugs in the above various dosage forms can be manufactured according to conventional methods in the pharmaceutical field.

Brief Description of the Drawings

[0059]

Figure 1

Figure 2

Modes for Carrying Out the Invention

[0060] The following examples are exemplary preferred embodiments of the present application and do not constitute any limitation of the present application.

[0061] Example 1: Test of Favipiravir in Reducing the Viral Nucleic Acid Load of Cells Infected with SARS-CoV-2 Virus (1) Drug Treatment of Virus-Infected Cells

[0062] Vero E6 cells (purchased from ATCC, catalog number 1586) were seeded on a 24-well plate, incubated for 24 hours, and then infected with the virus. Specifically, the SARS-CoV-2 (2019-nCoV) virus (nCoV-2019 BetaCoV / Wuhan / WIV04 / 2019 strain provided by the Wuhan Institute of Virology, Chinese Academy of Sciences) was diluted to the corresponding concentration with a 2% cell maintenance solution [formulation: FBS (purchased from Gibco, catalog number 16000044) was added to MEM (purchased from Gibco, product number 10370021) at a volume ratio of 2%, thereby obtaining a 2% cell maintenance solution], and then added to the 24-well plate so that each well contained a virus load of 100 TCID 50 50. Next, Favipiravir (purchased from Selleck Chemicals, catalog number S7975) was diluted to the corresponding concentration with a 2% cell maintenance solution, and it was added to the corresponding wells so that the final drug concentrations were 100 μM, 33 μM, 11 μM, 3.7 μM, 1.23 μM, 0.41 μM, and 0.14 μM, respectively. Then the plate was placed in an incubator at 37°C and 5% CO2 and continuously cultured for 48 hours. For the cells in the vehicle control group, only the 2% cell maintenance solution was added without adding any test drug. (2) RNA Extraction

[0063] An RNA extraction kit was purchased from Qiagen (catalog number 74106). All the consumables (spin columns, RNase-free 2 mL collection tubes, etc.) and reagents (RLT, RW1, RPE, RNase-free water, etc.) required for the following RNA extraction process were parts of the kit. The following extraction process was all as recommended in the kit's instruction manual.

[0064] 1) 100 μL of the supernatant was taken from the test plate and added to a nuclease-free EP tube, and then 350 μL of buffer RLT was added. It was completely dissolved by pipetting (moving the liquid up and down with a pipette to mix), and the supernatant was taken by centrifugation. 2) Ethanol at 70% volume equivalent to the supernatant obtained in step 1) was added and mixed well. 3) The mixed solution obtained in step 2) was transferred to an RNase-free spin column and centrifuged at 12,000 rpm for 15 seconds, and the waste liquid was discarded. 4) 700 μL of buffer RW1 was added to the spin column, followed by centrifugation at 12,000 rpm for 15 seconds to wash the spin column, and the waste liquid was discarded. 5) 500 μL of buffer RPE was added to the spin column, followed by centrifugation at 12,000 rpm for 15 seconds to wash the spin column, and the waste liquid was discarded. 6) 500 μL of buffer RPE was added to the spin column, followed by centrifugation at 12,000 rpm for 2 minutes to wash the spin column, and the waste liquid was discarded. 7) The spin column was placed in a new 2 mL RNase-free collection tube and centrifuged at 12,000 rpm for 1 minute to dry the spin column, and then the entire spin column was transferred to the 1.5 mL collection tube in step 8). 8) The spin column dried in step 7) was placed in a new 1.5 mL collection tube, 30 μL of RNase-free water (water without RNase) was added, centrifuged at 12,000 rpm for 2 minutes, and an RNase inhibitor (purchased from NEB, catalog number M0314L) was added to the resulting eluate containing the corresponding RNA, and each RNA concentration was determined by measurement with a Nano Drop (trademark; purchased from Thermo Scientific, Nano Drop One).

[0065] (3) RNA Reverse Transcription In this experiment, a reverse transcription kit (PrimeScript TM (trademark) RT reagent kit containing gDNA Eraser manufactured by TaKaRa Company, catalog number RR047Q) was used for RNA reverse transcription. It was carried out according to the following steps.

[0066] (i) Removal of gRNA: RNA samples were collected from each experimental group. 1 μg of each sample was taken and subjected to reverse transcription. First, 2 μL of 5× gDNA Eraser buffer was added to the RNA samples of each experimental group, and RNase-free water was added to the reaction system to make up to 10 μL. The mixture was thoroughly mixed and incubated in a 42°C water bath for 2 minutes to remove any possible gRNA present in the samples. (ii) Reverse transcription: An appropriate amount of enzyme, primer mix, and reaction buffer were added to the samples obtained in (i), and RNase-free water was added to make up a volume of 20 μL. The reaction was carried out in a 37°C water bath for 15 minutes, and then placed in an 85°C water bath for 5 seconds to obtain cDNA by transcription.

[0067] (4) Real-time PCR Fluorescent quantitative PCR was used to determine the copy number per 1 mL of the original virus solution.

[0068] The reaction system was mixed using TB Green (registered trademark) Premix (manufactured by TaKaRa, catalog number RR820A), and the amplification reaction and reading were performed using a StepOnePlus (trademark) real-time PCR instrument (ABI brand). The copy number per 1 mL of the original virus solution was calculated. The steps were as follows:

[0069] (i) Standard: Plasmid pMT-RBD (this plasmid was provided by the Wuhan Institute of Virology, Chinese Academy of Sciences) was diluted to 5×10 8 copies / μL, 5×10 7 copies / μL, 5×10 6 copies / μL, 5×10 5 copies / μL, 5×10 4 copies / μL, 5×10 3 copies / μL, 5×10 2 copies / μL. 2 μL of the standard substance or cDNA template was taken for the qPCR reaction.

[0070] (ii) The sequences of the primers used in this experiment were as follows (all in the 5′→3′ direction):

[0071] [Chemical formula]

[0072] (iii) The reaction procedure was as follows: Pre-denaturation: 5 minutes at 95°C; Cycle parameters: A total of 40 cycles of 15 seconds at 95°C, 15 seconds at 54°C, and 30 seconds at 72°C.

[0073] (5) Cytotoxicity test of the test drug The detection of the cytotoxicity of the drug was carried out using a CCK-8 kit (from Beyotime). The specific steps were as follows: (i) 1×10 4 Vero E6 (ATCC) cells were seeded into a 96-well plate and incubated at 37°C for 8 hours.

[0074] (ii) The drug was diluted with DMSO to an appropriate concentration of the stock solution, and then diluted to the same concentration as in the drug treatment with MEM medium (purchased from Gibco, catalog number 10370021) containing 2% FBS (purchased from Gibco, catalog number 16000044). The original medium in the 96-well plate was discarded, 100 μL of the drug-containing MEM medium was added to the cells, and three replicate wells were made for each concentration. A vehicle control (DMSO and medium were added to the cell wells without adding the drug) and a blank control (DMSO and medium were added to the wells without adding cells) were prepared. After adding the drug, the wells were cultured at 37°C for 48 hours.

[0075] (iii) 20 μL of the CCK-8 solution (manufactured by Beyotime) was added to the wells to be tested, gently mixed to avoid generating bubbles, and continuously incubated at 37°C for 2 hours. OD 450It was read on a microplate reader (manufactured by Molecular Devices, model: SupectraMax(™) M5), and the cell viability was calculated:

[0076] [Number]

[0077] Here, A is the reading of the microplate reader.

[0078] (6) Experimental results The results of the virus replication inhibition experiment showed that test compounds at concentrations of 100 μM, 33 μM, 11.1 μM, and 3.7 μM could effectively inhibit the replication of the SARS-CoV-2 virus genome in the infected cell supernatant (Table 1 and Figure 1).

[0079] [Table 1]

[0080] The results of the cytotoxicity test showed that treatment with the test compound (favipiravir) did not change the cell viability at all test concentrations, that is, the test compound had no toxic effect on cells at all concentrations (Table 2 and Figure 1).

[0081] [Table 2]

[0082] Example 2: Test of favipiravir for protecting cells infected with SARS coronavirus from death (1) Grouping and marking of mice

[0083] One hundred and twenty-nine 129 mice, 3 to 4 weeks old and weighing 9 to 13 g (provided by the Institute Pasteur of Shanghai, Chinese Academy of Sciences), were randomly assigned to four groups, namely, a virus control group, a high-dose administration group, a medium-dose administration group, and a low-dose administration group, with 10 mice in each group, and marked with ear tags (studs) for individual identification. (2) Preparation of drugs

[0084] 0.5% CMC-Na was used as the solvent for dissolving favipiravir. First, the drug favipiravir was accurately weighed, an appropriate amount of 0.5% CMC-Na solution was added, and the sample was treated for 15 minutes under vortex stirring and ultrasonic conditions until a uniform particle suspension was obtained. Then, it was diluted two-fold (2×) with 0.5% CMC-Na and diluted according to the dosages of 200 mg / kg, 100 mg / kg, and 50 mg / kg, respectively. The obtained drug solution was stored at 4°C until later use. (3) Challenge, dosing, and data collection in mice

[0085] The challenge was performed by intraperitoneal injection. That is, each mouse was intraperitoneally injected with 1×10 6 PFU of the challenge dose of SARS coronavirus (provided by the Academy of Military Medicine). Here, the virus was diluted to the required amount with physiological saline. Intraperitoneal administration was performed once 4 hours, 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, and 144 hours after the challenge. Here, the dosages were 200 mg / kg for the high dose, 100 mg / kg for the medium dose, and 50 mg / kg for the low dose, and the virus control group was administered the same volume of 0.5% CMC-Na solvent. Body weight measurement and recording were performed at a fixed time point (fixed hour) every day, the death of the mice was recorded at the same time, and the survival curve was drawn as shown in Figure 2. (4) Test results

[0086] The in vivo test results indicate that the final survival rate of the mice is in a dose- and dose-effect dependent relationship (Table 3 and Figure 2), suggesting that treatment with the test compound can effectively protect mice from death caused by SARS coronavirus infection.

[0087]

Table 3

Claims

1. A pharmaceutical composition for treating a disease or infection caused by a coronavirus, said pharmaceutical composition comprising a compound represented by Formula I: 【Chemical 1】 a pharmaceutically acceptable salt, solvate and / or hydrate thereof, wherein said coronavirus is SARS-CoV-2, pharmaceutical composition.

2. A pharmaceutical composition for inhibiting a coronavirus, said pharmaceutical composition comprising a compound represented by Formula I: [Chemical 2] a pharmaceutically acceptable salt, solvate and / or hydrate thereof, wherein said coronavirus is SARS-CoV-2, pharmaceutical composition.

3. A pharmaceutical composition for inhibiting the replication or propagation of a coronavirus in cells, said pharmaceutical composition comprising a compound represented by Formula I: 【Chemical Formula 3】 a pharmaceutically acceptable salt, solvate and / or hydrate thereof, wherein said coronavirus is SARS-CoV-2, pharmaceutical composition.

4. The pharmaceutical composition according to any one of claims 1 to 3, further comprising a pharmaceutically acceptable salt carrier or excipient.

5. The pharmaceutical composition according to any one of claims 1 to 4, which is a solid preparation, injection, external preparation, spray, liquid preparation, or composite preparation.

6. The pharmaceutical composition according to claim 1, wherein the disease caused by said coronavirus is a disease caused by SARS-CoV-2, i.e., COVID-19.

7. The pharmaceutical composition according to claim 1, wherein the disease caused by said coronavirus is a respiratory disease.

8. The pharmaceutical composition according to claim 7, wherein the disease caused by said coronavirus is a simple infection, pneumonia, acute respiratory infection, severe acute respiratory syndrome (SARI), hypoxemic respiratory failure, acute respiratory distress syndrome, sepsis, septic shock, or severe acute respiratory syndrome (SARS).

9. The pharmaceutical composition according to claim 8, wherein said simple infection is fever, cough and / or sore throat.

10. The pharmaceutical composition according to claim 3, wherein said cells are mammalian cells.

11. The pharmaceutical composition according to claim 10, wherein said mammals are Bovinae, Equidae, Caprinae, Suidae, Canidae, Felidae, rodents, primates.

12. The pharmaceutical composition according to claim 10, wherein said mammals are humans, cats, dogs or pigs.

Images