Combination drugs and treatment methods for treating coronavirus infection

Abstract

The present invention provides a combination drug comprising nitazoxanide or tizoxanide and ribavirin, and its use for treating or preventing coronavirus infection. The provided combination drug is particularly effective for treating diseases (COVID-19) caused by SARS-CoV-2 infection.

Description

Technical Field

[0001] The present invention relates to a combination drug and a treatment method for treating or preventing coronavirus infection, in particular acute respiratory infectious diseases or pneumonia-based diseases caused by SARS-CoV-2 (new coronavirus) infection. Background Art

[0002] Since the outbreak of the acute respiratory tract infection caused by SARS-CoV-2 at the end of 2019, COVID-19 has posed an unprecedented threat to the life and health of human beings around the world. SARS-CoV-2 belongs to the same subfamily of coronaviruses as SARSr-CoV and MERSr-CoV, but their genetic homology is not high. The new pneumonia caused by SARS-CoV-2 infection is highly contagious, and the general population is susceptible and difficult to control. It has become a long-term problem. Although the development of anti-SARS-CoV-2 drugs is actively promoted worldwide, there is no particularly effective therapeutic drug. Finding a treatment plan that can effectively treat or prevent diseases caused by its infection is still an urgent clinical need.

[0003] Nitazoxanide (NTZ) is a safe, orally bioavailable thiazolide approved in the United States for the treatment of Cryptosporidium parvum and Giardia lamblia infections, and is used to treat infectious gastroenteritis. The drug has been used in many countries in Africa and South America and has shown good anti-parasitic effects without adverse reactions. Tizoxanide (TIZ) is the main active metabolite of nitazoxanide in the body. As anti-protozoal infection drugs, nitazoxanide and tizoxanide were later found to affect the activity of inflammatory proteins COX-2 (cyclooxygenase), NF-κB, p65, IκBα, ERK1 / 2 or p38MAPK in the body's cells, thereby treating inflammatory-related diseases, including inflammatory diseases induced by bacterial endotoxins such as lipopolysaccharide (LPS). There are also studies that show that nitazoxanide has an inhibitory effect on the activity of acetyl-CoA carboxylase in HepG2 cells, and can play an anti-obesity, lipid-lowering, anti-fatty liver and anti-atherosclerotic role (CN110478357A). In addition, some studies believe that nitazoxanide and tizoxanide can reduce the duration of related symptoms caused by human respiratory syncytial virus (CN108289961A), and have a potential role in the clinical treatment of rotavirus, norovirus, HBV and HCV infections ( Rossignol, Romark Laboratories, LC, 2014; CN108289961A), but there is no data to support its antiviral activity in vivo. The mechanism of nitazoxanide's antiviral activity is still unclear.

[0004] Ribavirin is a nucleoside analog antiviral drug approved by the FDA for the treatment of hepatitis C, viral hemorrhagic fever, and severe lower respiratory tract infections caused by respiratory syncytial virus (RSV) (only nebulizer inhalation). Although it has been shown to have inhibitory effects on a variety of other DNA and RNA viruses (such as herpes simplex virus, influenza virus, etc.) in vitro, its indications are usually limited due to the lack of clinical evidence. For example, the FDA clearly stated in the approved drug instructions that ribavirin is not suitable for the treatment of influenza, etc. ( (ribavirin) Tablets Initial U.S. Approval: 2002). Ribavirin alone or in combination with systemic glucocorticoids has been tried for empirical treatment of severe acute respiratory syndrome (SARS) caused by SARS-CoV infection, but the clinical benefit was disappointing (WHO. Management of severe acute respiratory syndrome (SARS). 2003Apr 11.). SUMMARY OF THE INVENTION

[0006] The present invention provides a drug combination regimen for treating or preventing coronavirus infection, especially 2019 novel coronavirus (SARS-CoV-2) infection, which can inhibit coronavirus infection, alleviate or reverse pneumonia-based diseases caused by coronavirus infection, or prevent coronavirus infection.

[0007] In one aspect, the present invention relates to a novel combination drug and its use in treating or preventing coronavirus infection or diseases or conditions related thereto. The infection or related disease refers to a disease or condition caused by SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and / or HKU1 infection, in particular SARS-CoV-2 infection and pneumonia caused therefrom.

[0008] In one embodiment, the combination drug comprises at least one selected from nitazoxanide or tizoxanide or a pharmaceutically acceptable salt thereof, and at least one ribavirin or a prodrug, metabolite, derivative, enantiomer, diastereomer, tautomer, racemate or any pharmaceutically acceptable salt or ester of the foregoing.

[0009] In one embodiment, the combination comprises at least one nitazoxanide or a pharmaceutically acceptable salt thereof, and at least one ribavirin or a pharmaceutically acceptable salt or ester thereof.

[0010] In one feature, the nitazoxanide is administered in an oral formulation, including tablets, suspensions, or capsules.

[0011] In one feature, the ribavirin can be administered as an oral formulation, inhalation or injection. In one embodiment, ribavirin can be administered directly through the respiratory tract in the form of an aerosol inhalation.

[0012] In one embodiment, the combination drug disclosed in the present invention is used to treat, alleviate or relieve at least one symptom of the related disease or condition, wherein the symptom includes cough, decreased appetite, wheezing, dyspnea, fever, malaise, dizziness, nausea, decreased or loss of smell and pneumonia.

[0013] In one embodiment, the combination drug disclosed in the present invention is used in the preparation of a pharmaceutical composition for treating or preventing coronavirus infection or a disease or condition related thereto, wherein the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and / or HKU1, in particular SARS-CoV-2, and the related disease or condition is pneumonia.

[0014] In another aspect, the present invention relates to a method for treating or preventing coronavirus infection or a disease or condition associated therewith, the method comprising administering a therapeutically effective amount of the above-mentioned new combination drug to a subject in need thereof. The associated disease or condition is an acute respiratory infectious disease or pneumonia.

[0015] In one embodiment, the treatment or prevention method comprises administering to a subject in need thereof a therapeutically effective amount of at least one selected from nitazoxanide or tizoxanide or a pharmaceutically acceptable salt thereof, or any solvate thereof, and a therapeutically effective amount of at least one ribavirin or a prodrug, metabolite, derivative, enantiomer, diastereomer, tautomer, racemate or any pharmaceutically acceptable salt or ester of the foregoing.

[0016] In one embodiment, the method of treatment or prevention comprises administering to a subject in need thereof a therapeutically effective amount of at least one selected from nitazoxanide or a pharmaceutically acceptable salt thereof, and a therapeutically effective amount of at least one ribavirin or a pharmaceutically acceptable salt or ester thereof.

[0017] In some embodiments, nitazoxanide or tizoxanide and ribavirin can be administered to a subject simultaneously or sequentially.

[0018] In one feature, the nitazoxanide is administered in an oral formulation, including tablets, suspensions, or capsules.

[0019] In one feature, the ribavirin can be administered as an oral formulation, inhalation or injection. In one embodiment, ribavirin can be administered directly through the respiratory tract in the form of an aerosol inhalation.

[0020] In one feature, the subject is a patient with severe illness caused by coronavirus infection, and the ribavirin is administered directly through the respiratory tract in the form of a nebulizer inhaler.

[0021] In one embodiment, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and / or HKU1, in particular SARS-CoV-2, and the associated disease or condition is pneumonia.

[0022] In certain embodiments, the method for preventing a disease or condition caused by or associated with a coronavirus infection refers to chemoprevention / pharmacological prevention.

[0023] In some embodiments, the administration can be used to prevent the progression of mild illness to severe illness, or to alleviate at least one symptom, wherein the at least one symptom is selected from cough, decreased appetite, wheezing, dyspnea, fever, malaise, dizziness, nausea, decreased or loss of smell, and pneumonia.

[0024] The foregoing and other objects, aspects, features and advantages of the present invention will be further explained by the following description and claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 The results of testing the cytotoxicity of nitazoxanide (NTZ), ribavirin (RBV) and GH23 (NTZ+RBV) on Vero E6 cells using the standard CCK-8 method are shown.

[0027] Figure 2 The antiviral activity of NTZ, RBV and GH23 against SARS-CoV-2 in vitro was shown. DETAILED DESCRIPTION OF THE INVENTION

[0029] I. Definitions

[0030] As used in the specification and claims, the singular forms "a", "an" or "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

[0031] As used herein, "about" means within a range of ±10%. For example, "about 1" means "0.9 to 1.1", "about 2%" means "1.8% to 2.2%", "about 2% to 3%" means "1.8% to 3.3%", and "about 3% to about 4%" means "2.7% to 4.4%".

[0032] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0033] The term "effective amount" of an active agent refers to an amount sufficient to induce a desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of a compound of the invention may vary depending on factors such as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.

[0034] The term "treatment, treating or therapy" of a disease or disorder refers to a method of reducing, delaying or ameliorating such a condition before or after the condition occurs. Treatment may be directed to one or more effects or symptoms of the disease and / or underlying pathology. Treatment may be any reduction, and may be, but is not limited to, complete elimination of a disease or symptom of a disease. Such reduction or prevention, as measured by any standard technique, may be at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% or 100% compared to an equivalent untreated control.

[0035] The term "subject" refers to any animal (e.g., mammal), including but not limited to humans, non-human primates, rodents, etc., which are recipients of a specific treatment or prevention regimen. Generally, the terms "subject" and "patient" or "a person or high-risk group in need of infection prevention" are used interchangeably herein to refer to humans.

[0036] The term "and / or" should be understood as "one or both" of the connected elements, i.e., in some cases, they are present in combination and in some other cases, they are present separately. Thus, as a non-limiting example, when used in conjunction with open language (such as "comprising"), a reference to "A and / or B" may refer to only A (optionally including other elements in addition to B) in one embodiment; only B (optionally including other elements in addition to A) in another embodiment; and both A and B (optionally including other elements) in yet another embodiment; and so on. When a range of values ​​is listed herein, it is intended to cover every value and sub-range within that range. For example, "1-5 mg" is intended to include 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 1-2 mg, 1-3 mg, 1-4 mg, 1-5 mg, 2-3 mg, 2-4 mg, 2-5 mg, 3-4 mg, 3-5 mg, and 4-5 mg.

[0037] The term "administering" is used in its broadest sense herein. These terms refer to any method of administering to a subject a compound or combination drug described herein, and may include, for example, administering the compound to a subject systemically, locally, or in situ. Therefore, the compounds disclosed herein produced in a subject from a combination drug (whether or not it includes the present compound) are included in these terms. When these terms are used in conjunction with "systemic" or "systemically", they generally refer to systemic absorption or accumulation of a compound or combination drug in the blood, followed by distribution throughout the body.

[0038] As used herein, "combination" or "combination" therapy or treatment refers to the administration of at least two different drugs to treat a disorder, condition or symptom, for example, viral pneumonia, viral infection, coronavirus pneumonia, etc. Such combination therapy may include the administration of one drug before, during and / or after the administration of another drug. The interval between drug administrations may be up to several weeks, but more typically within 48 hours, and most typically within 24 hours.

[0039] The compounds of the present invention can form salts, which are also within the scope of the present invention. Unless otherwise indicated, the compounds of the present invention mentioned herein should be understood to include their salts. The term "salt" as used herein means acidic and / or basic salts formed with inorganic and / or organic acids and bases. In addition, when the compounds of the present invention contain a basic moiety (such as, but not limited to, pyridine or imidazole) and an acidic moiety (such as, but not limited to, carboxylic acid), zwitterions ("inner salts") can be formed in and included in the term "salt" used herein. Pharmaceutically acceptable (i.e., non-toxic, physiologically acceptable) salts are preferred, although other salts are also useful in, for example, separation or purification steps in the preparation process.

[0040] The chemoprevention described in this article refers to the administration of the combination drug regimen provided in this article to asymptomatic viral infected persons, or persons who test negative for viral infection but are considered to have the possibility of viral infection based on epidemiological history analysis, or patients who test negative for viral infection but are considered to have viral infection-like pneumonia based on other medical diagnoses (such as medical imaging analysis, etc.), as a preventive measure to prevent or reduce the pathogenicity of viral infection.

[0041] The terms "synergistically," "synergistic," "synergistically," or "enhancing" refer to the interaction or combination of two or more components to produce a sum (or "additive effect") of effects greater than their individual effects.

[0042] II. Combination drugs, uses and treatment methods

[0043] The present invention provides novel combination drugs and their use in treating or preventing coronavirus infection (especially SARS-CoV-2 infection) or diseases or conditions associated therewith (such as pneumonia, especially COVID-19). The present invention also provides a method for treating or preventing coronavirus infection (especially SARS-CoV-2 infection) or diseases or conditions associated therewith (such as pneumonia, especially COVID-19), comprising administering a therapeutically effective amount of the combination drug described herein to a subject in need thereof.

[0044] The new combination drug provided by the present invention, in one embodiment, comprises at least one thiazolyl compound selected from nitazoxanide, tizoxanide or its derivatives, any pharmaceutically acceptable salts of the foregoing, and any solvates of the foregoing, and at least one ribavirin or its prodrug, metabolite, derivative, enantiomer, diastereomer, tautomer, racemate, or any pharmaceutically acceptable salt or ester of the foregoing.

[0045] The present invention also provides the use of the combined drug in the preparation of a pharmaceutical composition for treating or preventing coronavirus infection or diseases or conditions related thereto, wherein the coronavirus includes but is not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and HKU1, in particular SARS-CoV-2, and the related diseases or conditions are acute respiratory infectious diseases, including pneumonia.

[0046] Nitazoxanide chemical name: o-[N-(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenol acetate), the chemical formula is as follows:

[0047]

[0048] The chemical name of Tizoxanide is 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide, and the chemical formula is as follows:

[0049]

[0050] Chemical name of ribavirin: 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-yl]-1H-1,2,4-triazole-3-carboxamide, also known as 1-(β-D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide, CAS registration number 36791-04-5, chemical formula is as follows:

[0051]

[0052] The present invention provides nitazoxanide or tizoxanide combined with ribavirin for synergistically inhibiting coronavirus infection, such as SARS-CoV-2 infection.

[0053] The above findings provide a feasible combination drug treatment regimen for the treatment of SARS-CoV-2 or other coronavirus infections and their related diseases or conditions, such as pneumonia, which can effectively treat coronavirus infections, or quickly relieve related symptoms, or reduce the duration of clinical symptoms, and reduce the possibility of coronavirus infection and retransmission.

[0054] In one embodiment, the combination drug disclosed herein shows no new adverse events and has a safety profile similar to that of each drug in the combination drug when used alone.

[0055] In one embodiment, the combination drug disclosed in the present invention can prevent the progression of mild symptoms to severe symptoms, or alleviate at least one symptom, the symptom comprising cough, decreased appetite, wheezing, dyspnea, fever, malaise, dizziness, nausea, decreased or loss of smell, and pneumonia.

[0056] On the other hand, the present invention relates to a method for treating or preventing coronavirus infection or a disease or condition associated therewith. The method comprises administering to a subject in need thereof a therapeutically effective amount of at least one thiazolide compound selected from nitazoxanide, tizoxanide or a derivative thereof, any of the foregoing pharmaceutically acceptable salts, and any of the foregoing solvates, and a therapeutically effective amount of at least one ribavirin or a prodrug, metabolite, derivative, enantiomer, diastereomer, tautomer, racemate, or any of the foregoing pharmaceutically acceptable salts or esters.

[0057] In one embodiment, the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 or HKU1, in particular SARS-CoV-2. In one embodiment, the related disease or disorder is pneumonia.

[0058] In one embodiment, the method comprises administering to a subject in need thereof a therapeutically effective amount of nitazoxanide, and a therapeutically effective amount of ribavirin.

[0059] In some embodiments, nitazoxanide and ribavirin are administered to a patient simultaneously, contemporaneously, separately, and / or sequentially.

[0060] In some embodiments, nitazoxanide is administered to a subject at a total daily dose ranging from about 100 mg to about 2000 mg. In some embodiments, nitazoxanide can be administered once daily (QD), twice daily (BID), three times daily (TID), or four times daily (QID).

[0061] In some embodiments, nitazoxanide is administered to a subject in a single dose ranging from about 100 mg to about 1000 mg. In some embodiments, nitazoxanide is administered to a subject in a single dose selected from about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 700 mg, about 800 mg, about 900 mg, and about 1000 mg.

[0062] In some embodiments, nitazoxanide is administered to a subject in a single dose of about 300 mg to about 500 mg twice daily (BID) or three times daily (TID).

[0063] In some embodiments, nitazoxanide is administered to a subject at a dose selected from about 200 mg BID, about 250 mg BID, about 300 mg BID, about 350 mg BID, about 400 mg BID, about 450 mg BID, about 500 mg BID, about 550 mg BID, about 600 mg BID, about 200 mg TID, about 250 mg TID, about 300 mg TID, about 350 mg TID, about 400 mg TID, about 450 mg TID, about 500 mg TID, about 550 mg TID, about 600 mg TID, about 200 mg QID, about 250 mg QID, about 300 mg QID, about 350 mg QID, about 400 mg QID, about 450 mg QID, and about 500 mg QID.

[0064] In some embodiments, nitazoxanide is administered to the subject at a dose of about 500 mg BID.

[0065] In some embodiments, nitazoxanide is administered to a subject at a dose of about 300 mg to about 350 mg TID.

[0066] In some embodiments, ribavirin is administered to a subject at a total daily dose ranging from about 20 mg to about 1200 mg. In some embodiments, nitazoxanide may be administered once daily (QD), twice daily (BID), or three times daily (TID).

[0067] In one embodiment, ribavirin is administered to a subject in a single dose ranging from about 20 mg to about 400 mg.

[0068] In some embodiments, nitazoxanide is administered to a subject in a single dose selected from about 20 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 250 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, and about 500 mg.

[0069] In some embodiments, ribavirin is administered to the subject in a single dose of about 50 mg to about 400 mg twice daily (BID) or three times daily (TID).

[0070] In some embodiments, ribavirin is administered twice daily (BID). In some embodiments, ribavirin is administered in an amount selected from about 20 mg BID, about 50 mg BID, about 60 mg BID, about 80 mg BID, about 100 mg BID, about 120 mg BID, about 140 mg BID, about 150 mg BID, about 160 mg BID, about 180 mg BID, about 200 mg BID, about 220 mg BID, about 240 mg BID, about 250 mg BID, about 260 mg BID, about 280 mg BID, about 300 mg BID, about 320 mg BID, about 330 mg BID, about 340 mg BID, about 350 mg BID, about 360 mg BID, about 380 mg BID, about 400 mg BID, about 420 mg BID, about 440 mg BID, about 450 mg BID, about 460 mg BID, about 480 mg BID, and about 500 mg BID. In some embodiments, ribavirin is administered to a subject at a dose of about 200 mg BID. In some embodiments, ribavirin is administered to a subject at a dose of about 400 mg BID.

[0071] In some embodiments, ribavirin is used three times a day (TID). In some embodiments, ribavirin is used to a subject with a dosage selected from 20mg TID, about 50mg TID, about 60mg TID, about 80mg TID, about 100mg TID, about 120mg TID, about 140mg TID, about 150mg TID, about 160mg TID, about 180mg TID, about 200mg TID, about 220mg TID, about 240mg TID, about 250mg TID, about 260mg TID, about 280mg TID, about 300mg TID, about 320mg TID, about 330mg TID, about 340mg TID, about 350mg TID, about 360mg TID, about 380mg TID and about 400mg TID. In some embodiments, ribavirin is used to a subject with a dosage of about 100mg TID. In some embodiments, ribavirin is administered to the subject at a dose of about 150 mg TID.

[0072] In some embodiments, the first nitazoxanide and ribavirin can be administered simultaneously. In some embodiments, the first administration interval of nitazoxanide and ribavirin is in the range of about 0 hours to about 16 hours. In some embodiments, the first administration interval of nitazoxanide and ribavirin is at least 0 hours, at least 0.1 hours, at least 0.2 hours, at least 0.2 hours, at least 0.3 hours, at least 0.4 hours, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 13 hours, at least 14 hours, at least 15 hours and / or at least 16 hours.

[0073] In some embodiments, nitazoxanide and ribavirin are administered to the subject twice daily (BID), respectively, the first nitazoxanide and ribavirin can be administered simultaneously or the interval between the first administrations of nitazoxanide and ribavirin is in the range of about 0 hours to about 8 hours, wherein nitazoxanide is administered in a single dose in the range of about 300 mg to about 500 mg, and wherein ribavirin is administered in a single dose in the range of about 200 mg to about 400 mg.

[0074] In some embodiments, nitazoxanide and ribavirin are administered to the subject three times a day (TID), respectively, the first nitazoxanide and ribavirin can be administered simultaneously or the interval between the first administrations of nitazoxanide and ribavirin is in the range of about 0 hours to about 4 hours, wherein nitazoxanide is administered in a single dose in the range of about 200 mg to about 350 mg, and wherein ribavirin is administered in a single dose of about 100 mg to about 200 mg.

[0075] In certain embodiments, the method of preventing a disease or condition caused by or associated with a coronavirus infection comprises chemoprevention or drug prevention.

[0076] In one embodiment, the administration prevents the progression of mild illness to severe illness, or alleviates at least one symptom, wherein the at least one symptom is selected from cough, decreased appetite, wheezing, dyspnea, fever, malaise, dizziness, nausea, decreased or loss of smell, and pneumonia.

[0077] III. Embodiment

[0078] Example 1 Cell preparation

[0079] Vero E6 cell line was obtained from the American Type Culture Collection (ATCC, No. 1586) and cultured in MEM medium (MEM; Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen) at 37°C and 5% CO2.

[0080] Example 2 Virus strain isolation

[0081] SARS-CoV-2 (strain HB-01) was obtained from the Chinese Center for Disease Control and Prevention (China CDC). The complete genome sequence of this SARS-CoV-2 has been submitted to GISAID (BetaCoV / Wuhan / IVDC-HB-01 / 2020|EPI_ISL_402119) and deposited in the National Microbiological Data Center of China (accession number NMDC10013001, genome accession number MDC60013002-01). The virus strain SARS-CoV-2 was propagated in Vero E6 cells, and the virus titer was determined by measuring the 50% tissue culture infectious dose (TCID50) using an immunofluorescence assay. All infection experiments were performed in a biosafety level 3 (BLS-3) laboratory.

[0082] Example 3 Test Drugs

[0083] Ribavirin (RBV; Catalog No.: R101754) and nitazoxanide (NTZ; Catalog No.: N159057) were purchased from ALADDIN. GH23 is a combination drug of NTZ and RBV.

[0084] Example 4 Viral RNA extraction and quantitative analysis (aRT-PCR)

[0085] SARS-CoV-2 viral RNA was extracted using the MiniBEST viral RNA / DNA extraction kit (Takara, catalog number 9766), and the specific operations were performed according to the manufacturer's operating manual.

[0086] Reverse transcription was performed using the PrimeScript RT Reagent Kit and gDNA Eraser (Takara, Cat. No. RR047A) kit; qRT-PCR experiments were performed using the StepOnePlus TM Real-time PCR system (Applied Biosystem) and TB Green Premix Ex Taq II (Takara, Cat. No. RR820A) kit.

[0087] SARS-CoV-2 viral RNA was taken from 100 μL of cell culture supernatant, eluted in 30 μL of RNase-free water, and 3 μL of total RNA was first digested using gDNA Eraser to remove genomic DNA contaminants, and the first strand of cDNA was synthesized in a 20 μL reaction volume. The cDNA (2 μL) was then used as a template for PCR. The receptor binding domain (RBD) of the spike gene was amplified from the cDNA template using PCR using the following primers:

[0088] RBD-Forward:5'-GCTCCATGGCCTAATATTACAAACTTGTGCC3'

[0089] RBD-Reverse:5'-TGCTCTAGACTCAAGTGTCTGTGGATCAC-3'.

[0090] The amplified PCR product was cloned into the pMT / BiP / V5-His vector (Invitrogen) and used as a standard after sequencing confirmation. The copy number of the plasmid in each dilution was determined (10 3 -10 9 The standard curve was obtained by quantitative real-time PCR using the following primers:

[0091] RBD-qF1:5'-CAATGGTTTAACAGGCACAGG-3'

[0092] RBD-qR1:5'-CTCAAGTGTCTGTGGATCACG-3'.

[0093] The PCR amplification method was as follows: denaturation at 95°C for 5 minutes, followed by 40 cycles of three steps including 95°C for 15 seconds, 54°C for 15 seconds, and 72°C for 30 seconds.

[0094] Example 5 Cytotoxicity test of experimental drugs

[0095] The cytotoxicity of the test drugs on Vero E6 cells was determined by a standard CCK8 assay, which was performed according to the manufacturer's instructions (Beyotime, China). In these studies, the test drugs (NTZ, RBV, GH23) were incubated with Vero E6 cells for 48 hours. The results of the standard CCK8 assay are shown in Figure 1 shown.

[0096] Incubation of Vero E6 cells with NTZ produced dose-dependent cytotoxicity, CC 50 When Vero E6 cells were treated with RBV, no cytotoxic effect was observed (CC 50 >400). CC of GH23 (NTZ+RBV) 50 The value was 37.2 μM, which was basically the same as when NTZ was used alone. This result showed that the combined use of NTZ and RBV had no synergistic toxicity.

[0097] Example 6 Test of Antiviral Activity of Experimental Drugs

[0098] To evaluate the antiviral efficacy of the combination drug of the present invention, Vero E6 cells were cultured at a density of 1×10 4 Cells / well were cultured overnight in 48-well cell culture dishes. Cells were pretreated with different concentrations of test compounds for 1 hour. DMSO was used as a negative control. Viruses were then added to infect cells for 2 hours at a multiplicity of infection (MOI) of 0.05. The virus-drug mixture was then removed and the cells were further cultured with fresh medium containing the test drug. 48 hours after viral infection, cell supernatants were collected and lysed in lysis buffer (Takara, Cat. No. 9766) for quantitative analysis of viral RNA levels. Dose-response curves of viral RNA copy number relative to drug concentration were plotted using GraphPad Prism 6 software to evaluate the ability of each drug to inhibit SARS-CoV-2 viral replication.

[0099] The results are as follows Figure 2 Treatment of Vero E6 cells with NTZ alone (0-30 μM) inhibited SARS-CoV-2 levels, EC 50 The EC of Vero E6 cells treated with RBV alone was 2.1 μM. 50The value was 108.8 μM. This finding is consistent with a previous study that found that although ribavirin can be clearly integrated into coronavirus RNA, it is also easily excised by the 3'-5' exon ribonuclease (ExoN) activity of the coronavirus protein nsp14 (Ferrona F et al. PNAS. 2017 115(2): E162-E171). However, combined treatment of Vero E6 cells with GH23 (NTZ and RBV) significantly synergistically inhibited SARS-CoV-2 levels, EC 50 Reaching 0.78μM.

[0100] Table 1 summarizes the CC of NTZ, RBV, and GH23 50 ,EC 50 and the corresponding selectivity index (SI) values. The selectivity indexes of NTZ, RBV, and GH23 were 17.5, >3.7, and 47.7, respectively. These data suggest that the safety window of combined NTZ and RBV treatment for SARS-CoV-2 patients is significantly higher than that of NTZ or RBV alone.

[0101] Table 1 Antiviral activity of GH23 against SARS-CoV-2

[0102]

Claims

1. A combination of drugs for use in the preparation of a pharmaceutical composition for treating or preventing a disease or condition caused by or associated with a coronavirus infection, wherein the combination of drugs comprises at least one selected from nitazoxanide or tizoxanide or a pharmaceutically acceptable salt thereof, and at least one ribavirin or a pharmaceutically acceptable salt or ester thereof, wherein the coronavirus is SARS-CoV-2.

2. The use according to claim 1, wherein the disease or condition caused by the coronavirus infection is an acute respiratory infectious disease or pneumonia.

3. The use according to claim 1, wherein nitazoxanide is administered in the form of an oral preparation.

4. The use according to claim 1, wherein ribavirin is administered in the form of an oral preparation, an inhalation or an injection.

5. The use according to claim 1, wherein the nitazoxanide or tizoxanide and the ribavirin can be administered to the subject simultaneously or sequentially.

6. The use according to claim 1, wherein the use can be used to prevent the progression of the disease or condition from a mild case to a severe case, or to alleviate at least one symptom.

7. The use of claim 6, wherein the symptom is selected from the group consisting of cough, loss of appetite, wheezing, dyspnea, fever, malaise, dizziness, nausea, reduced or lost sense of smell, and pneumonia.

8. The use of claim 5, wherein the subject is a critically ill patient caused by coronavirus infection, wherein ribavirin is administered to the subject in need in the form of an inhaler.

9. The use of claim 1, comprising administering nitazoxanide and ribavirin to a subject in need thereof, wherein nitazoxanide is administered to the subject at a total daily dose ranging from 100 mg to 2000 mg, wherein ribavirin is administered to the subject at a total daily dose ranging from 20 mg to 1200 mg, wherein nitazoxanide and ribavirin are administered twice daily (BID) or three times daily (TID), respectively.

10. The use of claim 9, wherein ribavirin is administered to the subject in a single dose ranging from 100 mg to 600 mg.

11. The use of claim 9, wherein ribavirin is administered to the subject in a single dose ranging from 20 mg to 400 mg.

12. The use of claim 1, further comprising chemoprevention, for preventing diseases or conditions caused by or associated with coronavirus infection.

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