WO2021180251A1 - 治疗冠状病毒感染的联用药物及治疗方法 - Google Patents

治疗冠状病毒感染的联用药物及治疗方法 Download PDF

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WO2021180251A1
WO2021180251A1 PCT/CN2021/092693 CN2021092693W WO2021180251A1 WO 2021180251 A1 WO2021180251 A1 WO 2021180251A1 CN 2021092693 W CN2021092693 W CN 2021092693W WO 2021180251 A1 WO2021180251 A1 WO 2021180251A1
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cov
ribavirin
administered
sars
nitazoxanide
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PCT/CN2021/092693
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English (en)
French (fr)
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李嘉强
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北京强新生物科技有限公司
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Priority to US17/910,779 priority Critical patent/US20230241037A1/en
Priority to CN202180020165.3A priority patent/CN115884770A/zh
Publication of WO2021180251A1 publication Critical patent/WO2021180251A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/426—1,3-Thiazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4178—1,3-Diazoles not condensed 1,3-diazoles and containing further heterocyclic rings, e.g. pilocarpine, nitrofurantoin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/425—Thiazoles
    • A61K31/427—Thiazoles not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4523—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems
    • A61K31/454—Non condensed piperidines, e.g. piperocaine containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. pimozide, domperidone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4965—Non-condensed pyrazines
    • A61K31/497—Non-condensed pyrazines containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses

Definitions

  • the present invention relates to a combination drug and a treatment method for treating or preventing coronavirus infections, especially acute respiratory infections or pneumonia-based diseases caused by SARS-CoV-2 (new coronavirus) infections.
  • SARS-CoV-2 The infectious disease (COVID-19), which is mainly caused by SARS-CoV-2, has been outbreak globally since the end of 2019, posing an unprecedented threat to human life and health worldwide.
  • SARS-CoV-2 belongs to the same subfamily of coronavirus as SARSr-CoV and MERSr-CoV, but the gene homology is not high.
  • the new type of pneumonia caused by SARS-CoV-2 infection is extremely infectious, and the population is generally susceptible and difficult to control, which has become a long-standing problem.
  • anti-SARS-CoV-2 drugs is actively advancing globally, there is still no particularly effective therapeutic drug. Finding a treatment plan that can effectively treat or prevent the disease caused by its infection is still an urgent clinical need.
  • Nitazoxanide is a safe, oral bioavailable thiazolide (thiazolide) approved in the United States for the treatment of Cryptosporidium parvum and Giardia lamblia infections. ), used to treat infectious gastroenteritis. The results of using the drug in many countries in Africa and South America show that its anti-parasitic infection effect is good, and there is no adverse reaction.
  • Tizoxanide is the main active metabolite of nitazoxanide in the body. Nitrazoxanide and tizoxanide are used as antiprotozoal infection drugs.
  • nitazoxanide has an inhibitory effect on the acetyl-Coenzyme A carboxylase activity of HepG2 cells, and can exert anti-obesity, hypolipidemic, anti-fatty liver and anti-atherosclerotic effects (CN110478357A).
  • nitazoxanide and tizoxanide can reduce the duration of related symptoms caused by human respiratory syncytial virus (CN108289961A), and it is useful for the clinical treatment of rotavirus, norovirus, HBV and HCV infections. Potential effect ( Rossignol, Romark Laboratories, LC, 2014; CN108289961A), but there is no data to support its antiviral activity in vivo. The mechanism of nitazoxanide against viral infections is still unclear.
  • Ribavirin is a nucleoside analogue 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) (fog only) Chemical inhalation). Although it has been proven in vitro to have inhibitory effects on many other DNA and RNA viruses (such as herpes simplex virus, influenza virus, etc.), due to the lack of clinically definite evidence, its indications are usually limited, such as the FDA's approved drug instructions It is clearly pointed out that ribavirin is not suitable for the treatment of influenza, etc. ( (ribavirin) Tablets Initial USApproval: 2002).
  • Ribavirin as a single agent or in combination with systemic glucocorticoids has been tried for the empirical treatment of severe acute respiratory syndrome (SARS) caused by SARS-CoV infection, but the clinical benefits have been disappointing (WHO. Management of severe acute respiratory syndrome(SARS). 2003 Apr 11.).
  • the present invention provides a drug combination program for the treatment or prevention of coronavirus infection, especially the 2019 novel coronavirus (SARS-CoV-2) infection, which can inhibit the coronavirus infection, alleviate or reverse the pneumonia-based diseases caused by the coronavirus infection, or Prevent coronavirus infection.
  • SARS-CoV-2 2019 novel coronavirus
  • the present invention relates to a new combination drug and its use in the treatment or prevention of coronavirus infection or diseases or disorders related to it.
  • said infection or related disease refers to the disease or disease caused by SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and/or HKU1 infection, especially SARS-CoV-2 infection and its Pneumonia caused by.
  • 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 or metabolite thereof , Derivatives, enantiomers, diastereomers, tautomers, racemates or any of the foregoing pharmaceutically acceptable salts or esters.
  • the combination drug comprises at least one nitazoxanide or a pharmaceutically acceptable salt thereof, and at least one ribavirin or a pharmaceutically acceptable salt or ester thereof.
  • the nitazoxanide is administered in an oral formulation, including tablets, suspensions, or capsules.
  • the ribavirin can be administered as an oral preparation, inhalation, or injection. In one embodiment, ribavirin can be administered directly through the respiratory tract in the form of an aerosol inhaler.
  • the combination drug disclosed in the present invention is used to treat, reduce or alleviate at least one symptom of the related disease or condition, and the symptom includes a symptom selected from the group consisting of cough, decreased appetite, wheezing, dyspnea, fever, General weakness, dizziness, nausea, weakened or lost sense of smell, and pneumonia.
  • the combination drug disclosed in the present invention is used in the preparation of a pharmaceutical composition for the treatment or prevention of coronavirus infection or diseases or conditions related thereto, the coronavirus being SARS-CoV-2, SARS -CoV, MERS-CoV, 229E, NL63, OC43 and/or HKU1, especially SARS-CoV-2, the related disease or condition is pneumonia.
  • the present invention relates to a method of treating or preventing a coronavirus infection or a disease or disorder related thereto, the method comprising administering a therapeutically effective amount of the aforementioned new combination drug to a subject in need.
  • the related disease or condition is acute respiratory infection or pneumonia.
  • the method of treatment or prevention comprises administering to a subject in need a therapeutically effective amount of at least one selected from nitazoxanide or tizoxanide or a pharmaceutically acceptable salt thereof, or any of them Solvate, and a therapeutically effective amount of at least one ribavirin or its prodrugs, metabolites, derivatives, enantiomers, diastereomers, tautomers, racemates or Any pharmaceutically acceptable salt or ester of the foregoing.
  • the method of treatment or prevention comprises administering to a subject in need 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 of Bavirin or a pharmaceutically acceptable salt or ester thereof.
  • nitazoxanide or tizoxanide and ribavirin can be administered to the subject simultaneously or sequentially.
  • the nitazoxanide is administered in an oral formulation, including tablets, suspensions, or capsules.
  • the ribavirin can be administered as an oral preparation, inhalation, or injection. In one embodiment, ribavirin can be administered directly through the respiratory tract in the form of an aerosol inhaler.
  • the subject is a severely ill patient caused by coronavirus infection, and the ribavirin is administered directly through the respiratory tract as an aerosol inhaler.
  • the coronavirus is SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and/or HKU1, especially SARS-CoV-2, and the related disease or condition is pneumonia.
  • the method of preventing diseases or conditions caused by or related to coronavirus infection refers to chemoprevention/drug prophylaxis.
  • the administration can be used to prevent the progression from mild to severe, or to relieve at least one symptom, wherein at least one symptom is selected from cough, loss of appetite, wheezing, dyspnea, fever, malaise, dizziness, Nausea, diminished or loss of smell, and pneumonia.
  • Figure 1 shows 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.
  • Figure 2 shows the antiviral activity of NTZ, RBV and GH23 against SARS-CoV-2 in vitro.
  • the singular form “a”, “an” or “the” includes plural references unless the context clearly dictates otherwise.
  • the term “cell” includes a variety of cells, including mixtures thereof.
  • an active agent refers to an amount sufficient to initiate the desired biological response.
  • the effective amount of the compound of the present invention may vary according to the desired biological endpoint, the pharmacokinetics of the compound, the disease to be treated, the mode of administration, the patient and other factors.
  • treatment refers to a method of reducing, delaying or ameliorating the condition before or after the occurrence of the condition.
  • Treatment can be directed to one or more effects or symptoms of the disease and/or underlying pathology.
  • the treatment can be any reduction, and can be, but is not limited to, the complete elimination of the disease or symptoms of the disease.
  • the degree of reduction or prevention measured by any standard technique is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95% % Or 100%.
  • subject refers to any animal (such as a mammal), including but not limited to humans, non-human primates, rodents, etc., which are recipients of a specific treatment or prevention regimen.
  • subject and patient or “persons in need of prevention of infection or high-risk groups” are used interchangeably herein to refer to humans.
  • references to "A and/or B" may in one embodiment refer to only A (optionally including Elements other than B); in another embodiment, only refers to B (optionally including other elements other than A); in yet another embodiment, refers to A and B (optionally including Other elements); etc.
  • references to "A and/or B” may in one embodiment refer to only A (optionally including Elements other than B); in another embodiment, only refers to B (optionally including other elements other than A); in yet another embodiment, refers to A and B (optionally including Other elements); etc.
  • 1-5mg is meant to include 1mg, 2mg, 3mg, 4mg, 5mg, 1-2mg, 1-3mg, 1-4mg, 1-5mg, 2-3mg, 2-4mg, 2-5mg, 3- 4mg, 3-5mg and 4-5mg.
  • administration is used herein in its broadest sense. These terms refer to any method of intervention to the subject of the compound or combination of drugs described herein, and may include, for example, systemic, local, or in situ intervention of the compound into the subject. Therefore, the compounds disclosed herein that are produced in a subject from a combination drug (whether or not it includes the compound) are included in these terms. When these terms are used in conjunction with “systemic” or “systemically”, they generally refer to the compound or combination drug in the blood that is absorbed or accumulated systemically in the body and then distributed throughout the body.
  • composition (combination) or “combination (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 Wait.
  • combination therapy may include the administration of one drug before, during, and/or after the administration of another drug.
  • the interval between drug administrations can be up to several weeks, but is more usually within 48 hours, and most commonly within 24 hours.
  • salts as used herein means acidic and/or basic salts formed with inorganic and/or organic acids and bases.
  • zwitterions inner salts
  • Pharmaceutically acceptable salts are preferred, although other salts are also useful in, for example, separation or purification steps in the preparation process.
  • Chemoprophylaxis mentioned in this article refers to those who are asymptomatically infected with the virus, or those who have a negative test for viral infection but have the possibility of viral infection through epidemiological history analysis, or those who have a negative test for viral infection but have been diagnosed by other medical treatments (such as medical Imaging analysis, etc.) It is believed that patients with viral infection-like pneumonia administer the combined drug regimen provided herein as a preventive measure to prevent or reduce the pathogenicity of viral infection.
  • the present invention provides a new combination drug and its use in the treatment or prevention of coronavirus infection (especially SARS-CoV-2 infection) or related diseases or conditions (such as pneumonia, especially COVID-19).
  • the present invention also provides a method for the treatment or prevention of coronavirus infection (especially SARS-CoV-2 infection) or related diseases or conditions (such as pneumonia, especially COVID-19), including administering effective treatment to a subject in need The amount of the combination drug described herein.
  • the combination drug comprises at least one thiazolide compound selected from the group consisting of nitazoxanide, tizoxanide or derivatives thereof, and any of the foregoing pharmaceuticals
  • thiazolide compound selected from the group consisting of nitazoxanide, tizoxanide or derivatives thereof, and any of the foregoing pharmaceuticals
  • the present invention also provides the use of the combination drug in the preparation of a pharmaceutical composition for the treatment or prevention of coronavirus infections or diseases or disorders related thereto.
  • the coronaviruses include but are not limited to SARS-CoV-2, SARS-CoV, MERS-CoV, 229E, NL63, OC43 and HKU1, especially SARS-CoV-2, related diseases or conditions are acute respiratory infectious diseases, including pneumonia.
  • Nitrazoxanide chemical name o-[N-(5-nitro-1,3-thiazol-2-yl)carbamoyl]phenol acetate), the chemical formula is as follows:
  • Tizoxanide chemical name 2-hydroxy-N-(5-nitro-2-thiazolyl)benzamide, the chemical formula is as follows:
  • Ribavirin chemical name 1-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolane-2-yl]-1H-1,2,4 -Triazole-3-carboxamide, also known as 1-( ⁇ -D-ribofuranosyl)-1H-1,2,4-triazole-3-carboxamide, CAS registration number 36791-04-5,
  • the chemical formula is as follows:
  • the present invention provides nitazoxanide or tizoxanide combined with ribavirin for synergistically inhibiting coronavirus infection, such as SARS-CoV-2 infection.
  • the above findings provide a feasible combination treatment plan for the treatment of SARS-CoV-2 or other coronavirus infections and related diseases or diseases, such as pneumonia, which can achieve effective treatment of coronavirus infections, or quickly alleviate related diseases, or reduce clinical The duration of symptoms and reduce the possibility of coronavirus infection and re-transmission.
  • the combination drug disclosed in the present invention can prevent the progression from mild to severe, or relieve at least one symptom, the symptom being selected from the group consisting of cough, decreased appetite, wheezing, dyspnea, fever, malaise, Dizziness, nausea, diminished or loss of smell, and pneumonia.
  • the present invention relates to methods of treating or preventing coronavirus infection or diseases or disorders related thereto.
  • the method comprises administering to a subject in need a therapeutically effective amount of at least one thiazolide compound selected from the group consisting of nitazoxanide, tizoxanide or derivatives thereof, any of the foregoing pharmaceutically acceptable salts, and the foregoing Any solvate of, and a therapeutically effective amount of at least one ribavirin or its prodrugs, metabolites, derivatives, enantiomers, diastereomers, tautomers, exogenous Rotate, or any of the foregoing pharmaceutically acceptable salts or esters.
  • the method comprises administering to a subject in need thereof a therapeutically effective amount of nitazoxanide, and a therapeutically effective amount of ribavirin.
  • nitazoxanide and ribavirin are administered to the patient simultaneously, simultaneously, separately, and/or sequentially.
  • nitazoxanide is administered to the subject in 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).
  • QD once daily
  • BID twice daily
  • TID three times daily
  • QID four times daily
  • nitazoxanide is administered to the subject in a single dose ranging from about 100 mg to about 1000 mg. In some embodiments, nitazoxanide is 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 Single doses of 900 mg and about 1000 mg are administered to the subject.
  • nitazoxanide is administered to the subject in a single dose of about 300 mg to about 500 mg, twice daily (BID) or three times daily (TID).
  • nitazoxanide is administered to the subject at a dose of about 500 mg BID.
  • nitazoxanide is administered to the subject in a dose of about 300 mg to about 350 mg TID.
  • nitazoxanide is 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 Single doses of 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 are administered to the subject.
  • 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).
  • ribavirin is administered twice daily (BID).
  • BID twice daily
  • ribavirin is 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 180mg BID, about 200mg BID, about 220mg BID, about 240mg BID, about 250mg BID, about 260mg BID, about 280mg BID, about 300mg BID, about 320mg BID, about 330mg BID, about 340mg BID, about 350mg BID, about 360mg BID , About 380mg BID, about 400mg BID, about 420mg BID, about 440mg BID, about 450mg BID, about 460mg BID, about 480mg BID, and about 500mg BID are administered to the subject.
  • BID
  • ribavirin is administered three times a day (TID).
  • ribavirin is selected from 20 mg TID, about 50 mg TID, about 60 mg TID, about 80 mg TID, about 100 mg TID, about 120 mg TID, about 140 mg TID, about 150 mg TID, about 160 mg TID, about 180 mg 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, A dose of about 380mg TID and about 400mg TID was administered to the subject.
  • ribavirin is administered to the subject at a dose of about 100 mg TID.
  • ribavirin is administered to the subject at a dose of about 150
  • the first nitazoxanide and ribavirin can be administered simultaneously.
  • the interval between the first dosing of nitazoxanide and ribavirin is in the range of about 0 hours to about 16 hours.
  • the first dosing interval of nitazoxanide and ribavirin is at least 0 hour, at least 0.1 hour, at least 0.2 hour, at least 0.2 hour, at least 0.3 hour, at least 0.4 hour, 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.
  • nitazoxanide and ribavirin are each administered twice daily (BID) to the subject, and the first time nitazoxanide and ribavirin can be administered simultaneously or the first time
  • the dosing interval of nitazoxanide and ribavirin is in the range of about 0 hours to about 8 hours, wherein nitazoxanide is administered in a single dose ranging from about 300 mg to about 500 mg, and ribavirin is administered in a range of about A single dose of 200 mg to about 400 mg is administered.
  • nitazoxanide and ribavirin are respectively administered to the subject three times a day (TID), and the first nitazoxanide and ribavirin can be administered simultaneously or the first time
  • the dosing interval of zoxanide and ribavirin is in the range of about 0 hours to about 4 hours, wherein nitazoxanide is administered in a single dose ranging from about 200 mg to about 350 mg, and ribavirin is administered at about 100 mg. A single dose of up to about 200 mg is administered.
  • the method of preventing a disease or condition caused by or related to coronavirus infection includes chemoprevention or drug prophylaxis.
  • the administration can prevent the progression from mild to severe, or relieve at least one symptom, wherein at least one symptom is selected from cough, loss of appetite, wheezing, dyspnea, fever, malaise, dizziness, nausea , Decreased or lost sense of smell, and pneumonia.
  • the African Green Monkey Kidney Vero E6 cell line was taken from the American Standard Bacteria (ATCC, No. 1586), and in MEM medium (MEM; Gibco Invitrogen) supplemented with 10% fetal bovine serum (FBS; Gibco Invitrogen) to Cultivate at 37°C and 5% CO 2.
  • MEM MEM medium
  • FBS fetal bovine serum
  • SARS-CoV-2 (strain HB-01) was obtained from the Chinese Center for Disease Control and Prevention (China CDC). The complete genome sequence of the SARS-CoV-2 has been submitted to GISAID (BetaCoV/Wuhan/IVDC-HB-01/2020
  • TCID50 tissue culture infectious dose
  • Ribavirin (RBV; catalog number: R101754) and nitazoxanide (NTZ; catalog number: N159057) were purchased from ALADDIN.
  • GH23 is a combination of NTZ and RBV.
  • SARS-CoV-2 virus RNA was extracted using MiniBEST virus RNA/DNA extraction kit (Takara, catalog number 9766), and the specific operation was performed in accordance with the manufacturer's operating manual.
  • Reverse transcription uses PrimeScript RT Reagent Kit and gDNA Eraser (Takara, article number RR047A) kit; qRT-PCR experiment uses StepOnePlus TM Real-time PCR system (Applied Biosystem) and TB Green Premix Ex Taq II (Takara, article number RR820A) kit .
  • SARS-CoV-2 viral RNA was taken from 100 ⁇ L of cell culture supernatant, eluted in 30 ⁇ L of ribonuclease-free water, and 3 ⁇ L of total RNA was digested for the first time with gDNA Eraser to remove genomic DNA contaminants. Synthesize the first strand of cDNA in the reaction volume. Then cDNA (2 ⁇ L) was used as a template for PCR. Use PCR to amplify the receptor binding domain (RBD) of the spike gene from the cDNA template with the following primers:
  • the amplified PCR product was cloned into the pMT/BiP/V5-His vector (invitrogen) and used as a standard product after confirmation by sequencing.
  • the standard curve was obtained by measuring the copy number (10 3 -10 9 copy number) of the plasmid in each dilution.
  • the primers used for quantitative real-time PCR are:
  • RBD-qR1 5'-CTCAAGTGTCTGTGGATCACG-3'.
  • the PCR amplification method is as follows: denaturation at 95°C for 5 minutes, and then 40 cycles including three steps of 95°C for 15 seconds, 54°C for 15 seconds, and 72°C for 30 seconds.
  • test drug The cytotoxicity of the test drug on Vero E6 cells was determined by standard CCK8 analysis, which was performed in accordance with 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 standard CCK8 analysis result is shown in Figure 1.
  • Vero E6 cells were cultured overnight in a 48-well cell culture dish with a density of 1 ⁇ 10 4 cells/well. The cells were pretreated with different concentrations of test compounds for 1 hour. Take DMSO as a negative control. Subsequently, virus was added to infect the cells with a multiplicity of infection (MOI) of 0.05 for 2 hours. The virus-drug mixture is then removed, and the cells are further cultured in fresh medium containing the test drug. At 48 hours after virus infection, the cell supernatant was collected and lysed in lysis buffer (Takara, Catalog No. 9766) for quantitative analysis of viral RNA levels. GraphPad Prism 6 software was used to draw a dose-response curve of viral RNA copy number versus drug concentration to evaluate the ability of each drug to inhibit SARS-CoV-2 virus replication.
  • MOI multiplicity of infection
  • Table 1 summarizes the CC 50 , EC 50 and the corresponding selectivity index (SI) values of NTZ, RBV and GH23.
  • the selectivity indexes of NTZ, RBV and GH23 are 17.5, >3.7 and 47.7, respectively.

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Abstract

包含硝唑尼特或替唑尼特和利巴韦林的联用药物，及其用于治疗或预防冠状病毒感染的用途。所提供的联用药物对于治疗SARS-CoV-2感染引起的疾病(COVID-19)特别有效。

Description

治疗冠状病毒感染的联用药物及治疗方法 技术领域
本发明涉及治疗或预防冠状病毒感染，特别是SARS-CoV-2(新型冠状病毒)感染引起的急性呼吸道传染病或肺炎为主的疾病的联用药物及治疗方法。
背景技术
由SARS-CoV-2引起的急性呼吸道感染为主的传染病(COVID-19)，自2019年底在全球爆发至今，对全球人类的生命健康构成前所未有的巨大威胁。SARS-CoV-2与SARSr-CoV和MERSr-CoV同属冠状病毒亚科，但基因同源性不高。由SARS-CoV-2感染引起的新型肺炎具有极强的传染力，人群普遍易感，难以控制，已成为会长期存在的问题。尽管抗SARS-CoV-2的药物研发在全球范围内积极推进，但还没有任何特别有效的治疗药物，找到能够有效治疗或预防其感染引起的疾病的治疗方案仍是临床的迫切需求。
硝唑尼特(Nitazoxanide，NTZ)是一种在美国获准用于治疗小隐孢子虫(Cryptosporidium parvum)和贾第鞭毛虫(Giardia lamblia)感染的安全的、口服上生物可利用的噻唑化物(thiazolide)，用于治疗传染性胃肠炎。该药在非洲及南美多个国家使用结果表明，其抗寄生虫感染效果好，且无不良反应。替唑尼特(Tizoxanide，TIZ)是硝唑尼特在体内的主要活性代谢产物。硝唑尼特及替唑尼特作为抗原生动物感染药物，后被研究发现其能够影响机体细胞炎症相关蛋白COX-2(环氧酶)，NF-κB，p65，IκBα，ERK1/2或p38MAPK的活性，进而治疗炎症性相关疾病，包括脂多糖(LPS)等细菌内毒素诱发的炎症性疾病。还有研究公开硝唑尼特对HepG2细胞乙酰辅酶A羧化酶活性具有抑制作用，能够发挥抗肥胖，降血脂，抗脂肪肝及抗动脉粥样硬化的作用(CN110478357A)。此外，一些研究认为硝唑尼特及替唑尼特可以减少由人呼吸道合胞病毒引起的相关症状的持续时间(CN108289961A)，对轮状病毒、诺如病毒、HBV和HCV感染的临床治疗有潜在作用(
Figure PCTCN2021092693-appb-000001
Rossignol,Romark Laboratories,L.C.,2014；CN108289961A)，但是尚没有数据支持其在体内的抗病毒活性。硝唑尼特抗病毒感染的机制尚不清楚。
利巴韦林(Ribavirin)是一种核苷类似物抗病毒药物，FDA批准用于治疗丙型肝炎、病毒性出血热、以及呼吸道合胞病毒(RSV)所致的严重下呼吸道感染(仅雾化吸入剂)。尽管体外被证明对其它多种DNA和RNA病毒(如单纯疱疹病毒、流感病毒等)具有抑制作用，由于缺乏临床上的确切证据，其适应症通常被限制，如FDA在已批准的药物说明书中明确指出利巴韦林不适合用于治疗流感等(
Figure PCTCN2021092693-appb-000002
(ribavirin)Tablets Initial U.S.Approval:2002)。利巴韦林单药或与全身性糖皮质激素联合曾被尝试用于由SARS-CoV感染引起的严重急性呼吸综合征(SARS)的经验性治疗，但临床获益却令人失望(WHO.Management of severe acute respiratory syndrome(SARS).2003 Apr 11.)。
发明概述
本发明提供治疗或预防冠状病毒感染的药物组合方案，尤其是2019新型冠状病毒(SARS-CoV-2)感染，可抑制冠状病毒感染，缓解或逆转冠状病毒感染引起的肺炎为主的疾病，或预防冠状病毒感染。
在一方面，本发明涉及一种新的联用药物，以及其在治疗或预防冠状病毒感染或与其相关的疾病或病症中的用途。其中所述感染或相关疾病是指SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43和/或HKU1感染所引起的疾病或病症，特别是SARS-CoV-2感染及其引起的肺炎。
在一个实施方案中，所述联用药物包含至少一种选自硝唑尼特或替唑尼特或其药学上可接受的盐，和至少一种利巴韦林或其前药、代谢物、衍生物、对映异构体、非对映异构体、互变异构体、外消旋体或前述的任何药学上可接受的盐或酯。
在一个实施方案中，所述联用药物包含至少一种硝唑尼特或其药学上可接受的盐，和至少一种利巴韦林或其药学上可接受的盐或酯。
在一个特征中，所述硝唑尼特以口服制剂给药，包括片剂、混悬剂、或胶囊剂。
在一个特征中，所述利巴韦林可以口服制剂、吸入剂或注射剂施用。在一个实施方案中，利巴韦林可以雾化吸入剂形式直接通过呼吸道给药。
在一个实施方案中，本发明公开的联用药物用于治疗、减轻或缓解所述 相关疾病或病症的至少一种症状，所述症状包括选自咳嗽、食欲下降、气喘、呼吸困难、发热、全身乏力、头晕、恶心、嗅觉减弱或丧失和肺炎。
在一个实施方案中，本发明公开的联用药物在用于制备治疗或预防冠状病毒感染或与其相关的疾病或病症的药物组合物中的用途，所述冠状病毒是SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43和/或HKU1，特别是SARS-CoV-2，所述相关疾病或病症是肺炎。
在另一方面，本发明涉及治疗或预防冠状病毒感染或与其相关的疾病或病症的方法，所述方法包含向有需要的对象施用治疗有效量的上述新的联用药物。所述相关的疾病或病症是急性呼吸道传染病或肺炎。
在一个实施方案中，所述治疗或预防方法包含向有需要的对象施用治疗有效量的至少一种选自硝唑尼特或替唑尼特或其药学上可接受的盐、或其任何的溶剂化物，和治疗有效量的至少一种利巴韦林或其前药、代谢物、衍生物、对映异构体、非对映异构体、互变异构体、外消旋体或前述的任何药学上可接受的盐或酯。
在一个实施方案中，所述治疗或预防方法包含向有需要的对象施用治疗有效量的至少一种选自硝唑尼特或其药学上可接受的盐，和治疗有效量的至少一种利巴韦林或其药学上可接受的盐或酯。
在一些实施方案中，硝唑尼特或替唑尼特和利巴韦林可以同时或者依次向对象施用。
在一个特征中，所述硝唑尼特以口服制剂给药，包括片剂、混悬剂、或胶囊剂。
在一个特征中，所述利巴韦林可以口服制剂、吸入剂或注射剂施用。在一个实施方案中，利巴韦林可以雾化吸入剂形式直接通过呼吸道给药。
在一个特征中，所述对象是由冠状病毒感染引起的重症患者，所述利巴韦林以雾化吸入剂直接通过呼吸道给药。
在一个实施方案中，所述冠状病毒是SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43和/或HKU1，特别是SARS-CoV-2，所述相关疾病或病症是肺炎。
在某些实施方案中，所述预防由冠状病毒感染引起或与其相关的疾病或 病症的方法是指化学预防/药物预防。
在一些实施方案中，所述施用可用于防止轻症向重症的进展，或缓解至少一种症状，其中至少一种症状选自咳嗽、食欲下降、气喘、呼吸困难、发热、全身乏力、头晕、恶心、嗅觉减弱或丧失和肺炎。
本发明的前述的或者其它的目的、方面、特征以及优势将通过如下的说明书以及权利要求书进一步阐释。
附图简述
图1示出了用标准CCK-8法测试硝唑尼特(Nitazoxanide，NTZ)、利巴韦林(Ribavirin，RBV)和GH23(NTZ+RBV)对Vero E6细胞的细胞毒性结果。
图2示出了NTZ、RBV和GH23体外抗SARS-CoV-2的抗病毒活性。
发明详述
I.定义
如说明书和权利要求书中所使用的，单数形式“一”、“一个”或“该”包括复数指代，除非上下文另有明确说明。例如，术语“细胞”包括多种细胞，包括其混合物。
如本文所用，“约”意指在±10％范围内。例如，“约1”表示“0.9至1.1”，“约2％”表示“1.8％至2.2％”，“约2％至3％”表示“1.8％至3.3％”，“约3％至约4％”表示“2.7％至4.4％”。
除非另外定义，否则本文使用的所有技术和科学术语均具有与本发明所属领域普通技术人员的通常理解相同的含义。
术语活性剂的“有效量”是指足以引发所需生物反应的量。如本领域普通技术人员所理解的，本发明化合物的有效量可以根据所需的生物学终点，化合物的药代动力学、所治疗的疾病、给药方式和病人等因素而变化。
术语“治疗(treatment,treating或therapy)”疾病或紊乱是指在病症发生之前或之后减少、延迟或改善这种病症的方法。治疗可以针对疾病和/或潜在病理的一种或多种作用或症状。治疗可以是任何减少，并且可以是但不限于疾病 或疾病的症状的完全消除。与等效的未处理对照相比，这种通过任何标准技术测量得到的降低或预防程度至少为5％、10％、20％、40％、50％、60％、80％、90％、95％或100％。
术语“对象(subject)”是指任何动物(例如哺乳动物)，包括但不限于人、非人灵长类动物、啮齿动物等，它们是特定治疗或预防方案的接受者。通常，术语“对象”和“患者”或“需要预防感染者或高危人群”在本文中可互换使用，以指代人。
术语“和/或”应当理解为相连元素的“其一或两者”，即在一些情况下是结合存在的，在一些其他情况下是分别存在的。因此，作为非限制性示例，当与开放式语言(如“包含”)结合使用时，对“A和/或B”的引用，在一个实施方案中可以仅指代A(可选地包括除B之外的其他元素)；在另一个实施方案中，仅指代B(可选地包括除A之外的其他元素)；在又一个实施方案中，指代A和B(可选地包括其他元素)；等等。当文中列出一系列数值时，它意在涵盖该范围内的每个数值和子范围。例如，“1-5mg”意在包括1mg、2mg、3mg、4mg、5mg、1-2mg、1-3mg、1-4mg、1-5mg、2-3mg、2-4mg、2-5mg、3-4mg、3-5mg和4-5mg。
术语“施用”在本文中以其最宽泛意义使用。这些术语是指介入给受试者本文所述的化合物或联用药物的任何方法，并且可以包括例如全身地，局部地或原位地将所述化合物介入给受试者。因此，从联用药物(无论其是否包括本化合物)在受试者体内产生的本文公开的化合物包含在这些术语中的。当这些术语与“全身的”或“全身地”结合使用时，它们通常指在血液中化合物或联用药物在体内全身吸收或积累，随后分布在整个身体中。
本文所用的“组合物(组合)”或者“组合(联合)”疗法或者治疗是指施用至少两种不同的药物治疗紊乱、病症或者症状，例如，病毒性肺炎，病毒性感染、冠状病毒性肺炎等。这种组合疗法可以包含在施用另一种药物之前、之时和/或之后施用一种药物。药物施用的间隔期最长可达数周，但更通常的情况是在48小时内，最通常的情况是在24小时内。
本发明化合物可以形成盐，这些盐也在本发明的范围内。除非另有说明，否则本文提及的本发明化合物应理解为包括其盐。本文所用的术语“盐”表示与 无机和/或有机酸和碱形成的酸性和/或碱性盐。此外，当本发明的化合物包含碱性部分(例如但不限于吡啶或咪唑)和酸性部分(例如但不限于羧酸)时，两性离子(“内盐”)可以形成于并包括在本文所用的术语“盐”中。药学上可接受的(即无毒的，生理学上可接受的)盐是优先的，虽然其它盐在例如制备过程中的分离或纯化步骤中也是有用的。
本文所述化学预防是指对无症状的病毒感染者，或病毒感染检测呈阴性但流行病学史分析认为有病毒感染可能性的人，或病毒感染检测呈阴性但经其他医学诊断(如医学影像学分析等)认为是具有病毒感染样肺炎的患者施用本文所提供的联用药物方案，作为预防措施，以预防或减轻病毒感染的致病力。
术语“协同”、“协同性”、“协同地”或“增强”是指两种或更多种组分相互作用或组合产生大于其单独作用的效果之和(或“加合效果”)。
II.联用药物、用途和治疗方法
本发明提供了新的联用药物及其在治疗或预防冠状病毒感染(尤其是SARS-CoV-2感染)或与其相关的疾病或病症(如肺炎，尤其是COVID-19)中的用途。本发明还提供了治疗或预防冠状病毒感染(尤其是SARS-CoV-2感染)或与其相关的疾病或病症(如肺炎，尤其是COVID-19)的方法，包含向有需要的对象施用治疗有效量的本文所述的联用药物。
本发明提供的新的联用药物，在一个实施方案中，所述联用药物包含至少一种噻唑化物化合物，选自硝唑尼特、替唑尼特或其衍生物、前述的任何的药学上可接受的盐、以及前述的任何的溶剂化物，和至少一种利巴韦林或其前药、代谢物、衍生物、对映异构体、非对映异构体、互变异构体、外消旋体、或前述的任何药学上可接受的盐或酯。
本发明还提供了所述联用药物在制备用于治疗或预防冠状病毒性感染或与其相关的疾病或病症的药物组合物中的用途，所述冠状病毒包括但不限于SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43和HKU1，特别是SARS-CoV-2，相关的疾病或病症是急性呼吸系统传染病，包括肺炎。
硝唑尼特化学名：邻[N-(5-硝基-1,3-噻唑-2-基)氨基甲酰]苯酚乙酸酯)，化学式如下：
Figure PCTCN2021092693-appb-000003
替唑尼特化学名：2-羟基-N-(5-硝基-2-噻唑基)苯甲酰胺，化学式如下：
Figure PCTCN2021092693-appb-000004
利巴韦林化学名：1-[(2R,3R,4S,5R)-3,4-二羟基-5-(羟甲基)氧戊环-2-基]-1H-1,2,4-三唑-3-羧酰胺，也可称为1-(β-D-呋喃核糖基)-1H-1,2,4-三唑-3-羧酰胺，CAS注册号36791-04-5，化学式如下：
Figure PCTCN2021092693-appb-000005
本发明提供硝唑尼特或替唑尼特与利巴韦林联合用于协同抑制冠状病毒感染，如SARS-CoV-2感染。
上述发现为治疗SARS-CoV-2或其他冠状病毒感染及其相关疾病或病症，如肺炎，提供了可行的联合用药治疗方案，可以实现有效治疗冠状病毒感染，或快速缓解相关病症，或减少临床症状持续时间，以及降低冠状病毒感染及再传播的可能性。
在一个实施方案中，本发明所公开的联用药物没有显示出新的不良事件，并且具有与联合药物中每个药物单独使用时类似的安全性特性。
在一个实施方案中，本发明公开的联用药物可防止轻症向重症的进展，或缓解至少一种症状，所述症状包括选自咳嗽、食欲下降、气喘、呼吸困难、发热、全身乏力、头晕、恶心、嗅觉减弱或丧失和肺炎。
另一方面，本发明涉及治疗或预防冠状病毒感染或与其相关的疾病或病症的方法。所述方法包含向有需要的对象施用治疗有效量的至少一种噻唑化物化 合物，选自硝唑尼特、替唑尼特或其衍生物、前述的任何的药学上可接受的盐、以及前述的任何的溶剂化物，和治疗有效量的至少一种利巴韦林或其前药、代谢物、衍生物、对映异构体、非对映异构体、互变异构体、外消旋体、或前述的任何药学上可接受的盐或酯。
在一个实施方案中，所述冠状病毒是SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43或HKU1，特别是SARS-CoV-2。在一个实施方案中，所述相关疾病或病症是肺炎。
在一个实施方案中，所述方法包含向有需要的对象施用治疗有效量的硝唑尼特，和治疗有效量的利巴韦林。
在一些实施方案中，硝唑尼特和利巴韦林被同时地、同期地、分别地、和/或依次地给患者施用。
在一些实施方案中，硝唑尼特以从约100mg至约2000mg的范围的总日剂量给受试者施用。在一些实施方案中，硝唑尼特可以每日一次(QD)、每日两次(BID)、每日三次(TID)或每日四次(QID)施用。
在一些实施方案中，硝唑尼特以从约100mg至约1000mg的范围的单次剂量给受试者施用。在一些实施方案中，硝唑尼特以选自约100mg、约150mg、约200mg、约250mg、约300mg、约350mg、约400mg、约450mg、约500mg、约550mg,约700mg，约800mg,约900mg和约1000mg的单次剂量给受试者施用。
在一些实施方案中，硝唑尼特以约300mg至约500mg的单次剂量，每日两次(BID)或每日三次(TID)给受试者施用。
在一些实施方案中，硝唑尼特以选自约200mg BID、约250mg BID、约300mg BID、约350mg BID、约400mg BID、约450mg BID、约500mg BID、约550mg BID、约600mg BID、约200mg TID、约250mg TID、约300mg TID、约350mg TID、约400mg TID、约450mg TID、约500mg TID、约550mg TID、约600mg TID、约200mg QID、约250mg QID、约300mg QID、约350mg QID、约400mg QID、约450mg QID和约500mg QID的剂量给受试者施用。
在一些实施方案中，硝唑尼特以约500mg BID的剂量给受试者施用。
在一些实施方案中，硝唑尼特以约300mg至约350mg TID的剂量给受 试者施用。
在一些实施方案中，利巴韦林以从约20mg至约1200mg的范围的总日剂量给受试者施用。在一些实施方案中，硝唑尼特可以每日一次(QD)、每日两次(BID)或每日三次(TID)施用。
在一个实施方案中，利巴韦林以从约20mg至约400mg的范围的单次剂量给受试者施用。
在一些实施方案中，硝唑尼特以选自约20mg、约50mg、约60mg、约70mg、约80mg、约90mg、约100mg、约110mg、约120mg、约130mg、约140mg、约150mg、约160mg、约170mg、约180mg、约190mg、约200mg、约250mg、约300mg、约350mg、约400mg、约450mg和约500mg的单次剂量给受试者施用。
在一些实施方案中，利巴韦林以约50mg至约400mg的单次剂量，每日两次(BID)或每日三次(TID)给受试者施用。
在一些实施方案中，利巴韦林以每日两次(BID)施用。在一些实施方案中，利巴韦林以选自约20mg BID、约50mg BID、约60mg BID、约80mg BID、约100mg BID、约120mg BID、约140mg BID、约150mg BID、约160mg BID、约180mg BID、约200mg BID、约220mg BID、约240mg BID、约250mg BID、约260mg BID、约280mg BID、约300mg BID、约320mg BID、约330mg BID、约340mg BID、约350mg BID、约360mg BID、约380mg BID、约400mg BID、约420mg BID、约440mg BID、约450mg BID、约460mg BID、约480mg BID和约500mg BID的剂量给受试者施用。在一些实施方案中，利巴韦林以约200mg BID的剂量给受试者施用。在一些实施方案中，利巴韦林以约400mg BID的剂量给受试者施用。
在一些实施方案中，利巴韦林以每日三次(TID)施用。在一些实施方案中，利巴韦林以选自20mg TID、约50mg TID、约60mg TID、约80mg TID、约100mg TID、约120mg TID、约140mg TID、约150mg TID、约160mg TID、约180mg TID、约200mg TID、约220mg TID、约240mg TID、约250mg TID、约260mg TID、约280mg TID、约300mg TID、约320mg TID、约330mg TID、约340mg TID、约350mg TID、约360mg TID、约380mg TID和约400mg TID 的剂量给受试者施用。在一些实施方案中，利巴韦林以约100mg TID的剂量给受试者施用。在一些实施方案中，利巴韦林以约150mg TID的剂量给受试者施用。
在一些实施方案中，第一次硝唑尼特和利巴韦林可以同时给药。在一些实施方案中，第一次硝唑尼特和利巴韦林的给药间隔在约0小时至约16小时的范围内。在一些实施方案中，第一次硝唑尼特和利巴韦林的给药间隔为至少0小时、至少0.1小时、至少0.2小时、至少0.2小时、至少0.3小时、至少0.4小时、至少1小时、至少2小时、至少3小时、至少4小时、至少4小时、至少5小时、至少6小时、至少7小时、至少8小时、至少9小时、至少10小时、至少11小时、至少12小时、至少13小时、至少14小时、至少15小时和/或至少16小时。
在一些实施方案中，硝唑尼特和利巴韦林分别以每日两次(BID)给受试者施用，第一次硝唑尼特和利巴韦林可以同时给药或第一次硝唑尼特和利巴韦林的给药间隔在约0小时至约8小时的范围内，其中硝唑尼特以约300mg至约500mg范围的单次剂量施用，其中利巴韦林以约200mg至约400mg的单次剂量施用。
在一些实施方案中，硝唑尼特和利巴韦林分别以每日三次(TID)给受试者施用，第一次硝唑尼特和利巴韦林可以同时给药或第一次硝唑尼特和利巴韦林的给药间隔在约0小时至约4小时的范围内，其中硝唑尼特以约200mg至约350mg范围的单次剂量施用，其中利巴韦林以约100mg至约200mg的单次剂量施用。
在某些实施方案中，所述预防由冠状病毒感染引起或与其相关的疾病或病症的方法包括化学预防或药物预防。
在一个实施方案中，所述施用可防止轻症向重症的进展，或缓解至少一种症状，其中至少一种症状选自咳嗽、食欲下降、气喘、呼吸困难、发热、全身乏力、头晕、恶心、嗅觉减弱或丧失和肺炎。
III.实施例
实施例1细胞制备
非洲绿猴肾Vero E6细胞系取自美国标准菌库(ATCC，No.1586)，并 在添加了10％胎牛血清(FBS；Gibco Invitrogen)的MEM培养基(MEM；Gibco Invitrogen)中，以37℃，5％CO 2条件下培养。
实施例2病毒株分离
SARS-CoV-2(株HB-01)从中国疾病预防控制中心(中国CDC)获得。该SARS-CoV-2的完整基因组序列已提交GISAID(BetaCoV/Wuhan/IVDC-HB-01/2020|EPI_ISL_402119)，并保存在中国国家微生物数据中心(登录号NMDC10013001，基因组登录号MDC60013002-01)。病毒株SARS-CoV-2在Vero E6细胞中繁殖，采用免疫荧光测定法通过测定50％组织培养物感染剂量(TCID50)来确定病毒滴度。所有感染实验均在生物安全三级(BLS-3)实验室中进行。
实施例3试验药物
利巴韦林(RBV；货号：R101754)和硝唑尼特(NTZ；货号：N159057)自ALADDIN购买。GH23为NTZ与RBV两药联合的联用药物。
实施例4病毒RNA提取及定量分析(aRT-PCR)
SARS-CoV-2病毒RNA的提取采用MiniBEST病毒RNA/DNA提取试剂盒(Takara，货号9766)，具体操作按照生产商操作手册执行。
逆转录使用PrimeScript RT Reagent Kit以及gDNA Eraser(Takara，货号RR047A)试剂盒；qRT-PCR实验使用StepOnePlus TM Real-time PCR system(Applied Biosystem)以及TB Green Premix Ex Taq II(Takara，货号RR820A)试剂盒。
SARS-CoV-2病毒RNA取自100μL细胞培养上清液，在30μL无核糖核酸酶的水中洗脱，使用gDNA Eraser对3μL总RNA进行第一次消化，以移除基因组DNA污染物，在20μL的反应体积中合成cDNA的第一链。然后cDNA(2μL)作为PCR的模板。使用PCR从cDNA模板上用以下引物扩增出刺突基因的受体结合域(RBD):
RBD-Forward:5’-GCTCCATGGCCTAATATTACAAACTTGTGCC3’
RBD-Reverse:5’-TGCTCTAGACTCAAGTGTCTGTGGATCAC-3’。
。扩增的PCR产物被克隆到pMT/BiP/V5-His载体(invitrogen)中，经测序确认后作为标准品使用。通过测定各稀释液中质粒的拷贝数(10 3-10 9拷贝 数)得到标准曲线。用于定量实时PCR的引物为:
RBD-qF1:5’-CAATGGTTTAACAGGCACAGG-3’
RBD-qR1:5’-CTCAAGTGTCTGTGGATCACG-3’。
PCR扩增方式如下：95℃5分钟变性，然后进行包括95℃持续15秒，54℃持续15秒，72℃持续30秒三个步骤的40个循环。
实施例5试验药物的细胞毒性测试
试验药物对Vero E6细胞的细胞毒性通过标准CCK8分析测定，CCK8分析按照制造商说明书(Beyotime，中国)执行。在这些研究中，试验药物(NTZ、RBV、GH23)与Vero E6细胞共同孵育48小时。标准CCK8分析结果如图1所示。
用NTZ孵育Vero E6细胞可产生剂量依赖性细胞毒性，CC 50为36.8μM。当Vero E6细胞被RBV处理时，没有观察到细胞毒性作用(CC 50>400)。GH23(NTZ+RBV)的CC 50值为37.2μM，与单独使用NTZ时基本相同。这一结果表明，NTZ和RBV的联合使用没有协同毒性。
实施例6试验药物抗病毒活性测试
为评估本发明联用药物的抗病毒效力，将Vero E6细胞在密度为1×10 4细胞/孔的48孔细胞培养皿中培养过夜。用不同浓度的试验化合物预处理细胞1小时。以DMSO为阴性对照。随后添加病毒以感染复数(multiplicity of infection,MOI)为0.05感染细胞2小时。然后除去病毒-药物混合物，用含试验药物的新鲜培养基进一步培养细胞。在病毒感染后48小时，收集细胞上清液，并在裂解缓冲液(Takara，货号9766)中裂解，以进行病毒RNA水平的定量分析。使用GraphPad Prism 6软件绘制病毒RNA拷贝数相对于药物浓度的剂量反应曲线，以评价每种药物抑制SARS-CoV-2病毒复制的能力。
结果如图2所示。单独用NTZ处理Vero E6细胞(0-30μM)抑制SARS-CoV-2水平，EC 50为2.1μM，而单独用RBV处理Vero E6细胞的EC 50值为108.8μM。该发现与一项先前的研究发现一致，尽管利巴韦林可以明显整合入冠状病毒RNA，但它也容易被冠状病毒蛋白nsp14的3'-5'外显子核糖核酸酶(ExoN)活性切除(Ferrona F et al.PNAS.2017 115(2):E162-E171)。然而，用GH23(NTZ和RBV)联合治疗Vero E6细胞，可显著协同抑制SARS-CoV-2 水平，EC 50达到0.78μM。
表1总结了NTZ、RBV和GH23的CC 50、EC 50和相应的选择性指数(SI)值。NTZ、RBV和GH23的选择性指数分别为17.5、>3.7和47.7。这些数据表明，联合应用NTZ和RBV治疗SARS-CoV-2患者的安全窗比单独使用NTZ或RBV治疗显著更高。
表1 GH23抗SARS-CoV-2的抗病毒活性
Figure PCTCN2021092693-appb-000006

Claims (18)

  1. 一种联用药物，其包含至少一种选自硝唑尼特或替唑尼特或其药学上可接受的盐，和至少一种利巴韦林或其药学上可接受的盐或酯。
  2. 权利要求1的联用药物在制备用于治疗或预防冠状病毒感染所引起的疾病或病症的药物组合物中的用途。
  3. 权利要求2所述的用途，所述冠状病毒是SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43和/或HKU1，特别是SARS-CoV-2。
  4. 权利要求2所述的用途，所述冠状病毒感染所引起的疾病或病症是急性呼吸道传染病或肺炎。
  5. 权利要求2所述的用途，所述冠状病毒是SARS-CoV-2，所述冠状病毒感染所引起的疾病或病症是SARS-CoV-2感染引起的疾病(COVID-19)。
  6. 权利要求1的联用药物，其中硝唑尼特以口服制剂施用。
  7. 权利要求1的联用药物，其中利巴韦林可以口服制剂、吸入剂或注射剂施用。
  8. 一种用于治疗或预防由冠状病毒感染引起或与其相关的疾病或病症的方法，其包含向有需要的对象施用治疗有效量的至少一种选自硝唑尼特或替唑尼特或其药学上可接受的盐和治疗有效量的至少一种利巴韦林或其药学上可接受的盐或酯，所述硝唑尼特或替唑尼特和利巴韦林可以同时或者依次向对象施用。
  9. 权利要求8所述的方法，其中所述冠状病毒是SARS-CoV-2、SARS-CoV、MERS-CoV、229E、NL63、OC43和/或HKU1，特别是SARS-CoV-2。
  10. 权利要求8所述的方法，其中所述冠状病毒感染引起的疾病或病症是由SARS-CoV-2感染引起呼吸道疾病或肺炎。
  11. 权利要求8所述的方法，其中所述方法可用于防止轻症向重症的进展，或缓解至少一种症状，其中至少一种症状选自咳嗽、食欲下降、气喘、呼吸困难、发热、全身乏力、头晕、恶心、嗅觉减弱或丧失和肺炎。
  12. 权利要求8所述的方法，其中硝唑尼特以口服制剂施用。
  13. 权利要求8所述的方法，其中利巴韦林可以口服制剂、吸入剂或注射剂施用。
  14. 权利要求8所述的方法，所述对象是由冠状病毒感染引起的重症患者时，其中利巴韦林以吸入剂向有需要的对象施用。
  15. 权利要求8所述的方法，其包含向有需要的对象施用硝唑尼特和利巴韦林，其中硝唑尼特以从约100mg至约2000mg的范围的总日剂量给受试者施用，其中利巴韦林以从约20mg至约1200mg的范围的总日剂量给受试者施用，其中硝唑尼特和利巴韦林可分别以每日两次(BID)或每日三次(TID)施用。
  16. 权利要求15所述的方法，其中利巴韦林以从约100mg至约600mg的范围的单次剂量给受试者施用。
  17. 权利要求15所述的方法，其中利巴韦林以从约20mg至约400mg的范围的单次剂量给受试者施用。
  18. 权利要求8所述的方法，所述预防由冠状病毒感染引起或与其相关的疾病或病症的方法包括化学预防方法或药物预防。
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