WO2021164689A1 - Application de nelfinavir dans la préparation d'un médicament pour la prévention et le traitement d'une nouvelle pneumonie à coronavirus - Google Patents

Application de nelfinavir dans la préparation d'un médicament pour la prévention et le traitement d'une nouvelle pneumonie à coronavirus Download PDF

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WO2021164689A1
WO2021164689A1 PCT/CN2021/076585 CN2021076585W WO2021164689A1 WO 2021164689 A1 WO2021164689 A1 WO 2021164689A1 CN 2021076585 W CN2021076585 W CN 2021076585W WO 2021164689 A1 WO2021164689 A1 WO 2021164689A1
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nelfinavir
ncov
active ingredient
protease
coronavirus
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PCT/CN2021/076585
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English (en)
Chinese (zh)
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徐志建
姚航平
许叶春
吴南屏
彭诚
郑敏
卢翔云
苏海霞
程林芳
刘福民
吴海波
靳昌忠
吴志刚
沈敬山
朱维良
蒋华良
李兰娟
郑永唐
韩建保
宋天章
冯小丽
田仁荣
李川
杨军令
宫丽崑
吴春晖
蒋翔锐
王震
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中国科学院上海药物研究所
浙江大学医学院附属第一医院
海南海药股份有限公司
中国科学院昆明动物研究所
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Publication of WO2021164689A1 publication Critical patent/WO2021164689A1/fr

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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
    • A61K31/435—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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • 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
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/4706—4-Aminoquinolines; 8-Aminoquinolines, e.g. chloroquine, primaquine
    • 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
    • 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

Definitions

  • the invention relates to the field of medicine, in particular to the application of nelfinavir in the preparation of drugs for the prevention and treatment of new coronary pneumonia.
  • Severe Acute Respiratory Syndrome Coronavirus 2 SARS-CoV-2 or 2019 Novel Coronavirus, 2019-nCoV infection can cause new coronary pneumonia (Covid-19).
  • the transmission route of the 2019-nCoV virus is not fully grasped. It is known to be transmitted through droplets and contact, and there is human-to-human transmission, medical staff infection, a certain risk of community transmission, and the virus may mutate. At present, there is no specific prevention and treatment method for the disease caused by the new coronavirus.
  • the 2019-nCoV coronavirus belongs to the genus Coronavirus of the Coronavirus family, and is a single-stranded positive-sense RNA virus with an envelope. Similar to other known coronaviruses, the 2019-nCoV coronavirus also completes the proliferation of progeny viruses through several processes such as adsorption, penetration, uncoating, biosynthesis, and assembly and release of progeny viruses.
  • the infection of host cells by the 2019-nCoV coronavirus starts when the spike glycoprotein on the surface of the virus envelope binds to the receptor on the surface of the host cell, and then membrane fusion occurs. The virus enters the host cell and is released under the action of cell lysosomes and other organelles.
  • the single-stranded sense RNA of the virus’s genetic material is translated to produce polyprotein under the action of the host cell’s mitochondria, ribosomes and other protein synthesis elements and necessary raw materials.
  • the two essential cysteamines of the 2019-nCoV coronavirus Acid protease: papain-like protease (PL pro ) and 3C-like protease (3C-like protease, 3CL pro ) cleave and process polyprotein precursors at specific sites, resulting in multiple important to the virus life cycle Non-structural protein.
  • the viral RNA replicates the nucleic acid material of the progeny virus, and a large number of required structural proteins are translated to complete the assembly and release of the progeny virus.
  • Any link or key enzyme in the life cycle of cells infected by the 2019-nCoV coronavirus can be used as research targets for antiviral drugs, such as the cysteine proteases PL pro and 3CL pro that hydrolyze and cleave polyprotein precursors, which are responsible for the completion of the product.
  • 3CL protease (3chymotrypsin-like protease, 3CL pro ), also known as the main protease (M pro ), is the key protease in the process of producing multiple non-structural proteins after the coronavirus RNA is translated into polyproteins pp1a and pp1ab. Replication and infection are essential. Inhibiting the catalytic function of 3CL protease can effectively inhibit the cleavage of viral polyprotein precursors, block viral replication, and inhibit the generation of progeny viruses.
  • 3CL pro is a cysteine protease, which is a key protease that catalyzes the proteolysis of single positive-strand RNA virus precursors, and plays an important role in the replication activity of coronaviruses such as 2019-nCoV. Therefore, 3CL pro is currently recognized as an ideal target for the development of anti-coronavirus drugs.
  • the purpose of the present invention is to provide an active ingredient that can effectively inhibit the Coronavirus 3CL protease and its new use in inhibiting the Coronavirus.
  • the present invention provides the use of nelfinavir and its composition in anti-coronavirus, especially novel coronavirus (2019-nCoV).
  • the active ingredient or the preparation containing the active ingredient is used to prepare (a) inhibitors of the 2019 novel coronavirus (2019-nCoV) 3CL protease; and/or (b) treatment and/or prevention, Drugs to relieve related diseases caused by 2019 new coronavirus (2019-nCoV) infection.
  • the medicine also contains one or more additional active ingredients selected from the following group:
  • RNA replicase inhibitor such as Remdesivir (Remdesivir or GS-5734)
  • the related disease caused by the 2019 novel coronavirus infection is selected from the following group: respiratory tract infection, pneumonia and its complications, or a combination thereof.
  • the active ingredient is selected from the group consisting of nelfinavir and nelfinavir mesylate.
  • the drug further includes an additional component selected from the group consisting of: anti-retroviral drugs or immunity-enhancing drugs.
  • the composition or medicine includes: oral preparations and non-oral preparations.
  • the preparation includes: powder, granule, capsule, injection, tincture, oral liquid, tablet, lozenge, or dripping pill.
  • the drug is used to reduce the SARS-CoV-2 viral load in nasal swabs and anal swabs of susceptible subjects.
  • the susceptible objects include humans and non-human primates (such as macaques).
  • a pharmaceutical composition which contains:
  • the first active ingredient is selected from the group consisting of nelfinavir, or a pharmaceutically acceptable salt thereof; or a combination thereof;
  • the pharmaceutical composition is a pharmaceutical composition for inhibiting the 2019 novel coronavirus (2019-nCoV) 3CL protease.
  • the pharmaceutical composition contains:
  • the first active ingredient, the first active ingredient is selected from the group consisting of nelfinavir, or a pharmaceutically acceptable salt thereof; or a combination thereof;
  • the second active ingredient is selected from the group consisting of: (Y1) RNA replicase inhibitor (such as Remdesivir (Remdesivir or GS-5734)); (Y2) Lopinavir (Lopinavir); (Y3) Ritonavir (Ritonavir); (Y4) Favipiravir; (Y5) Chloroquine (Chloroquine, Sigma-C6628) or a pharmaceutically acceptable salt (such as chloroquine phosphate), (Y6 ) Any combination of the above Y1 ⁇ Y5;
  • the dosage form of the drug is oral administration or non-oral administration.
  • the oral administration dosage form is tablet, powder, granule or capsule, or emulsion or syrup.
  • the non-oral administration dosage form is injection, injection, aerosol, spray, eye drops, or ear drops.
  • the concentration of the first active ingredient (a single compound or the sum of multiple compounds) in the drug is 0.01-100 mg/ml, preferably 0.1-20 mg/ml (liquid dosage form).
  • the concentration of the first active ingredient (a single compound or the sum of multiple compounds) in the drug is 0.01-1000 mg/g, preferably 0.1-50 mg/g (solid dosage form).
  • the use of the pharmaceutical composition described in the second aspect of the present invention is provided, which is used to prepare (a) an inhibitor for inhibiting the 2019 novel coronavirus (2019-nCoV) 3CL protease; and / Or (b) Drugs for the treatment and/or prevention and alleviation of related diseases caused by 2019 novel coronavirus (2019-nCoV) infection.
  • the related disease caused by the 2019 novel coronavirus infection is selected from the following group: respiratory tract infection, pneumonia and its complications, or a combination thereof.
  • a method for inhibiting the 2019 novel coronavirus (2019-nCoV) 3CL protease comprises the steps of: combining the first active ingredient or the preparation containing the first active ingredient with the 2019 novel coronavirus (2019-nCoV) 3CL protease contact, thereby inhibiting the activity of the 3CL protease;
  • the first active ingredient is nelfinavir or a pharmaceutically acceptable salt thereof.
  • the inhibition method is an in vitro method and is a non-therapeutic and non-diagnostic method.
  • the inhibition method is an in vivo method and a therapeutic method.
  • the method is non-therapeutic and non-diagnostic.
  • the method is in vitro.
  • the 3CL protease of 2019-nCoV is a recombinant or 2019-nCoV expressed 3CL protease.
  • the first active ingredient is selected from the group consisting of nelfinavir and nelfinavir mesylate.
  • a method for treating, preventing, and/or alleviating related diseases caused by 2019 novel coronavirus (2019-nCoV) infection including the steps of: administering a safe and effective amount of The first active ingredient or the preparation containing the first active ingredient, wherein the first active ingredient is nelfinavir or a pharmaceutically acceptable salt thereof.
  • the method further comprises: administering a safe and effective amount of the second active ingredient to the subject in need, wherein
  • the second active ingredient is selected from the following group: (Y1) RNA replicase inhibitor (such as Remdesivir (Remdesivir or GS-5734)); (Y2) Lopinavir (Lopinavir); (Y3) Rib Tonavir (Ritonavir); (Y4) Favipiravir; (Y5) Chloroquine (Chloroquine, Sigma-C6628) or a pharmaceutically acceptable salt thereof (such as chloroquine phosphate), (Y6) any combination of the above Y1-Y5 ;
  • the subject is a mammal, preferably a primate mammal, more preferably a human.
  • the first active ingredient is selected from the following group: nelfinavir, or nelfinavir mesylate.
  • Figure 1 shows the docking mode of nelfinavir for 2019-CoV M pro.
  • the white surface is the binding pocket of 2019-nCoV M pro.
  • Cyan is nelfinavir
  • off-white is TG-0205221 (a SARS-CoV M pro protease inhibitor in the protein crystal structure (PDB ID: 2GX4));
  • TG-0205221 off-white and nelfinavir (Cyan) superimposition;
  • the binding mode of TG-0205221 and SARS M pro (PDB ID: 2GX4);
  • the docking mode of nelfinavir and 2019-nCoV M pro The yellow dashed line shows the critical range
  • Figure 2 shows the experimental results of Nelfinavir Mesylate against 2019 new coronavirus (2019-nCoV) in vitro. The results show that 3-30 ⁇ M nelfinavir mesylate has a significant antiviral effect in vitro.
  • Figure 4 shows that the Nelfinavir Mesylate administration group can significantly reduce the body temperature of monkeys (equivalent to improving clinical symptoms); it has no effect on body weight.
  • Figure 5 shows that the Nelfinavir Mesylate administration group can significantly reduce the viral load of nasal swabs (Figure 5a) and anal swabs (Figure 5c); but it cannot significantly reduce the viral load of throat swabs (Figure 5c). 5b) viral load. Can significantly reduce the viral load of the lungs and kidneys (Figure 5d).
  • Figure 6 shows that the Nelfinavir Mesylate administration group can improve the pathological tissue damage of major organs throughout the body.
  • Figure 7 shows the blood drug concentration of the Nelfinavir Mesylate administration group. Day 0, 1, 3, and 7 were the highest blood drug concentration (average value 0.55 ⁇ M), and the 5th day was the lowest blood drug concentration (0.26 ⁇ M). For comparison, in the case of a clinical administration dose of 1250 mg BID, the highest blood concentration of humans is 7.04 ⁇ M, and the lowest blood concentration (Ctrough, Morning) is 3.87 ⁇ M (VIRACEPT FDA Package Insert Reference ID: 3986049).
  • the inventors unexpectedly developed for the first time a class of active ingredients that can effectively inhibit the 3CL protease of the 2019 novel coronavirus (2019-nCoV) and other coronaviruses.
  • the active ingredient of the present invention Nafinavir or its pharmaceutically acceptable salt
  • Virus replication and viability are examples of viruses that can effectively inhibit the 3CL protease of the 2019 novel coronavirus (2019-nCoV) and other coronaviruses.
  • the active ingredient of the present invention can unexpectedly significantly reduce the SARS-CoV-2 viral load of nasal swabs and anal swabs; it can significantly reduce the SARS-CoV-2 viral load of lungs and kidneys. It can improve the pathological tissue damage of major organs throughout the body.
  • the present invention has been completed on this basis.
  • the present invention discloses the use of nelfinavir and its composition in anti-coronavirus, especially in anti-2019-nCoV virus therapy.
  • Nefinavir and its composition have excellent inhibitory effects on the highly conserved 3CL hydrolase, which is indispensable for coronavirus replication, and show good effects in the macaque new coronary pneumonia model, and have good clinical application prospects.
  • the active compound of the present invention and “the active compound of the present invention that inhibit 3CL protease” are used interchangeably, and refer to a compound having excellent 3CL protease inhibitory activity, especially nelfinavir or its pharmaceutically acceptable compounds. Accepted salt, or a combination thereof.
  • the formulation of the present invention refers to a formulation containing the active compound of the present invention.
  • new coronavirus As used herein, the terms “new coronavirus”, “2019-nCoV” or “SARS-CoV-2” are used interchangeably.
  • the 2019 new coronavirus is the seventh type of coronavirus that is known to infect humans and causes new coronary pneumonia. (COVID-19) is one of the serious infectious diseases that threaten human health around the world.
  • Coronavirus belongs to the Nidovirales (Nidovirales) Coronaviridae (Coronaviridae), which is an enveloped positive-stranded RNA virus, and its subfamily includes four genera of ⁇ , ⁇ , ⁇ and ⁇ .
  • HCoV-229E and HCoV-NL63 belong to the ⁇ genus coronavirus
  • HCoV-OC43, SARS-CoV, HCoV-HKU1, MERS-CoV and 2019-nCoV are all ⁇ genus coronavirus.
  • the new coronavirus (2019-nCoV) that broke out at the end of 2019 has about 80% similarity with SARS-CoV and 40% similarity with MERS-CoV, and it also belongs to the beta coronavirus.
  • the genome of this type of virus is a single-stranded positive-stranded RNA, which is one of the largest RNA viruses in the genome.
  • the codes include replicase, spike protein, envelope protein, envelope protein, and nucleocapsid protein.
  • the genome is translated into two peptide chains of several thousand amino acids, the precursor polyprotein (Polyprotein), and then the precursor protein is cleaved by proteases to produce non-structural proteins (such as RNA polymerase and unwinding). Enzymes) and structural proteins (such as spike proteins) and accessory proteins.
  • 3CL protease (3 Chymotrypsin-like protease, 3CLpro) is the main protease responsible for cleaving the precursor protein in the coronavirus (so it is also called M pro ), which is indispensable for virus replication.
  • 3CLpro is a cysteine hydrolase, which is highly conserved in various types of coronaviruses. It is also similar to the 3C protease in picornaviruses, but there is no similar protease in the human body. Therefore, it is a broad-spectrum anti-single positive-stranded RNA. Ideal target for viral drugs.
  • an active ingredient that can effectively inhibit the replication of coronaviruses such as 2019 novel coronavirus (2019-nCoV) is provided.
  • the active ingredient is selected from the group consisting of nelfinavir, or its pharmaceutically acceptable salt, or its crystal, or its solvate.
  • the compound has a molecular formula of C 32 H 45 N 3 O 4 S, a white powder, a molecular weight of 567.78, and a CAS number of 159989-64-7.
  • a preferred pharmaceutically acceptable salt of nelfinavir is its mesylate (ie nelfinavir mesylate):
  • the molecular formula of the mesylate is C 33 H 49 N 3 O 7 S 2 , a white powder, a molecular weight of 663.89, and a CAS number of 159989-65-8.
  • the general name of this compound is Nelfinavir mesilate.
  • active compound of the present invention and “active compound of the present invention that inhibit 3CL protease” are used interchangeably, and refer to compounds having excellent 3CL protease inhibitory activity, including nelfinavir, or its pharmaceutically acceptable compounds. Accepted salts, or crystals, or solvates thereof.
  • the active ingredient of the present invention includes the active compound of the present invention that inhibits 3CL protease, or a pharmaceutically acceptable salt, enantiomer, diastereomer or racemate, or prodrug thereof . It should be understood that the active ingredient of the present invention also includes the crystal form, amorphous compound, and deuterated compound of the active compound of the present invention.
  • the "pharmaceutically acceptable salt” refers to the reaction of the active compound of the present invention with an inorganic acid or an organic acid to form a conventional non-toxic salt.
  • conventional non-toxic salts can be prepared by reacting the active compound of the present invention with inorganic or organic acids.
  • the inorganic acids include hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, aminosulfonic acid, and phosphoric acid.
  • Acids include citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, benzenesulfonic acid, p-toluenesulfonic acid, methanesulfonic acid, naphthalenesulfonic acid, ethanesulfonic acid, naphthalene disulfonic acid, maleic acid, malic acid, malonic acid , Fumaric acid, succinic acid, propionic acid, oxalic acid, trifluoroacetic acid, stearic acid, pamoic acid, hydroxymaleic acid, phenylacetic acid, benzoic acid, salicylic acid, glutamic acid, ascorbic acid, p-aminobenzenesulfonate Acid, 2-acetoxybenzoic acid, isethionic acid, etc.; or the active compound of the present invention is combined with propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic
  • the active ingredient of the present invention is also particularly suitable for combined use with other antiviral drugs.
  • Representative other antiviral drugs include (but are not limited to): reverse transcriptase inhibitors, protease inhibitors, co-receptor antagonists, retroviral integrase inhibitors, virus adsorption inhibitors, specific viral transcription inhibitors Agents, antibodies, or combinations thereof.
  • the active ingredient of the present invention can inhibit the infectious activity of new coronaviruses such as 2019-nCoV. Therefore, when the active ingredient of the present invention is administered or administered therapeutically, the 3CL protease activity can be inhibited, thereby inhibiting the infection of the 2019 novel coronavirus (2019-nCoV) and achieving an antiviral effect.
  • new coronaviruses such as 2019-nCoV.
  • the present invention also provides the active compound of the present invention that inhibits 3CL protease, or a pharmaceutically acceptable salt thereof, or a prodrug, or an extract thereof, or a mixture of one or more of its medicinal materials as an active ingredient Use in the preparation of drugs for the treatment and/or prevention and alleviation of respiratory tract infections, pneumonia and other related diseases caused by the 2019 new coronavirus infection.
  • the pharmaceutical composition provided by the present invention preferably contains the active ingredient in a weight ratio of 0.001-99wt%, and the preferred ratio is that the active compound of the present invention as the active ingredient accounts for 0.1wt% to 90wt% or 1wt% to 50wt% of the total weight,
  • the remaining part is a pharmaceutically acceptable carrier, diluent or solution or salt solution.
  • the carrier includes conventional diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants and the like in the pharmaceutical field.
  • the compounds and pharmaceutical compositions provided by the present invention can be in various forms, such as tablets, capsules, powders, syrups, solutions, suspensions and aerosols, etc., and can be present in suitable solid or liquid carriers or diluents. Neutralization is suitable for sterilization equipment for injection or drip infusion.
  • Various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional preparation methods in the pharmaceutical field.
  • the unit measurement of the preparation formula usually contains 0.05-400 mg of the active compound of the present invention.
  • the unit measurement of the preparation formula contains 1 mg-500 mg of the active compound of the present invention.
  • the compounds and pharmaceutical compositions of the present invention can be used clinically on mammals, including humans and animals, and can be administered via oral, nose, skin, lung, or gastrointestinal tract. Most preferred is oral administration.
  • the most preferred daily dose is 0.01-400 mg/kg body weight, taken in one time, or 0.01-200 mg/kg body weight in divided doses. Regardless of the method of administration, the individual's optimal dosage should be determined based on the specific treatment. Usually, start with a small dose and gradually increase the dose until the most suitable dose is found.
  • the drugs or inhibitors of the present invention can be administered in various ways, for example, can be introduced into the body by injection, spraying, nose drops, eye drops, penetration, absorption, physical or chemically mediated methods such as muscle, intradermal, subcutaneous, and intravenous methods. , Mucosal tissue; or mixed or wrapped by other substances into the body.
  • the active compound of the present invention can effectively inhibit 2019-nCoV 3CL protease, with an IC 50 value of about 3 ⁇ M and an IC 90 value of about 6 ⁇ M.
  • the active compound of the present invention has low toxic and side effects and good druggability.
  • Nefinavir is an old anti-AIDS drug with high safety.
  • the recommended dose is 1250mg twice a day or 750mg three times a day.
  • the recommended dose for children 2-13 years old is 25-35 mg/kg 3 times a day or 45-55 mg/kg twice a day. This suggests that it has a good medicinal prospect in the field of anti-new coronary pneumonia.
  • Example 1 Computational simulation of nelfinavir against 2019-nCoV M pro protease
  • SWISS-MODEL was used to model the three-dimensional structure of the protease of 2019-nCoV M pro protease, and nelfinavir was docked.
  • the three-dimensional similarity of the target protein binding mode of action is 70.2%.
  • the docking complex was simulated using Amber16 for 5 nanoseconds, and then the binding free energy was calculated using the MM/GBSA and SIE methods.
  • the obtained energies were -24.69 ⁇ 0.52kcal/mol and -9.42 ⁇ 0.04kcal/mol, respectively.
  • the specific results are shown in Table 1 and Table 2.
  • the statistical results come from the molecular dynamics trajectory of 0.5-5 nanoseconds. Extract 500 frames from it for SIE calculation.
  • Example 2 Experimental test of nelfinavir against 2019-nCoV M pro protease
  • the inhibitory activity of the compound against 2019-nCoV M pro protease was determined by using fluorescence resonance energy transfer (fluorescence resonance energy transfer FRET) technology to determine the inhibitor of 3CL protease
  • fluorescence resonance energy transfer FRET fluorescence resonance energy transfer FRET
  • the level of enzymes inhibits activity.
  • the volume of the entire enzymatic reaction system is 120 ⁇ L, the final concentration of protease is 30 nM, and the final concentration of substrate is 20 ⁇ M.
  • the buffer of the reaction system includes 50mM Tris pH7.3, 1mM EDTA.
  • nelfinavir or its salt is an inhibitor of 2019-nCoV M pro protease.
  • Example 3 Inhibition experiment of nelfinavir on 2019-nCoV at the cellular level
  • the cells were infected with the 2019 new coronavirus strain 100TCID50 for 3 hours.
  • the culture medium containing the virus was aspirated and the cells were washed twice with PBS, and then added with different concentrations.
  • the culture medium of the drug is continued for 48 hours. Then aspirate the cell culture supernatant, extract the viral nucleic acid by the magnetic bead method, and use the new coronavirus fluorescent quantitative PCR kit to analyze the virus content in the cell culture fluid of the nelfinavir treatment group.
  • nelfinavir can significantly inhibit 2019-nCoV at the cellular level, with an IC 50 of 3.06 ⁇ M and an IC 90 of about 6.0 ⁇ M.
  • the experiment used 6 young healthy macaques (male, age 3-6 years old), the challenge route was a combination of nasal drops (0.4mL/nostril) and intratracheal (1.2mL, fiberoptic bronchoscopy), and the total virus titer was 1 ⁇ 10 7 TCID50mL, diluted with sterile 0.9% normal saline.
  • the experimental animals were anesthetized by injection of Shutai 50 into the thigh muscle, and then the body temperature and weight were measured in order; nasal swabs, throat swabs, anal swabs and peripheral venous blood samples were collected in sequence, and the venous blood was anticoagulated with heparin.
  • Time of detection and sample collection They are the 0th day before the challenge and 1, 3, 5, and 7 days after the challenge. Euthanasia was performed on the 8th day after the challenge, and tissues of heart, liver, spleen, left lower lobe, kidney, stomach, jejunum, colon, bladder, thymus and biceps were collected.
  • the experimental animals were divided into the nelfinavir mesylate administration group (3) and the control group (3).
  • the dosage was based on body weight and the dose was 200 mg/kg.
  • the control group was given 0.5% CMC-Na suspension Liquid, 5mL/kg.
  • the administration time is 10:00 in the morning and 20:00 in the evening, with an interval of 10 hours.
  • the frequency of administration is 2 times/day.
  • the method of administration is gastric administration via a nasogastric tube. Before each administration, 25ml of apple oatmeal is given in advance, then the medicine is given, and finally 5ml of apple oatmeal is given.
  • the administration group was administered 3 times before the challenge, and the continuous administration period was the first day and the 0th day before the challenge, and the first to 7 days after the challenge; the control group was given 6 hours after the challenge, and the continuous administration period
  • the drug cycle is once on the 0th day before the challenge, and on the 1st to 7th days after the challenge.
  • the virus used in the experiment was the new coronavirus 107 strain (http://nmdc.cn/resource/ncov/genome/detail/NMDCN0000HUI), provided by the Guangdong Provincial Center for Disease Control and Prevention, China.
  • the virus strain was expanded by the Vero-E6 cell strain.
  • Half of the tissue culture infection dose determination method is Reed-Muench method.
  • RNA of the swab was extracted with the kit (Roche Germany), and the RNA of the tissue sample was extracted with the TRIzol reagent method (Thermo USA). Viral RNA was detected using a probe one-step real-time quantitative PCR kit (TOYOBO, Japan). The dilution of each test sample refers to the standard of the Chinese Academy of Metrology, and finally the copy number of each sample is calculated.
  • the infected rhesus macaque tissue samples were fixed with 4% paraformaldehyde for 7 days, embedded in paraffin, sectioned (slices 4 microns) and other procedures for HE staining.
  • the concentration of nelfinavir in monkey plasma samples was determined by liquid chromatography-mass spectrometry.
  • the samples were pre-treated with 1:3 absolute ethanol precipitation and quantified by external standard method.
  • API4000 Q trap mass spectrum using positive ion MRM scanning, nelfinavir 568.3 ⁇ 330.2 (breaking energy 45V). Standard song concentration: 1.37 ⁇ 3000nM.
  • Statistical analysis software R is used for data analysis. For body temperature, weight, swab viral load, etc., two-way mixed ANOVA is used for comparison between groups; if the conditions of ANOVA are not met, Mann-Whitney U test is used for comparison. For tissue viral load and histopathology, use t-test or Mann-Whitney U test for comparison. P value ⁇ 0.05 was judged as statistically different.
  • the nelfinavir mesylate administration group can significantly reduce the body temperature of the monkeys (equivalent to improving the clinical symptoms), but at the same time does not affect the weight of the rhesus monkeys, which has a good clinical application prospect.
  • the nelfinavir mesylate administration group can significantly reduce the viral load of nasal swabs and anal swabs; but it cannot significantly reduce the viral load of throat swabs; it can also significantly reduce the lung and kidneys Viral load.
  • the nelfinavir mesylate administration group can improve the pathological tissue damage of major organs throughout the body.
  • the blood concentration test of the nelfinavir mesylate administration group found that, in this example, when the rhesus monkeys were prophylactically administered with a concentration of 200 mg/kg of nelfinavir mesylate, the blood The drug concentration is only one tenth of the clinical drug concentration.
  • nelfinavir can inhibit the replication of SARS-CoV at the cellular level (Biochem.Biophys.Res.Commun.2004,318(3),719-25). In 2005, it was discovered that nelfinavir was against SARS-CoV M The IC 50 of pro is 46 ⁇ M (Biochem. Biophys. Res. Commun. 2005, 333(1), 194-199).
  • nelfinavir is ineffective against the SARS-CoV animal model (Antivir. Chem. Chemother. 2006, 17(5), 275-84), mainly due to poor cell activity.
  • IC 50 3.5 ⁇ M
  • CC 50 9.5 ⁇ M
  • the IC 90 of nelfinavir against SARS-CoV is 40 ⁇ M, while the experiment of the present invention shows that the IC 90 of nelfinavir against 2019-nCoV is only 6 ⁇ M, which is more than 6 times the difference between the two.
  • nelfinavir has an unexpectedly high therapeutic index and a better IC 90 value for 2019-nCoV.
  • the 2019 novel coronavirus (2019-nCoV) has more significant antiviral activity and drug safety.
  • these results also suggest that the 2019 novel coronavirus (2019-nCoV) is different from the proteins (especially 3CL hydrolase) of previous coronaviruses (such as SARS) in terms of structure and activity.
  • the active ingredient of the present invention can unexpectedly Significantly reduce the SARS-CoV-2 viral load of nasal swabs and anal swabs, and significantly reduce the SARS-CoV-2 viral load of lungs and kidneys, and can improve the pathological tissue damage of major organs throughout the body.
  • nelfinavir or a pharmaceutically acceptable salt thereof has a significant inhibitory effect on 2019-nCoV-3CLpro, with IC 50 and IC 90 reaching single-digit micromolar levels.
  • the inventor’s research also suggests that nelfinavir can also effectively inhibit the infection and replication of SARS-CoV-2 by acting on multiple targets, especially SARS-CoV from nasal swabs and anal swabs.
  • the decrease of CoV-2 viral load on the one hand indicates that the active ingredient of the present invention has a significant therapeutic effect, and on the other hand, it can also reduce the risk of further infection of the SARS-CoV-2 virus.
  • nelfinavir or its pharmaceutically acceptable salt has excellent anti-2019 novel coronavirus (2019-nCoV) activity and has good clinical application prospects.

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Abstract

La présente invention concerne une application de nelfinavir dans la préparation d'un médicament pour prévenir et traiter une nouvelle pneumonie à coronavirus. Spécifiquement, la présente invention concerne une utilisation de nelfinavir et d'une composition pharmaceutique de celui-ci en tant qu'inhibiteurs de la protéinase 3CL des nouveaux coronavirus 2019 (2019-nCov) dans la préparation d'un médicament pour le traitement et/ou la prévention et le soulagement de maladies associées provoquées par une infection à nouveau coronavirus 2019, telle qu'une infection des voies respiratoires et une pneumonie.
PCT/CN2021/076585 2020-02-17 2021-02-10 Application de nelfinavir dans la préparation d'un médicament pour la prévention et le traitement d'une nouvelle pneumonie à coronavirus WO2021164689A1 (fr)

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