WO2019041239A1 - 大麻二酚在制备抗流感的药物中的用途 - Google Patents

大麻二酚在制备抗流感的药物中的用途 Download PDF

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WO2019041239A1
WO2019041239A1 PCT/CN2017/099956 CN2017099956W WO2019041239A1 WO 2019041239 A1 WO2019041239 A1 WO 2019041239A1 CN 2017099956 W CN2017099956 W CN 2017099956W WO 2019041239 A1 WO2019041239 A1 WO 2019041239A1
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influenza
subtype
virus
inhibitor
cannabidiol
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PCT/CN2017/099956
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English (en)
French (fr)
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张可
谭昕
李向东
于朝晖
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汉义生物科技（北京）有限公司
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Priority to JP2020530715A priority Critical patent/JP6963719B2/ja
Priority to CA3073576A priority patent/CA3073576C/en
Priority to EP17923262.4A priority patent/EP3677258B1/en
Priority to PCT/CN2017/099956 priority patent/WO2019041239A1/zh
Priority to US16/639,858 priority patent/US11452707B2/en
Priority to AU2017429312A priority patent/AU2017429312B2/en
Publication of WO2019041239A1 publication Critical patent/WO2019041239A1/zh

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
    • A61K31/05—Phenols
    • 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
    • A61K31/13—Amines
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
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    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • 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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • 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
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/4045—Indole-alkylamines; Amides thereof, e.g. serotonin, melatonin
    • 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
    • A61K31/7012—Compounds having a free or esterified carboxyl group attached, directly or through a carbon chain, to a carbon atom of the saccharide radical, e.g. glucuronic acid, neuraminic acid
    • 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
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/70—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in livestock or poultry

Definitions

  • the invention belongs to the field of biomedicine and relates to the use of cannabidiol in the preparation of anti-influenza drugs.
  • Influenza is an acute respiratory infection caused by influenza virus (Influenza Virus). It is caused by rapid transmission, high incidence and serious complications. A disease that threatens human health.
  • Influenza virus is an influenza virulence factor transmitted through the acute respiratory tract. It belongs to the Orthomyxoviridae family and is a single-stranded negative-strand RNA virus. According to the antigenicity of its nuclear protein (NP) and matrix protein (M), the influenza virus can be divided into three types: A (A), B (B), and C (C) (Hay et al. , 2001). Among them, Influenza A Virus (IAV) is the most common and poses the greatest threat to humans, causing seasonal and regional outbreaks of influenza. In addition to infecting people, it can also cause infections in a variety of animals such as poultry, pigs and horses.
  • NP nuclear protein
  • M matrix protein
  • IAV Influenza A Virus
  • IAV is a single-strand negative-chain 8-segment RNA virus. Each RNA fragment is coated with a nuclear protein (NP) to form a nuclear protein complex (RNPs).
  • the IAV genome encodes at least 13 proteins, PB2, PB1, PB1-F2, N40, PA, PA-X, HA, NP, NA, M1, M2, NS1 and NS2. There are three kinds of proteins on the surface of the IAV capsule, which are HA, NA and M2.
  • the 8 RNA fragments of IAV are all coated with NP.
  • viral RNA is coated into RNPs by three subunits of NP and polymerase, Polymerase 1 (PB1), Polymerase 2 (PB2), and Polymerase A (PA).
  • PB1 functions as an RNA-dependent RNA polymerase.
  • PB2 binds to and cleaves the host mRNA cap as a primer to synthesize viral RNA.
  • PA can perform host RNA endonuclease function and may also have proteolytic activity.
  • NPs function as single-stranded RNA binding proteins and act as structural proteins in RNPs.
  • HA hemagglutinin
  • NA neuraminidase
  • H1N1, H2N2, and H3N2 mainly infect humans, and many other subtypes of natural hosts are a variety of birds and animals.
  • H5, H7 and H9 subtype strains are the most harmful to birds.
  • Highly pathogenic H5NI, Avian influenza viruses such as H7N9 and H9N2 once mutated and have human-to-human transmission ability, will lead to the epidemic of human avian influenza, indicating that the avian influenza virus has a great potential threat to humans.
  • RNA polymerase RNA polymerase
  • ALI acute lung injury
  • ARDS Acute respiratory distress syndrome
  • SIRS systemic inflammatory response syndrome
  • the anti-influenza drugs approved by the countries in the world are mainly: inosine monophosphate dehydrogenase (IMPDH) inhibitor ribavirin, interferon inducer arcidol hydrochloride, M2 Ion channel protein inhibitors amantadine hydrochloride and rimantadine hydrochloride, neuraminidase inhibitors osehamivir phosphate and zanamivir 4 classes 6 Varieties (Glezen, 2006; Kolocouris et al., 1996).
  • IMPDH inosine monophosphate dehydrogenase
  • interferon inducer arcidol hydrochloride M2 Ion channel protein inhibitors amantadine hydrochloride and rimantadine hydrochloride
  • neuraminidase inhibitors osehamivir phosphate and zanamivir 4 classes 6 Varieties (Glezen, 2006; Kolocouris et al., 1996).
  • M2 ion channel blockers including amantadine and rimantadine, block the viral shelling by blocking the M2 ion channel protein, preventing viral RNA from being released into the cytoplasm, disrupting early replication of the virus, thereby The role of the flu virus.
  • amantadine drugs are not effective against influenza B, and they are neurotoxic. Insomnia, attention distraction and neurotic side effects may occur several hours after taking, and amantadine drugs are easy to use under experimental conditions and clinical applications. Produce resistant strains. However, most of the current influenza strains have been resistant to these two drugs, and only the influenza A virus has M2 ion channel protein, so its clinical use is not extensive.
  • NA inhibitors currently only zanamivir and oseltamivir (Duffy), and another intravenous injection of peramivir was approved in Japan in January 2010.
  • Duffy zanamivir and oseltamivir
  • They prevent the sialic acid on the surface of infected cells from being cleaved, resulting in the release of the new virus from the surface of the infected cells, thereby preventing the virus from further infecting other cells.
  • H5N1 poultry Influenza patients also have a high mortality rate, and Duffy-resistant strains are constantly being isolated.
  • the current popular IAV has developed resistance to neuraminidase inhibitors.
  • Tamiflu can cause serious adverse reactions such as sudden breathing difficulties. For example, a study by the Japanese non-profit organization Medical Alert Center found that 38 of the 119 death patients who took Tamiflu were taking 12 hours. There is a serious state or death within.
  • Cannabidiol is one of the cannabinoids, and its structural formula is as shown in the following formula I:
  • Cannabinol is extracted from natural plant cannabis, has no psychic effect, and has therapeutic effects on anxiety, depression, convulsions and tumors. Studies have shown that CBD has a good anti-inflammatory effect. In 2000, Proceedings of the National Academy of Sciences reported that Dr. M. Feldman of the Kennedy Institute of Rheumatology in London, England, found that oral and systemic use of CBD can significantly reduce joint damage. Severity and the acute and chronic course of arthritis. In the 2015 issue of Immunopharmacol Immunotoxicology, CBD inhibits LPS-induced acute lung injury (Ribeiro et al., 2015).
  • cannabidiol can effectively inhibit the influenza virus and the RNA polymerase of influenza virus, and has the potential to prevent and treat influenza.
  • One aspect of the present invention relates to the use of any one of the following (1) to (3) for the preparation of a medicament for treating or preventing influenza or for ameliorating flu symptoms:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and One or more pharmaceutically acceptable excipients.
  • influenza is caused by one or more influenza viruses selected from the group consisting of influenza A virus, influenza B virus, and influenza C virus; preferably, the influenza A virus is Influenza A virus of H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the subject of the influenza is a mammal (eg, a human, a donkey, a monkey, a pig, a cow, or a sheep) or a bird (eg, a poultry such as a chicken, duck, or goose, or, for example, a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow, or a sheep
  • a bird eg, a poultry such as a chicken, duck, or goose, or, for example, a wild bird.
  • the flu symptom is at least one selected from the group consisting of the following symptoms:
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase (IMPDH) inhibitor Inosine monophosphate dehydrogenase (IMPDH) inhibitor, interferon inducer, M2 ion channel inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is arcidol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is osehamivir phosphate, oseltamivir (Tamiflu), zanamivir or peramivir.
  • Another aspect of the invention relates to the use of any one of (1)-(3) selected below for the preparation of a medicament against an influenza virus, for example, inhibiting replication of an influenza virus in a host cell:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza virus is selected from one or more of influenza A virus, influenza B virus, and influenza C virus; preferably, the influenza A virus is H1N1 subtype Influenza A virus of H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the host cell is a cell of a mammal (eg, a human, a donkey, a monkey, a pig, a cow, or a sheep) or a bird (eg, such as a poultry such as a chicken, duck, or goose, or, for example, a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow, or a sheep
  • a bird eg, such as a poultry such as a chicken, duck, or goose, or, for example, a wild bird.
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • a further aspect of the invention relates to a medicament selected from any one of (1) to (3) below for the preparation of a medicament for inhibiting replication of influenza virus RNA polymerase, for inhibiting the expression level of influenza virus RNA polymerase or for inhibiting influenza Use of drugs for the activity of viral RNA polymerase:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza virus RNA polymerase is selected from one or more of influenza A virus RNA polymerase, influenza B virus RNA polymerase, and influenza C virus RNA polymerase;
  • influenza A virus RNA polymerase is an RNA polymerase of influenza A virus of H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the inhibition of the expression level of influenza virus RNA polymerase refers to, for example, the protein level of influenza virus RNA polymerase or the level of mRNA encoding influenza virus RNA polymerase.
  • the expression level of the influenza virus RNA polymerase can be determined by the expression level of any one or more of the three subunits PB1, PB2 and PA of influenza virus RNA polymerase.
  • a further aspect of the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • the cannabinol is the sole active ingredient in the pharmaceutical composition.
  • the cannabidiol is used in combination with one or more other known active ingredients for the control of influenza.
  • the pharmaceutical composition further comprises an effective amount of one or more ingredients selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the pharmaceutical composition may be in the form of any pharmaceutically acceptable dosage form, including: tablets, sugar-coated tablets, film-coated tablets, enteric coated tablets, capsules, hard capsules, soft capsules, Oral liquid, oral preparation, granules, granules, pills, powders, ointments, dans, suspensions, powders, solutions, injections, suppositories, ointments, plasters, creams, sprays, drops, stickers Oral dosage forms such as capsules, tablets, oral liquids, granules, pills, powders, granules, ointments and the like.
  • the oral dosage form may contain conventional excipients such as a binder, a filler, a diluent, a tablet, a lubricant, a disintegrant, a coloring agent, a flavoring agent, and a humectant, and if necessary, a tablet Carry out the coating.
  • Suitable fillers include cellulose, mannitol, lactose and the like.
  • Fillers; suitable disintegrants include starch, polyvinylpyrrolidone and starch derivatives such as sodium starch glycolate;
  • suitable lubricants include, for example, magnesium stearate.
  • Suitable pharmaceutically acceptable wetting agents include sodium lauryl sulfate.
  • the pharmaceutical composition is an oral preparation.
  • the dose of cannabidiol administered to the subject is between 0.1 and 50 mg/kg body weight/day, more preferably between 0.5 mg/kg and 30 mg/kg body weight/day, and 0.5 mg/kg to 20 mg/kg body weight/day. 5 mg/kg to 30 mg/kg body weight/day or 5 mg/kg to 20 mg/kg body weight/day, further preferably 0.5 mg/kg to 10 mg/kg body weight/day, particularly preferably 0.5 mg/kg to 5 mg/kg body weight/ day.
  • a further aspect of the invention relates to a combination product comprising individually packaged product 1 and product 2,
  • the product 1 is selected from any one of the following (1) to (3):
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients;
  • the product 2 comprises an effective amount of one or more components selected from the group consisting of an inosine monophosphate dehydrogenase inhibitor, an interferon inducer, an M2 ion channel protein inhibitor, and a neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the invention also relates to the following aspects:
  • the present invention relates to a product selected from any one of (1) to (3) for treating or preventing influenza or alleviating flu symptoms:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably Ground, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza is caused by one or more influenza viruses selected from the group consisting of influenza A virus, influenza B virus, and influenza C virus; preferably, the influenza A virus is Influenza A virus of H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the subject of the influenza is a mammal (eg, a human, a donkey, a monkey, a pig, a cow, or a sheep) or a bird (eg, a poultry such as a chicken, duck, or goose, or, for example, a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow, or a sheep
  • a bird eg, a poultry such as a chicken, duck, or goose, or, for example, a wild bird.
  • the flu symptom is at least one selected from the group consisting of the following symptoms:
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the present invention also relates to a product selected from any one of (1) to (3) for use against an influenza virus (for example, inhibiting replication of an influenza virus in a host cell):
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza virus is selected from one or more of influenza A virus, influenza B virus, and influenza C virus; preferably, the influenza A virus is H1N1 subtype , H2N2 subtype, Influenza A virus of H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the host cell is a cell of a mammal (eg, a human, a donkey, a monkey, a pig, a cow, or a sheep) or a bird (eg, such as a poultry such as a chicken, duck, or goose, or, for example, a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow, or a sheep
  • a bird eg, such as a poultry such as a chicken, duck, or goose, or, for example, a wild bird.
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the present invention also relates to a product selected from any one of (1) to (3) for inhibiting replication of influenza virus RNA polymerase, inhibiting expression level of influenza virus RNA polymerase or inhibiting polymerization of influenza virus RNA Enzyme activity:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza virus RNA polymerase is selected from one or more of influenza A virus RNA polymerase, influenza B virus RNA polymerase, and influenza C virus RNA polymerase;
  • influenza A virus RNA polymerase is an RNA polymerase of influenza A virus of H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the present invention also relates to a method of treating or preventing influenza or ameliorating flu symptoms, comprising the step of administering to a subject in need thereof an effective amount of a product selected from any one of (1) to (3) below:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza is caused by one or more influenza viruses selected from the group consisting of influenza A virus, influenza B virus, and influenza C virus; preferably, the influenza A virus is Influenza A virus of H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the subject is a mammal (eg, a human, a donkey, a monkey, a pig, a cow or a sheep) or a bird (eg a poultry such as a chicken, duck or goose, or such as a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow or a sheep
  • a bird eg a poultry such as a chicken, duck or goose, or such as a wild bird.
  • the flu symptom is at least one selected from the group consisting of the following symptoms:
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the present invention also relates to a method of combating influenza virus (e.g., inhibiting replication of an influenza virus in a host cell) comprising administering to a subject in need thereof an effective amount of any one of (1) to (3) selected from the group consisting of The steps of a product:
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza virus is selected from one or more of influenza A virus, influenza B virus, and influenza C virus; preferably, the influenza A virus is H1N1 subtype Influenza A virus of H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the subject is a mammal (eg, a human, a donkey, a monkey, a pig, a cow or a sheep) or a bird (eg a poultry such as a chicken, duck or goose, or such as a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow or a sheep
  • a bird eg a poultry such as a chicken, duck or goose, or such as a wild bird.
  • the host cell is a cell of a mammal (eg, a human, a donkey, a monkey, a pig, a cow, or a sheep) or a bird (eg, such as a poultry such as a chicken, duck, or goose, or, for example, a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow, or a sheep
  • a bird eg, such as a poultry such as a chicken, duck, or goose, or, for example, a wild bird.
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the present invention also relates to a method of inhibiting replication of influenza virus RNA polymerase, inhibiting the expression level of influenza virus RNA polymerase or inhibiting the activity of influenza virus RNA polymerase, comprising administering to a subject in need thereof an effective amount selected from the group consisting of The steps of the product of any of the following (1) to (3):
  • a plant extract containing cannabidiol preferably, a cannabis extract containing cannabidiol; preferably Ground, an industrial cannabis extract containing cannabidiol;
  • a pharmaceutical composition comprising an effective amount of cannabidiol or a pharmaceutically acceptable salt or ester thereof, and one or more pharmaceutically acceptable excipients.
  • influenza virus RNA polymerase is selected from one or more of influenza A virus RNA polymerase, influenza B virus RNA polymerase, and influenza C virus RNA polymerase;
  • influenza A virus RNA polymerase is an RNA polymerase of influenza A virus of H1N1 subtype, H2N2 subtype, H3N2 subtype, H5NI subtype, H7N9 subtype or H9N2 subtype.
  • the subject is a mammal (eg, a human, a donkey, a monkey, a pig, a cow or a sheep) or a bird (eg a poultry such as a chicken, duck or goose, or such as a wild bird).
  • a mammal eg, a human, a donkey, a monkey, a pig, a cow or a sheep
  • a bird eg a poultry such as a chicken, duck or goose, or such as a wild bird.
  • the pharmaceutical composition further comprises an effective amount of one or more components selected from the group consisting of:
  • Inosine monophosphate dehydrogenase inhibitor interferon inducer, M2 ion channel protein inhibitor and neuraminidase inhibitor;
  • the inosine monophosphate dehydrogenase inhibitor is ribavirin;
  • the interferon inducer is abidedol hydrochloride
  • the M2 ion channel protein inhibitor is amantadine hydrochloride or rimantadine hydrochloride;
  • the neuraminidase inhibitor is oseltamivir phosphate, oseltamivir, zanamivir or peramivir.
  • the dosage and method of administration of the active ingredient cannabidiol depends on a number of factors, including the patient's age, weight, sex, natural health status, nutritional status, activity intensity of the compound, time of administration, rate of metabolism, and condition of the disease. Severity and subjective judgment of the doctor.
  • the dose of cannabidiol is between 0.1 and 50 mg/kg body weight/day, more preferably between 0.5 mg/kg and 30 mg/kg body weight/day, 0.5 mg/kg- 20 mg/kg body weight/day, 5 mg/kg-30 mg/kg body weight/day or 5 mg/kg-20 mg/kg body weight/day, further preferably 0.5 mg/kg to 10 mg/kg body weight/day, particularly preferably 0.5 mg/day. Kg-5 mg/kg body weight/day.
  • the cannabidiol a compound of formula I
  • cannabidiol can be purchased commercially (for example from Sigma, etc.) or synthesized using commercially available starting materials by the prior art. After synthesis, it can be passed through column chromatography, liquid-liquid extraction, and molecular distillation. Further purification by methods such as crystallization or crystallization. In addition, cannabidiol can also be extracted from cannabis, especially industrial cannabis.
  • compositions of cannabidiol include, but are not limited to, organic ammonium salts, alkali metal salts (sodium salts, potassium salts), alkaline earth metal salts (magnesium salts, barium salts, calcium salts), and the like.
  • the pharmaceutically acceptable salt of cannabidiol may be cannabinol (CBD) with sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, aluminum hydroxide, or hydroxide.
  • CBD cannabinol
  • the pharmaceutically acceptable ester of cannabidiol may be a monoester of cannabidiol and a C 0 -C 6 alkyl carboxylic acid, or may be the same as two of cannabidiol or different diesters of C 0- C 6 alkyl carboxylic acids, the C 0 -C 6 alkyl carboxylic acids may be straight-chain alkyl carboxylic acid, branched alkyl or cycloalkyl carboxylic acid,
  • the cannabis extract may be cannabinoid containing cannabis, especially an extract of industrial cannabis, such as an ethanol extract, an extract, or the like.
  • an extract of industrial cannabis such as an ethanol extract, an extract, or the like.
  • the content of cannabidiol is not particularly limited, and the content of cannabidiol in the cannabis extract can be further increased by means known to those skilled in the art such as concentration.
  • the cannabis extract is an extract, preferably wherein the content of cannabidiol is from 18% to 25%.
  • the cannabis extract is an extract obtained by using any one or more selected from the group consisting of stems, leaves, fruits, husks, roots, and flowers of cannabis.
  • the cannabis extract is a cannabis leaf extract.
  • the term "effective amount” refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of the disease or condition of the invention in a subject.
  • subject can refer to a patient or other animal that receives the composition of the invention to treat, prevent, ameliorate and/or alleviate the disease or condition of the invention, particularly a mammal, such as a human, a dog, a monkey, a cow, Horse and so on.
  • disease and/or condition refers to a physical state of the subject, the physical state being described in the present invention. Related to a disease and/or condition.
  • the product 1 and the product 2 are merely for the sake of clarity and do not have the meaning of order unless otherwise specified.
  • the cannabis is preferably industrial cannabis unless otherwise specified; the cannabis extract is preferably an industrial cannabis extract.
  • Cannabidiol is effective in inhibiting influenza virus and has the potential to prepare or as a drug for treating or preventing influenza.
  • Figure 3A day 6 after H1N1 infection.
  • Figure 3B day 8 after H1N1 infection.
  • Figure 5 Changes in lung index of mice infected with WSN. The rats on the 8th day of infection were taken and the lung infection was detected. Data are expressed as mean ⁇ standard error, *p ⁇ 0.05.
  • Figure 6 Changes in lung pathology in mice infected with WSN. The rats on the 8th day of infection were taken and the pathological analysis of the lungs was examined.
  • Figure 6A wild control
  • Figure 6B lung control for influenza infection
  • Figure 6C Tamiflu treatment group
  • Figure 6D CBD treatment group.
  • Figure 7 Changes in lung index of mice infected with WSN. Taking the 8th day of infection, detecting pulmonary vascular permeability Detection. Data are expressed as mean ⁇ standard error, *p ⁇ 0.05; ** p ⁇ 0.01.
  • Figure 8 Changes in neutrophils in alveolar perfusate of WSN-infected mice. The number of neutrophils in the alveolar perfusate was measured in mice with DPI for 8 days. Data are expressed as mean ⁇ standard error, *** p ⁇ 0.001.
  • Figure 9 Changes in lymphocytes in alveolar perfusate of WSN-infected mice. The number of lymphocytes in the alveolar perfusate was measured in mice with DPI for 8 days. Data are expressed as mean ⁇ standard error, **p ⁇ 0.01.
  • Figure 10 Changes in macrophages in alveolar perfusate of WSN-infected mice. The number of macrophages in the alveolar perfusate was measured in mice with DPI for 8 days. Data are expressed as mean ⁇ standard error, **p ⁇ 0.01, ***p ⁇ 0.001.
  • Figure 11A Changes in PA gene expression in CBD-treated WSN-infected mouse BMDM cells.
  • the expression of the PB1 gene was changed after CBD-treated H1N1-infected mouse BMDM cells for 24 hours. Data are expressed as mean ⁇ standard error, *p ⁇ 0.05.
  • Figure 11B Changes in expression of the PB1 gene in CBD-treated WSN-infected mouse BMDM cells.
  • the expression of the PB1 gene was changed after CBD-treated H1N1-infected mouse BMDM cells for 24 hours. Data are expressed as mean ⁇ standard error, *p ⁇ 0.05.
  • Figure 12A Expression changes of NP gene in CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • the expression of the NP gene was changed 24 hours after the CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • Data are expressed as mean ⁇ standard error, *** p ⁇ 0.001.
  • FIG. 12B Expression changes of PA gene in CBD-treated H1N1 infected human lung epithelial cell line A 549.
  • the expression of the PA gene was changed 24 hours after the CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • Data are expressed as mean ⁇ standard error, **p ⁇ 0.01.
  • Figure 12C Changes in expression of the PB1 gene in the CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • the expression of the PB1 gene was changed 24 hours after the CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • Data are expressed as mean ⁇ standard error, **p ⁇ 0.01.
  • Figure 12D Expression changes of the PB2 gene in the CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • the expression of the PB2 gene was changed 24 hours after the CBD-treated human lung epithelial cell line A 549 infected with H1N1.
  • Data are expressed as mean ⁇ standard error, **p ⁇ 0.01.
  • Figure 17A Expression changes of NP gene in CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • the expression of the NP gene was changed 24 hours after the CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • Data are expressed as mean ⁇ standard error, *p ⁇ 0.05.
  • FIG. 17B Expression changes of PA gene in CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • the expression of the PA gene was changed 24 hours after the CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • Data are expressed as mean ⁇ standard error, *p ⁇ 0.05.
  • Figure 17C Changes in expression of the PB1 gene in the CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • the expression of the PB1 gene was changed after CBD-treated human lung epithelial cell line A 549 infected with H5N1 for 24 h.
  • Data are expressed as mean ⁇ standard error, *p ⁇ 0.05, **p ⁇ 0.01.
  • Figure 17D Expression changes of PB2 gene in CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • the expression of the PB2 gene was changed 24 hours after CBD-treated human lung epithelial cell line A 549 infected with H5N1.
  • Data are expressed as mean ⁇ standard error, *p ⁇ 0.05, **p ⁇ 0.01.
  • H1N1WSN A/WSN/33) strain of IAV and the H5N1A/great black-headed gull/Qinghai/2009 (H5N1) strain were provided by the Institute of Microbiology, Chinese Academy of Sciences.
  • Example 1 Animal experiment of cannabidiol anti-influenza virus H1N1
  • the 6-week-old Kunming White Male Mouse was purchased from Beijing Weitong Lihua Experimental Technology Co., Ltd.
  • the 9-11 day old SPF chicken embryo was purchased from Beijing Meriale Laboratory Animal Technology Co., Ltd.
  • the egg shell was disinfected with iodophor and 70% ethanol; the hole was punched 2-3 mm above the scribe line, and 200 ⁇ l of the PBS moderately diluted PBS was injected into the allantoic cavity of the chicken embryo; the small hole was sealed with wax;
  • the eggs were cultured at 37 ° C for 48h-72h, and the survival of the chicken embryos was observed every 24h. If the chicken embryos were observed to die, the chicken embryos were placed at 4 ° C overnight and the allantoic fluid was collected. By 72 h, the remaining chicken embryos were collected. The allantoic fluid was collected after overnight at 4 °C.
  • Method of collecting allantoic fluid use a sterile tweezers to break the eggshell above the egg chamber, then tear the peritoneal capsule above the air chamber, and slowly absorb the allantoic fluid with a 1 ml pipette.
  • the harvested allantoic fluid was centrifuged at 2000-3000 rpm for 10 min, and the supernatant was aspirated to obtain the virus. Store at -80 ° C for later use.
  • MDCK cells (Madin-Daby canine kidney cells, purchased from ATCC, USA) were cultured, and the confluence reached 95%.
  • the H1N1 strain of the virus amplified by the chicken embryo was infected and amplified, centrifuged, and collected. Clear, virus stock solution was obtained, and virus titer was measured.
  • the results of the preliminary pre-test of the present inventors showed that the Tamiflu group (20 mg/kg/d) and the two groups of CBD injection groups (20 mg/kg/d and 60 mg/kg/d) were able to significantly inhibit IAV infection.
  • Mortality of mice Inhalation of H1N1 (12000 pfu) caused 95% of mice to die, and the two groups of CBD injection groups (20 mg/kg/d and 60 mg/kg/d) inhibited the mortality of mice caused by IAV infection.
  • H5N1 (1000 pfu) was inoculated with nasal drops, which caused 95% of mice to die, and the two groups of CBD injection groups (20 mg/kg/d and 60 mg/kg/d) inhibited the mortality of mice caused by IAV infection.
  • the formal experimental group administration and model preparation are based on the H1N1 WSN virus.
  • Kunming white male rats were randomly divided into 4 groups, 20 mice in each group, and administered intraperitoneally once a day from the 4th day of infection for 5 consecutive days.
  • the blank group and the 12,000 pfu influenza group were injected with the same amount of normal saline.
  • the specific grouping is as follows:
  • Blank group wild type: no infection, the same amount of normal saline was injected on the fourth day.
  • Tamiflu group Dosing on the 4th day of infection (Duffy, 20 mg/kg/d).
  • CBD group administration on the 4th day of infection (Cannabinol, 20mg/kg/d)
  • mice After mildly anesthetizing the mice with diethyl ether, the mice were intranasally inoculated with H1N1 influenza virus solution (previously stored in -80 °C spare virus stock solution diluted 1:120 in PBS), 0.05 ml / each, blank group Drop the nose with the same amount of normal saline.
  • H1N1 influenza virus solution previously stored in -80 °C spare virus stock solution diluted 1:120 in PBS
  • mice The body weight and temperature of the mice at different times were recorded, and the symptoms of the mice were observed, and the time of death and the number of deaths of the mice were recorded. Observed until 14 days after infection.
  • H1N1 (WSN) infected mice Day of post infection (DPI 0) to 10 days after infection (DPI10), weighed daily, subtracted DPI 0 body weight from the day's weight, divided by DPI 0 body weight, calculated body weight Percent change.
  • mice were grasped by the correct operation method, and even if the abdomen was facing the operator, the electronic thermometer was used to sense the head vegetable oil, which was inserted straight into the anus of the mouse, and was measured when the probe just entered the anus completely. Mouse body temperature was measured every 24 hours.
  • mice The number of surviving mice in each group was observed and recorded every day.
  • mice in each group were tested for lung index and spleen index.
  • the mice were anesthetized with pentobarbital 150-200 ⁇ l, the body weight of the mice was measured, blood was taken from the eyeballs, and then the mice were fixed on an anatomical plate, and the mice were dissected along the midline of the abdomen, and the hearts were removed.
  • the lung index and inhibition rate are calculated according to the following formula:
  • the lung tissue was immersed in 10% formaldehyde and then immersed in a concentration gradient of alcohol for 2-4 h to remove the water in the tissue block, and placed in the clearing agent xylene for 0.5-2 h.
  • the transparent tissue block was embedded in paraffin and fixed on a microtome and cut into thin slices (about 4-5 ⁇ m thick).
  • the paraffin in the section was removed with xylene, stained with hematoxylin and eosin (HE), and the pathological changes of lung tissue were observed under an optical microscope Olympus CX 41 (Olympus, Japan).
  • mice in each group were tested for permeability of pulmonary blood vessels.
  • the mice were anesthetized, and the Evan blue solution was injected through the tail vein. After 5 minutes, the alveolar perfusate was taken, centrifuged at 1500 rpm for 5 min, and the supernatant was taken. The absorbance OD at 590 nm was measured by a spectrophotometer.
  • mice per group were tested for the number of inflammatory cells in the alveolar perfusate.
  • the CBD group (20 mg/kg/d) significantly reduced the number of neutrophils in the alveolar perfusate of mice induced by H1N1 WSN infection.
  • mice were anesthetized, the eyes were bled or the heart was taken.
  • the supernatant was transferred to a new 10 mL centrifuge tube at -80 ° C for cryopreservation.
  • the cell pellet was resuspended in 450 ⁇ l of ddH 2 O and gently shaken for no more than 1 min.
  • the red blood cells were rapidly lysed and immediately added 50 ⁇ L of 10 ⁇ PBS.
  • the supernatant was centrifuged, the cells were resuspended in serum, and smeared.
  • Diff-Quick staining The smear was wet (ie, not dried) and immersed in ethanol ether solution for 15 s. The fixing solution at the edge was removed, and Diff-Quick I and Diff-Quick II solutions were inserted for 15 s each time, and the excess dye was washed away with running water.
  • SPSS 12.0.1 data processing software SPSS Inc., Chicago, IL was used for one-way analysis of variance. The data was in accordance with the normal distribution, and the significance test was performed by t test. Data are expressed as mean ⁇ standard error, p ⁇ 0.05 The difference was significant; p ⁇ 0.01 was the most significant difference.
  • the CBD injection group can significantly inhibit the weight loss caused by H1N1
  • mice There was a significant difference in body temperature changes in DPI 7-9 mice, with the greatest change in DPI 7-8. It can be seen from the figure that the mice began to have a significant decrease in body temperature on the 4th day after infection with IAV, reached the lowest point on the 7th to 8th day, and then began to rise, and the body temperature of the individual who did not die for 7 days began to rise significantly. Compared with the high toxicity group, the Tamiflu group (20 mg/kg/d) and the CBD group (20 mg/kg/d) significantly inhibited the decrease in body temperature.
  • the CBD group can significantly reduce the flu symptoms and behavioral discomfort in the H1N1 infection group.
  • CBD injection group can significantly reduce the mortality rate of H1N1 infection group
  • the Tamiflu group (20 mg/kg/d) and the CBD group (20 mg/kg/d) significantly reduced the mortality of mice caused by H1N1 WSN infection (Duffy group, P ⁇ 0.001; CBD) Group, P ⁇ 0.001).
  • Duffy group P ⁇ 0.001; CBD
  • the CBD group showed very obvious therapeutic effects. There were no significant differences in mortality between the Tamiflu group (9/20) and the CBD group (10/20).
  • CBD group can significantly reduce lung injury in H1N1 infection group
  • the Tamiflu group (20 mg/kg/d) and the CBD group (20 mg/kg/d) significantly reduced the lung injury index of mice induced by H1N1 WSN infection (Duffy group, P ⁇ 0.05; CBD) Group, P ⁇ 0.05), CBD group It shows a very obvious therapeutic effect. There were no significant differences in lung index between the Tamiflu group (9/20) and the CBD group (10/20).
  • the Tamiflu and CBD treatment groups significantly reduced the number of inflammatory cell invasiveness.
  • CBD group can significantly reduce the permeability of pulmonary blood vessels in H1N1 infected group
  • the Tamiflu group (20 mg/kg/d) and the CBD group (20 mg/kg/d) significantly reduced the permeability of the lung capillary of mice induced by H1N1 WSN infection and reduced lung damage.
  • Tamiflu group *P ⁇ 0.05; CBD group, **P ⁇ 0.01.
  • the CBD group can significantly reduce the number of inflammatory cells in the alveolar perfusate of the H1N1 infected group.
  • CBD group P ⁇ 0.001.
  • the CBD group showed a very significant reduction in the number of neutrophils in the alveolar perfusate.
  • CBD can significantly reduce the flu symptoms and discomfort in mice caused by H1N1WSN infection; it can significantly inhibit the decrease of body weight and hypothermia in mice; and reduce the mortality of mice caused by influenza. CBD also reduces the permeability of pulmonary blood vessels, reduces the infiltration of inflammatory cells (neutrophils, lymphocytes, macrophages) in the lung tissue of influenza-infected mice, and reduces ALI in acute lung injury.
  • Example 2 In vitro experiment of cannabinol inhibiting RNA polymerase of influenza A virus H1N1
  • BMDM mouse bone marrow-derived macrophage
  • CCL-185 TM human non-small cell lung cancer cell line A549
  • control group high-sugar medium DMEM, the manufacturer is Sigma, the article number is D5648-1L, diluted to 1 liter with purified water before the experiment),
  • CBD group (5 ⁇ M)
  • the supernatant was aspirated, and each group was added with the same volume of serum-free medium containing PBS, Tamiflu and CBD, respectively, wherein each serum-free medium contained the same concentration of PBS, Tamiflu and CBD; Incubate for 24 h at 37 ° C in a CO 2 incubator.
  • RNA was extracted, reverse transcribed, and qRT-PCR was used to detect the expression of viral mRNA.
  • the NP, PA, PB1 and PB2 gene expressions of H1N1 were detected, and the RT-PCR primers were as follows:
  • CBD inhibits the expression of influenza virus RNA-dependent RNA polymerase, which may inhibit the replication of IAV in host cells and reduce influenza infection.
  • the present invention provides a potential broad spectrum anti-influenza virus drug.
  • Example 1 Consistent with the conditions of Example 1. Animals were routinely raised at the P2 and P3 animal centers of the Institute of Microbiology, Chinese Academy of Sciences (Biosafety level 2 and 3, P2/P3). The photoperiod was 12 hours light/12 hours dark, and the animals were free to eat and free to drink. Animals were acclimated to the new environment for 1 day and related experiments were performed. China Agricultural University and Agricultural Biotechnology Animal experiments were approved by the National Key Laboratory Animal Ethics Committee (SKLAB-2017-3-002).
  • C57BL/6 male rats were randomly divided into 2 groups, 10 mice in each group, and administered intraperitoneally once a day for 5 days from the fourth day of infection.
  • the 1000 pfu influenza group was injected with the same amount of normal saline.
  • the specific grouping is as follows:
  • Influenza group H5N1 1000 pfu: The same amount of normal saline was injected on the fourth day of infection.
  • CBD group H5N1 1000 pfu infection was started on the 4th day (Cannabinol, 20 mg/kg/d).
  • the amplification method and titer detection method of the H5N1 virus were identical to those of Example 1, except that the virus subtype was H5N1.
  • mice The observation and recording of the physiological characteristics and onset symptoms of the mice were identical to those of Example 1.
  • H5N1 infected mice Day of post infection (DPI 0) to 11 days after infection (DPI 10), weighed daily, the body weight minus DPI 0 body weight, divided by DPI 0 body weight, calculate the percentage change in body weight .
  • H5N1 infected with 1000pfu dose, monitored for changes in body temperature
  • mice On the 9th day of the venom (DPI), the activity status of the mice was recorded, detected and quantified. The changes in the behavior of the mice were divided into main movements and modification actions (standing, sitting, standing up, walking, lying down). , head rotation, watching, smelling, washing your face, etc.) See the Experimental Materials and Methods section for details. Take a representative activity activity within 1 minute and count.
  • the CBD group can significantly inhibit the weight loss caused by H5N1
  • the CBD group can significantly inhibit the hypothermia caused by H5N1
  • the CBD group can significantly reduce the flu symptoms and behavioral discomfort in the H5N1 infection group.
  • CBD group can significantly prolong the survival time of H5N1 infection group
  • Example 4 In vitro experiment of cannabinol inhibiting RNA polymerase of influenza A virus H5N1
  • control group high-sugar medium DMEM, the manufacturer is Sigma, the article number is D5648-1L, diluted to 1 liter with purified water before the experiment),
  • the supernatant was aspirated, and each group was added with the same volume of serum-free medium containing PBS, Tamiflu and CBD, respectively, wherein each serum-free medium contained the same concentration of PBS, Tamiflu and CBD; Incubate for 24 h at 37 ° C in a CO 2 incubator.
  • RNA was extracted, reverse transcribed, and qRT-PCR was used to detect the expression of viral mRNA.
  • the expression of NP, PA, PB1 and PB2 genes of H5N1 was detected, and the RT-PCR primers were as follows:
  • CBD inhibits the expression of influenza virus RNA-dependent RNA polymerase, which may inhibit the replication of IAV in host cells and reduce influenza infection.
  • the present invention provides a potential broad spectrum anti-influenza virus drug.

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Abstract

本发明属于生物医药领域，涉及大麻二酚在制备抗流感的药物中的用途。具体地，本发明涉及选自如下的(1)－(3)中的任一项在制备治疗或预防流感的药物或者缓解流感症状的药物中的用途：(1)大麻二酚或其药学上可接受的盐或酯；(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。大麻二酚能够有效地抑制流感病毒，具有制备或者作为治疗或预防流感的药物的潜力。

Description

大麻二酚在制备抗流感的药物中的用途 技术领域
本发明属于生物医药领域，涉及大麻二酚在制备抗流感的药物中的用途。
背景技术
流行性感冒简称流感(Influenza)，是由流感病毒(Influenza Virus)引起的一种人、禽、畜共患的急性呼吸道传染病，它因传播速度快、发病率高和严重并发症而成为严重威胁人类健康的疾病。
流感病毒是经急性呼吸道传播的流感致病因子，属于正粘病毒科，单股负链RNA病毒。根据其核蛋白(Nucleoprotein，NP)和基质蛋白(Matrix protein，M)抗原性的不同，可将流感病毒分为甲(A)、乙(B)、丙(C)三型(Hay et al.,2001)。其中甲型流感病毒(Influenza A Virus，IAV)最为常见，对人类威胁最大，可引起流感季节性和地区性爆发。除感染人外，还可引起禽、猪、马等多种动物感染。
IAV为单股负链8节段的RNA病毒，每个RNA片段均由核蛋白(Nucleoprotein，NP)包被形成核蛋白复合体(Ribonucleoprotein，RNPs)。IAV基因组至少编码13种蛋白质，分别为PB2、PB1、PB1-F2、N40、PA、PA-X、HA、NP、NA、M1、M2、NS1和NS2。IAV囊膜表面分布着3种蛋白质，分别是HA、NA和M2。IAV的8个RNA片段都由NP包被。病毒粒子中，病毒RNA被NP和聚合酶的3个亚基即Polymerase 1(PB1)、Polymerase 2(PB2)和Polymerase A(PA)包被成RNPs。PB1发挥RNA依赖的RNA聚合酶功能。PB2结合并剪切宿主mRNA帽子作为引物合成病毒RNA。PA可以行使宿主RNA核酸内切酶功能，还可能具有蛋白酶水解活性。NP行使单链RNA结合蛋白的功能，并在RNPs中充当结构蛋白。
在IAV基因组中，编码PA、PBl、PB2和NP蛋白的基因在进化上具有高度的保守性，而IAV的抗原性极易发生变异。因此，根据其所携带的外膜蛋白血凝素抗原(Hemagglutinin，HA)和神经氨酸酶(Neuraminidase，NA)的不同，可将其分成若干亚型，目前已发现了16种HA亚型和10种NA亚型，它们不断变异，发生排列组合，实现自我进化，也不断带给人类新的威胁。
其中仅H1N1、H2N2、H3N2主要感染人类，其它许多亚型的自然宿主是多种禽类和动物。其中对禽类危害最大的为H5、H7和H9亚型毒株。具有高致病性的H5NI、 H7N9、H9N2等禽流感病毒，一旦变异而具有人与人的传播能力，会导致人间禽流感流行，预示着禽流感病毒对人类已具有很大的潜在威胁。
IAV进入细胞后，其基因组需要依赖其RNA聚合酶(RNA dependent RNA polymerase，RdRP)的作用进行复制和转录，进而翻译成具有感染性的病毒颗粒。如果该聚合酶失去活性，病毒便无法进行复制转录以生成完整的病毒颗粒。
临床研究结果显示，IAV潜伏期一般为3至4天，最长可达7天。患者一般表现为发热、咳嗽、少痰，可伴有头痛、肌肉酸痛、腹泻等全身症状。该病如不及时治疗，病情发展迅速，重症患者主要表现为重症急性肺损伤(Acute lung injury,ALI)，可引致严重的并发症，例如急性肺炎、支气管炎、充血性心力衰竭、肠胃炎、晕厥、出现幻觉等，严重者可导致死亡。ALI是指严重感染后，出现以肺泡毛细血管损伤导致的肺水肿和微肺不张等病理特征。急性呼吸窘迫综合征(Acute respiratory distress syndrome,ARDS)是一种严重的ALI(Bernard et al.,1987)，此外还能诱发全身的炎症反应综合征(Systemic inflammatory response syndrome，SIRS)。由于ARDS病死率高达40％－50％，这也是IAV致死的一个重要原因。
目前，经世界各国批准上市的抗流感病毒药主要有：肌苷单磷酸脱氢酶(IMPDH)抑制药利巴韦林(ribavirin)、干扰素诱导剂盐酸阿比朵尔(arbidol hydrochloride)、M2离子通道蛋白抑制药盐酸金刚烷胺(amantadine hydrochloride)和盐酸金刚乙胺(rimantadine hydrochloride)、神经氨酸酶抑制药磷酸奥司他韦(osehamivir phosphate)和扎那米韦(zanamivir)4大类6个品种(Glezen,2006；Kolocouris et al.,1996)。
M2离子通道阻断剂，包括金刚烷胺和金刚乙胺，它们通过阻断M2离子通道蛋白来阻止病毒脱壳，使病毒RNA不能释放到细胞质中，病毒的早期复制被中断，从而起到抗流感病毒的作用。但金刚烷胺类药物对B型流感无效，而且存在神经毒性、在服用后数小时，可出现失眠、注意力分散和神经质等副作用，且金刚烷胺类药物在实验条件下和临床应用时易产生抗药性毒株。但目前大多数流感病毒株已经对这两个药物耐药，而且只有A型流感病毒才有M2离子通道蛋白，因而其临床使用并不广泛。
NA抑制剂，目前上市的只有扎那米韦和奥塞米韦(Oseltamivir，达菲)两个药物，另外一个静脉注射剂型的帕拉米韦(peramivir)于2010年1月在日本获批。它们通过抑制NA活性，使感染细胞表面的唾液酸不能被切割，导致新生病毒不能从感染细胞表面释放，从而防止病毒进一步感染其它细胞。但即使应用达菲，H5N1型禽 流感的病人也具有很高的死亡率，对达菲具有耐药性的病毒株也不断被分离到。目前流行的IAV已经出现了对于神经氨酸酶抑制剂的耐药性。此外，达菲可引起突发呼吸困难等严重不良反应，例如，日本非盈利机构“医药警戒中心”的一项研究发现，服用了达菲的119名死亡患者中，有38人在服药12小时内出现重症状态或死亡。
大麻二酚(Cannabidiol,CBD)是大麻素类物质中的一种，其结构式如下面的式I所示：
Figure PCTCN2017099956-appb-000001
大麻二酚提取于天然植物大麻，无精神效应，对焦虑、抑郁、惊厥和肿瘤等均具有治疗作用。有研究表明，CBD具有良好的抗炎效果。在2000年，《美国科学院学报》(Proceedings of the National Academy of Sciences)报道，英国伦敦肯尼迪风湿病研究所的M·费尔德曼博士等发现，口服和全身使用CBD都可明显降低关节损坏的严重性以及关节炎的急性和慢性病程。在2015年的《Immunopharmacol Immunotoxicology》杂志报道，CBD抑制LPS诱导的急性肺损伤(Ribeiro et al.,2015)。
因此目前，亟需研发成新的抗流感病毒药物。
发明内容
本发明人经过深入的研究和创造性的劳动，惊奇地发现，大麻二酚能够有效地抑制流感病毒以及流感病毒的RNA聚合酶，具有防治流感的潜力。由此提供了下述发明：
本发明的一个方面涉及选自如下的(1)－(3)中的任一项在制备治疗或预防流感的药物或者缓解流感症状的药物中的用途：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一 种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感由选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种流感病毒引起；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
在本发明的一个实施方案中，所述流感的患病对象为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
在本发明的一个实施方案中，所述流感症状为流感引起的选自如下症状中的至少一种：
发热、咳嗽、头痛、肌肉酸痛以及腹泻。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶(IMPDH)抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林(ribavirin)；
优选地，所述干扰素诱导剂为盐酸阿比朵尔(arbidol hydrochloride)；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺(amantadine hydrochloride)或盐酸金刚乙胺(rimantadine hydrochloride)；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦(osehamivir phosphate)、奥塞米韦(Oseltamivir，达菲)、扎那米韦(zanamivir)或帕拉米韦(peramivir)。
本发明的另一方面涉及选自如下的(1)－(3)中的任一项在制备抗流感病毒(例如抑制流感病毒在宿主细胞中的复制)的药物中的用途：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感病毒选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
在本发明的一个实施方案中，所述宿主细胞为哺乳动物(例如人、猿、猴、猪、牛或羊)的细胞或者禽类(例如例如家禽如鸡、鸭或鹅，或者例如野生禽类)的细胞。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明的再一方面涉及选自如下的(1)－(3)中的任一项在制备抑制流感病毒RNA聚合酶的复制的药物、抑制流感病毒RNA聚合酶的表达水平的药物或者抑制流感病毒RNA聚合酶的活性的药物中的用途：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感病毒RNA聚合酶选自甲型流感病毒RNA聚合酶、乙型流感病毒RNA聚合酶和丙型流感病毒RNA聚合酶中的一种或多种；优选地，所述甲型流感病毒RNA聚合酶为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒的RNA聚合酶。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
在本发明的一个实施方案中，所述抑制流感病毒RNA聚合酶的表达水平是指，例如流感病毒RNA聚合酶的蛋白水平或编码流感病毒RNA聚合酶的mRNA水平。
在本发明的一个实施方案中，所述流感病毒RNA聚合酶的表达水平，可以通过流感病毒RNA聚合酶的3个亚基PB1、PB2和PA中的任意一个或者多个的表达水平来确定。
本发明的再一方面涉及一种药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述的药物组合物中，大麻二酚是唯一活性成分。在本发明的另一个实施方案中，大麻二酚与一种或多种其它已知的用于防治流感的活性成分联用。
在本发明的一个实施方案中，所述的药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
所述药物组合物的制剂形式可以是任何可药用的剂型，这些剂型包括：片剂、糖衣片剂、薄膜衣片剂、肠溶衣片剂、胶囊剂、硬胶囊剂、软胶囊剂、口服液、口含剂、颗粒剂、冲剂、丸剂、散剂、膏剂、丹剂、混悬剂、粉剂、溶液剂、注射剂、栓剂、软膏剂、硬膏剂、霜剂、喷雾剂、滴剂、贴剂；优选口服剂型，如：胶囊剂、片剂、口服液、颗粒剂、丸剂、散剂、丹剂、膏剂等。所述的口服剂型可含有常用的赋形剂，诸如粘合剂、填充剂、稀释剂、压片剂、润滑剂、崩解剂、着色剂、调味剂和湿润剂，必要时可对片剂进行包衣。适宜的填充剂包括纤维素、甘露糖醇、乳糖和其它类似的 填充剂；适宜的崩解剂包括淀粉、聚乙烯吡咯烷酮和淀粉衍生物，例如羟基乙酸淀粉钠；适宜的润滑剂包括，例如硬脂酸镁。适宜的药物可接受的湿润剂包括十二烷基硫酸钠。
优选地，所述药物组合物为口服制剂。
优选的给予受试者的大麻二酚的剂量介于0.1－50mg/kg体重/天，更优选为0.5mg/kg－30mg/kg体重/天、0.5mg/kg－20mg/kg体重/天、5mg/kg－30mg/kg体重/天或者5mg/kg－20mg/kg体重/天，进一步优选为0.5mg/kg－10mg/kg体重/天，特别优选为0.5mg/kg－5mg/kg体重/天。
本发明的再一方面涉及一种组合产品，其包含独立包装的产品1和产品2，
其中，
所述产品1选自如下的(1)－(3)中的任意一种：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；和
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料；
所述产品2包含有效量的选自如下的一种或者多种成分：肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明还涉及如下方面：
本发明涉及选自如下的(1)－(3)中的任一项的产品，其用于治疗或预防流感或者缓解流感症状：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选 地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感由选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种流感病毒引起；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
在本发明的一个实施方案中，所述流感的患病对象为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
在本发明的一个实施方案中，所述流感症状为流感引起的选自如下症状中的至少一种：
发热、咳嗽、头痛、肌肉酸痛以及腹泻。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明还涉及选自如下的(1)－(3)中的任一项的产品，其用于抗流感病毒(例如抑制流感病毒在宿主细胞中的复制)：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感病毒选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、 H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
在本发明的一个实施方案中，所述宿主细胞为哺乳动物(例如人、猿、猴、猪、牛或羊)的细胞或者禽类(例如例如家禽如鸡、鸭或鹅，或者例如野生禽类)的细胞。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明还涉及选自如下的(1)－(3)中的任一项的产品，其用于抑制流感病毒RNA聚合酶的复制、抑制流感病毒RNA聚合酶的表达水平或者抑制流感病毒RNA聚合酶的活性：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感病毒RNA聚合酶选自甲型流感病毒RNA聚合酶、乙型流感病毒RNA聚合酶和丙型流感病毒RNA聚合酶中的一种或多种；优选地，所述甲型流感病毒RNA聚合酶为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒的RNA聚合酶。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明还涉及一种治疗或预防流感或者缓解流感症状的方法，包括给予有需求的受试者以有效量的选自如下的(1)－(3)中的任一项的产品的步骤：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感由选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种流感病毒引起；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
在本发明的一个实施方案中，所述受试者为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
在本发明的一个实施方案中，所述流感症状为流感引起的选自如下症状中的至少一种：
发热、咳嗽、头痛、肌肉酸痛以及腹泻。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明还涉及一种抗流感病毒(例如抑制流感病毒在宿主细胞中的复制)的方法，包括给予有需求的受试者以有效量的选自如下的(1)－(3)中的任一项的产品的步骤：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感病毒选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
在本发明的一个实施方案中，所述受试者为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
在本发明的一个实施方案中，所述宿主细胞为哺乳动物(例如人、猿、猴、猪、牛或羊)的细胞或者禽类(例如例如家禽如鸡、鸭或鹅，或者例如野生禽类)的细胞。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
本发明还涉及一种抑制流感病毒RNA聚合酶的复制、抑制流感病毒RNA聚合酶的表达水平或者抑制流感病毒RNA聚合酶的活性的方法，包括给予有需求的受试者以有效量的选自如下的(1)－(3)中的任一项的产品的步骤：
(1)大麻二酚或其药学上可接受的盐或酯；
(2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选 地，为含有大麻二酚的工业大麻提取物；
(3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
在本发明的一个实施方案中，所述流感病毒RNA聚合酶选自甲型流感病毒RNA聚合酶、乙型流感病毒RNA聚合酶和丙型流感病毒RNA聚合酶中的一种或多种；优选地，所述甲型流感病毒RNA聚合酶为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒的RNA聚合酶。
在本发明的一个实施方案中，所述受试者为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
在本发明的一个实施方案中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
优选地，所述干扰素诱导剂为盐酸阿比朵尔；
优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
需要指出的是，活性成分大麻二酚的使用剂量和使用方法取决于诸多因素，包括患者的年龄、体重、性别、自然健康状况、营养状况、化合物的活性强度、服用时间、代谢速率、病症的严重程度以及诊治医师的主观判断。根据本发明中任一项所述的方法，其中，大麻二酚的剂量介于0.1－50mg/kg体重/天，更优选为0.5mg/kg－30mg/kg体重/天、0.5mg/kg－20mg/kg体重/天、5mg/kg－30mg/kg体重/天或者5mg/kg－20mg/kg体重/天，进一步优选为0.5mg/kg－10mg/kg体重/天，特别优选为0.5mg/kg－5mg/kg体重/天。
根据本发明中任一项所述的方法，其中，通过口服给药。
本发明中，
所述大麻二酚即式I化合物可通过商业途径购买(例如购自Sigma等)或者利用市售原料，通过现有技术合成。合成后可以通过柱色谱法、液液萃取法、分子蒸馏方 法或结晶等方式进一步纯化。此外，大麻二酚还可以从大麻，尤其是工业大麻中提取得到。
大麻二酚的药学上可接受的盐，包括但不限于：有机铵盐、碱金属盐(钠盐、钾盐)、碱土金属盐(镁盐、锶盐、钙盐)等。
在本发明的一些实施方式中，大麻二酚的药学上可接受的盐可以为大麻二酚(CBD)与氢氧化钠、氢氧化钾、氢氧化钙、氢氧化镁、氢氧化铝、氢氧化锂、氢氧化锌、氢氧化钡、氨、甲胺、二甲胺、二乙胺、甲基吡啶、乙醇胺、二乙醇胺、三乙醇胺、乙二胺、赖氨酸、精氨酸、鸟氨酸、胆碱、N，N′-二苯甲基乙二胺、氯普鲁卡因、二乙醇胺、普鲁卡因、N-苯甲基苯乙胺、N-甲基葡糖胺哌嗪、三(羟基甲基)-氨基甲烷等形成的盐。
在本发明的一些实施方式中，大麻二酚药学上可接受的酯可以为大麻二酚与一个C0-C6烷基羧酸形成的单酯，也可以为大麻二酚与两个相同或不同的C0-C6烷基羧酸形成的二酯，所述的C0-C6烷基羧酸可以是直链烷基羧酸、支链烷基羧酸或环烷基羧酸，例如HCOOH、CH3COOH、CH3CH2COOH、CH3(CH2)2COOH、CH3(CH2)3COOH、CH3(CH2)4COOH、(CH3)2CHCOOH、(CH3)3CCOOH、(CH3)2CHCH2COOH、(CH3)2CH(CH2)2COOH、(CH3)2CH(CH3)CHCOOH、(CH3)3CCH2COOH、CH3CH2(CH3)2CCOOH、环丙烷羧酸、环丁烷羧酸、环戊烷羧酸。
所述的大麻提取物，可以是含有大麻二酚的大麻，尤其是工业大麻的提取物，例如乙醇提取液、浸膏等。其中，大麻二酚的含量并不特别限定，而且可以通过本领域技术人员知悉的手段例如浓缩等来进一步提高大麻提取物中大麻二酚的含量。在本发明的一个实施方案中，所述的大麻提取物，其为浸膏，优选地，其中大麻二酚的含量为18％－25％。
在本发明的一些实施方式中，所述大麻提取物为以选自大麻的茎、叶、果实、果壳、根和花中的任意一种或者多种为原料得到的提取物。优选地，所述大麻提取物为大麻叶提取物。
本发明中，术语“有效量”是指可在受试者中实现治疗、预防、减轻和/或缓解本发明所述疾病或病症的剂量。
术语“受试者”可以指患者或者其它接受本发明组合物以治疗、预防、减轻和/或缓解本发明所述疾病或病症的动物，特别是哺乳动物，例如人、狗、猴、牛、马等。
术语“疾病和/或病症”是指所述受试者的一种身体状态，该身体状态与本发明所述 疾病和/或病症有关。
本发明中，如果没有特别说明，所述产品1、产品2仅仅是为了指代清楚，并不具有次序的含义。
本发明中，如果没有特别说明，大麻优选为工业大麻；大麻提取物优选为工业大麻提取物。
发明的有益效果
大麻二酚能够有效地抑制流感病毒，具有制备或者作为治疗或预防流感的药物的潜力。
附图说明
图1：WSN感染的小鼠体重变化。小鼠感染WSN后，连续监测体重11天。数据以平均值±标准误差表示。每个点代表小鼠体重变化当天平均值，(n＝20/组)。使用post hoc方法对差异进行显著性分析，显著性差异以*表示，**，p<0.01。
图2：WSN感染的小鼠体温变化。小鼠感染WSN后，连续监测体温12天。数据以平均值±标准误差表示。每个点代表小鼠体重变化当天平均值，(n＝15－20/组)。使用post hoc方法对差异进行显著性分析，显著性差异以*表示，*p<0.05。
图3：WSN感染的小鼠行为变化。小鼠感染WSN后，连续监测行为10天。数据以平均值±标准误差表示(n＝5/组)。使用post hoc方法对差异进行显著性分析，显著性差异以*表示，**p<0.01，***p<0.001。图3A，H1N1感染后第6天。图3B，H1N1感染后第8天。
图4：WSN感染的小鼠死亡率的变化。小鼠感染WSN后，连续监测存活数12天。数据以平均值±标准误差表示。每个点代表小鼠存活数，(n＝20/组)。使用post hoc方法对差异进行显著性分析。
图5：WSN感染的小鼠肺指数的变化。取感染第8天的鼠，检测肺部感染情况。数据以平均值±标准误表示，*p<0.05。
图6：WSN感染的小鼠肺病理的变化。取感染第8天的鼠，检测肺病理分析情况。图6A，野生对照；图6B，流感感染的肺对照；图6C，达菲处理组；图6D，CBD处理组。
图7：WSN感染的小鼠肺指数的变化。取感染第8天的鼠，检测肺部血管通透性 检测。数据以平均值±标准误表示，*p<0.05；**p<0.01。
图8：WSN感染的小鼠肺泡灌注液中中性粒细胞的变化。取DPI8天的鼠，检测肺泡灌注液中性粒细胞的数目。数据以平均值±标准误表示，***p<0.001。
图9：WSN感染的小鼠肺泡灌注液中淋巴细胞的变化。取DPI8天的鼠，检测肺泡灌注液中淋巴细胞数目。数据以平均值±标准误表示，**p<0.01。
图10：WSN感染的小鼠肺泡灌注液中巨噬细胞的变化。取DPI8天的鼠，检测肺泡灌注液中巨噬细胞数目。数据以平均值±标准误表示，**p<0.01,***p<0.001。
图11A：CBD处理的WSN感染的小鼠BMDM细胞中PA基因的表达变化。CBD处理的感染H1N1的小鼠BMDM细胞24h之后，PB1基因的表达变化。数据以平均值±标准误表示，*p<0.05。
图11B：CBD处理的WSN感染的小鼠BMDM细胞中PB1基因的表达变化。CBD处理的感染H1N1的小鼠BMDM细胞24h之后，PB1基因的表达变化。数据以平均值±标准误表示，*p<0.05。
图12A：CBD处理的H1N1感染的人肺上皮细胞系A 549中NP基因的表达变化。CBD处理的感染H1N1的人肺上皮细胞系A 549 24h之后，NP基因的表达变化。数据以平均值±标准误表示，***p<0.001。
图12B：CBD处理的H1N1感染的人肺上皮细胞系A 549中PA基因的表达变化。CBD处理的感染H1N1的人肺上皮细胞系A 549 24h之后，PA基因的表达变化。数据以平均值±标准误表示，**p<0.01。
图12C：CBD处理的H1N1感染的人肺上皮细胞系A 549中PB1基因的表达变化。CBD处理的感染H1N1的人肺上皮细胞系A 549 24h之后，PB1基因的表达变化。数据以平均值±标准误表示，**p<0.01。
图12D：CBD处理的H1N1感染的人肺上皮细胞系A 549中PB2基因的表达变化。CBD处理的感染H1N1的人肺上皮细胞系A 549 24h之后，PB2基因的表达变化。数据以平均值±标准误表示，**p<0.01。
图13：H5N1感染的小鼠体重变化。小鼠感染H5N1后，连续监测体重12天。数据以平均值(n＝10/组)。
图14：H5N1感染的小鼠体温变化。小鼠感染H5N1后，连续监测体温11天。数据以平均值±标准误差表示(n＝10/组)。
图15：H5N1感染的小鼠行为变化。小鼠感染IAV后，监测第9天行为。数据以 平均值±标准误差表示(n＝5/组)。使用post hoc方法对差异进行显著性分析，显著性差异以*表示，***p<0.001。
图16：H5N1感染的小鼠死亡率。
图17A：CBD处理的H5N1感染的人肺上皮细胞系A 549中NP基因的表达变化。CBD处理的感染H5N1的人肺上皮细胞系A 549 24h之后，NP基因的表达变化。数据以平均值±标准误表示，*p<0.05。
图17B：CBD处理的H5N1感染的人肺上皮细胞系A 549中PA基因的表达变化。CBD处理的感染H5N1的人肺上皮细胞系A 549 24h之后，PA基因的表达变化。数据以平均值±标准误表示，*p<0.05。
图17C：CBD处理的H5N1感染的人肺上皮细胞系A 549中PB1基因的表达变化。CBD处理的感染H5N1的人肺上皮细胞系A 549 24h之后，PB1基因的表达变化。数据以平均值±标准误表示，*p<0.05,**p<0.01。
图17D：CBD处理的H5N1感染的人肺上皮细胞系A 549中PB2基因的表达变化。CBD处理的感染H5N1的人肺上皮细胞系A 549 24h之后，PB2基因的表达变化。数据以平均值±标准误表示，*p<0.05，**p<0.01。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是本领域技术人员将会理解，下列实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体条件者，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
下面的实施例中，如果没有特别说明：
IAV的H1N1WSN(A/WSN/33)毒株和H5N1A/great black-headed gull/Qinghai/2009(H5N1)毒株由中国科学院微生物研究所提供。
涉及H1N1的实验均在生物安全二级实验室内进行，涉及H5N1的实验均在生物安全三级实验室内进行。
实施例1：大麻二酚抗甲型流感病毒H1N1的动物实验
1.实验动物和前期准备
6周龄的昆明白雄鼠，购自北京维通利华实验技术有限公司。
动物常规饲养于中国科学院微生物研究所P2和P3动物中心(Biosafety level 2and3,P2/P3)，光照周期为12小时光照/12小时黑暗，动物自由采食，自由饮水。动物适应新环境1天后进行相关实验。中国农业大学和农业生物技术国家重点实验室动物伦理委员会(SKLAB-2017-3-002)批准了动物实验。
9－11日龄SPF鸡胚购于北京梅里亚维实验动物技术有限公司。
2.实验方法
(1)鸡胚扩增H1N1流感病毒
9－11日龄SPF级鸡胚；照蛋，划出气室位置，并在血管较少处划线；
用碘伏和70％乙醇将鸡蛋外壳进行消毒；在划线处2－3mm上方打孔，取PBS适度稀释的流感病毒200μl注入到鸡胚的尿囊腔；用蜡将所打小孔封口；
将鸡蛋在37℃下培养48h－72h，每24h观察一次鸡胚存活情况，若观察到鸡胚死亡，将鸡胚置于4℃过夜后收集尿囊液，到72h时，将剩余鸡胚均置于4℃过夜后收集尿囊液。
收集尿囊液方法：用无菌的镊子将鸡蛋气室上方的蛋壳敲碎，再撕开气室上方的尿囊膜，用1ml的移液器慢慢吸取尿囊液。
将收获的尿囊液以2000－3000rpm离心10min，吸取上清，即可收获病毒。保存于-80℃备用。
(2)MDCK细胞系进行H1N1流感病毒滴度检测
培养MDCK细胞(Madin-Daby canine kidney cell，犬肾细胞，购自美国ATCC)，待汇合度达到95％，使用上面鸡胚扩增的病毒的H1N1毒株感染并进行扩增，离心，收集上清，得到病毒原液，进行病毒滴度测定。
(3)动物分组
本发明人前期的6次预试验的结果表明，达菲组(20mg/kg/d)和两组CBD注射液组(20mg/kg/d和60mg/kg/d)能够显著地抑制IAV感染引起的小鼠的死亡率。H1N1(12000pfu)滴鼻染毒，能够引起95％的小鼠死亡，且两组CBD注射液组(20mg/kg/d和60mg/kg/d)抑制IAV感染引起的小鼠的死亡率相同。H5N1(1000pfu)滴鼻染毒，能够引起95％的小鼠死亡，且两组CBD注射液组(20mg/kg/d和60mg/kg/d)抑制IAV感染引起的小鼠的死亡率相同。
因此，为了安全起见，正式实验分组给药及模型制备以H1N1WSN病毒为感染株， 将昆明白雄鼠随机分为4组，每组20只小鼠，从感染第4天起，每天腹腔注射给药1次，连续给药5天。空白组、12000pfu流感组用等量生理盐水注射。具体分组如下：
空白组(野生型)：不感染，第4天开始注射等量生理盐水。
12000pfu流感组(高毒组)：感染第4天开始注射等量生理盐水。
达菲组：感染第4天开始给药(达菲，20mg/kg/d)。
CBD组：感染第4天开始给药(大麻二酚，20mg/kg/d)
感染操作步骤：以乙醚轻度麻醉小鼠后，以H1N1流感病毒液(前面保存于-80℃备用的病毒原液按照1∶120稀释于PBS)滴鼻感染小鼠，0.05ml/只，空白组以等量生理盐水滴鼻。
(4)小鼠生理特征和发病症状的观察和记录
记录不同时间的小鼠体重、温度、观察小鼠发病症状，记录小鼠死亡时间和死亡数。观察至感染后14d。
1)体重的测量：在电子称上放一个合适的烧杯，将读数归零，将小鼠放入其中，待读数稳定时记录。小鼠体重每24小时称一次。
H1N1(WSN)感染小鼠当天(Day of post infection,DPI 0)至感染后10天(DPI10)，每天进行称重，以当天体重减去DPI 0体重，再除以DPI 0体重，计算出体重变化百分数。
2)体温的测量：H1N1引起的体温降低
同时，另外设置8000pfu流感组，仅用于体温的测量。WSN以8000pfu剂量感染，监测体温的变化。感染小鼠每组5只，DPI 0至DPI 12，每天进行肛温测量，以当天肛温减去DPI0肛温，再除以DPI0肛温，计算出体温变化百分数，
用正确的操作方法抓取小鼠，即使其腹部正对操作者，将电子体温计感应头蘸植物油，由小鼠肛门径直插入，待探测头刚好完全进入肛门时测量。小鼠体温每隔24小时测一次。
3)定期录制各组视频(每组2分钟，n＝6)。小鼠活动状况的量化参照下面的表1。
表1 RAPID行为动作分类法的行为动作操作说明
Figure PCTCN2017099956-appb-000002
在攻毒的第6天(DPI)和第8天(DPI)分别对小鼠的活动状况的进行了录像，检测和量化，小鼠行为的变化分为主动作和修饰动作(站立、坐，站起、行走、躺下、头转动、观望、闻、洗脸等)，详细情况参见实验材料和方法部分。取有代表性的2分钟内的行为活动。
4)存活率的计算
每天观察并记录各组小鼠的存活个数。
每组的存活率＝每组存活的个体数(只)/每组总个体数(只)×100％，计算出每组小鼠每天的存活率之后进行汇总作图，分析其死亡趋势。
(5)小鼠肺指数和脾指数
在感染DPI 8时，每组各取5只小鼠进行肺指数和脾指数检测。在IAV感染第8天，将小鼠用戊巴比妥150－200μl麻醉，测量小鼠体重，眼球取血，随后将小鼠固定于解剖板上，沿腹中线解剖小鼠，依次取下心、肺、肝、脾和淋巴结，其中取半肺测量肺重，脾测全重(测量时用吸水纸将肺、脾表面水分尽量除尽)，随后固定于多聚甲醛中用于切片。
肺指数与抑制率按以下公式计算：
Figure PCTCN2017099956-appb-000003
(6)肺组织病理切片观察
称量肺重量后，将肺组织浸于10％甲醛固定后再在浓度梯度的酒精中各浸泡2－4h中脱去组织块中的水分，置于透明剂二甲苯中0.5－2h。将已透明的组织块用石蜡包埋后固定于切片机上，切成薄片(厚约4－5μm)。再用二甲苯脱去切片中的石蜡后，用苏木精伊红(HE)染色，光学显微镜Olympus CX 41(Olympus，日本)下观察肺组织形态学病理改变。
(7)血管通透性检测
在感染DPI 8时，每组各取5只小鼠进行肺血管的通透性检测。麻醉小鼠，通过尾静脉注射伊文蓝溶液，5min后取肺泡灌注液，1500rpm离心5min，取上清液，利用分光光度计检测590nm处测吸光度OD。
(8)小鼠肺泡灌注液的获取及Diff-Quick染色
在感染DPI 8时，每组各取5只小鼠测肺泡灌注液的炎症细胞数。与高毒处理组相比，CBD组(20mg/kg/d)能显著地降低H1N1WSN感染引起的小鼠肺泡灌注液中中性粒细胞的数目。
麻醉小鼠，眼球放血或心脏取血。
剪开小鼠颈部皮肤，暴露出气管。
在气管上剪开一小口，注入1mL含0.1mM EDTA的PBS，吸回后收集在10mL离心管中，重复三次收集，共得约3mL灌注液。
1500rpm离心5min，上清转移至一新的10mL离心管-80℃冻存，细胞沉淀用450μl ddH2O重悬，轻轻振摇，不超过1min，红细胞即快速裂解，立即加入50μL10×PBS。
1500rpm，5min，去上清，PBS重悬。
血球计数板计数。
计数结束后，离心去上清，血清重悬细胞，涂片。
Diff-Quick染色：涂片带湿(即不进行干燥)浸入乙醇乙醚溶液固定15s，稍去掉边缘处固定液，插入Diff-QuickⅠ、Diff-QuickⅡ溶液各15s，流水冲去多余染料。
带湿(即不进行干燥)在镜下检测计数，统计各细胞比例。
(9)以上实验结果的数据统计学分析
采用SPSS 12.0.1数据处理软件(SPSS Inc.,Chicago,IL)单因素方差分析进行，数据符合正态分布，显著性检验采用t检验。数据用平均值±标准误差表示，p<0.05为 差异显著；p<0.01为差异极显著。
3.实验结果
(1)CBD注射组能显著地抑制H1N1引起的体重降低
如图1所示。
从图中可以看出，最开始小鼠体温波动，是由于小鼠购买后用于实验，适应新环境时出现的应激反应，但由于各组小鼠初期体重百分比曲线大致相当，可忽略该应激反应造成的变化。小鼠在感染流感病毒后第3天开始有明显体重下降，约第7－8天到达最低点，然后开始回升，7天不再死亡的个体体重开始明显回升。与高毒组相比，达菲组(20mg/kg/d)和CBD组(20mg/kg/d)能显著地抑制体重的降低(p<0.01)。
(2)CBD注射组能显著地抑制H1N1引起的体温降低
如图2所示。
DPI 7－9小鼠体温变化具有显著性差异，在DPI 7－8达到最大变化。从图中可以看出，小鼠在感染IAV后第4天开始有明显体温下降，约第7－8天到达最低点，然后开始回升，7天不再死亡的个体体温开始明显回升。与高毒组相比，达菲组(20mg/kg/d)和CBD组(20mg/kg/d)能显著地抑制体温的降低。
(3)CBD组能显著地降低H1N1感染组的流感症状和行为不适
如图3A和图3B所示。
在感染6－9DPI时，小鼠行为变化具有显著性差异，在DPI 8达到最大变化。与高毒处理组相比，达菲组(20mg/kg/d)和CBD注射液组(20mg/kg/d)能显著的增加感染鼠的活动能力，减轻流感引起的不适度。
(4)CBD注射组能显著地降低H1N1感染组的死亡率
如图4所示。
与高毒处理组相比，达菲组(20mg/kg/d)和CBD组(20mg/kg/d)能显著地降低H1N1WSN感染引起的小鼠死亡率(达菲组，P<0.001；CBD组，P<0.001)。达菲处理组死亡9只(9/20)，CBD组死亡10只(10/20)，CBD组体现出非常明显的治疗效果。达菲组(9/20)和CBD组(10/20)死亡率没有显著性差异。
(5)CBD组能显著地降低H1N1感染组的肺损伤
如图5和图6A－6D所示。
与高毒组相比，达菲组(20mg/kg/d)和CBD组(20mg/kg/d)能显著地降低H1N1WSN感染引起的小鼠肺损伤指数(达菲组，P<0.05；CBD组，P<0.05)，CBD组 体现出非常明显的治疗效果。达菲组(9/20)和CBD组(10/20)肺指数无显著性差异。达菲和CBD处理组显著减少炎症细胞数侵润。
(6)CBD组能显著地降低H1N1感染组肺血管的通透性
如图7所示。
与高毒组相比，达菲组(20mg/kg/d)和CBD组(20mg/kg/d)能显著地降低H1N1WSN感染引起的小鼠肺毛细管的通透性，减少肺的损伤。达菲组，*P<0.05；CBD组，**P<0.01。
(7)CBD组能显著地降低H1N1感染组肺泡灌注液的炎症细胞数
如图8所示。CBD组，P<0.001。与达菲组相比，CBD组体现出非常明显的减少肺泡灌注液中中性粒细胞的数目。
如图9所示。与IAV处理组相比，CBD组，P<0.01显著的减少肺泡灌注液中淋巴细胞的数目。
如图10所示。达菲组，P<0.01；CBD组，P<0.001显著的减少肺泡灌注液中巨噬细胞的数目。与达菲组相比，CBD组也体现出明显的减少肺泡灌注液中巨噬细胞的数目，P<0.05。
以上实验结果表明：
CBD能显著地降低H1N1WSN感染引起的小鼠的流感症状和不适；能够显著地抑制小鼠体重、低体温的降低；降低了流感引起的小鼠死亡率。CBD还降低肺血管的通透性，减少炎症细胞(中性粒细胞、淋巴细胞、巨噬细胞)在流感感染的小鼠肺组织的侵润，降低急性肺损伤ALI。
实施例2：大麻二酚抑制甲型流感病毒H1N1的RNA聚合酶的体外实验
传代的小鼠骨髓来源的巨噬细胞(bone-marrow derived macrophage，BMDM)和人非小细胞肺癌细胞系A549(
Figure PCTCN2017099956-appb-000004
CCL-185TM)，将其接种于6孔板，并分为如下4组：
对照组(高糖培养基DMEM，厂商是Sigma，货号是D5648-1L，在实验前用纯净水稀释成1升)、
H1N1感染对照组、
CBD组(5μM)和
达菲组(10μM)。
以上4组均在37℃过夜培养，18－24h细胞汇合度达100％且细胞间无间隙时感染H1N1WSN。MOI＝0.01感染细胞1h。
将上清液吸弃，各组分别对应加入相同体积的含PBS、达菲和CBD的无血清的培养基，其中各无血清的培养基中含的PBS、达菲和CBD的浓度相同；在CO2培养箱中37℃下孵育24h。
提取RNA，反转录，qRT-PCR检测病毒mRNA的表达情况。检测的H1N1的NP、PA、PB1和PB2基因表达，RT-PCR引物如下：
Figure PCTCN2017099956-appb-000005
实验结果分别如图11A和图11B(小鼠的BMDM细胞)以及图12A－图12D(人肺上皮细胞系A 549)所示。
结果显示，达菲组(10μM)对H1N1WSN的NP、PA、PB1和PB2基因的表达没有显著的抑制作用；而令人吃惊的是，CBD组(5μM)能显著地降低H1N1WSN的NP、PA、PB1和PB2基因的表达。结果表明，CBD能够有效地抑制流感病毒H1N1的RNA聚合酶的复制。
CBD抑制流感病毒RNA依赖的RNA聚合酶的表达，可能抑制IAV在宿主细胞中的复制，降低流感的感染。本发明提供了一个潜在的广谱抗流感病毒的药物。
实施例3：大麻二酚抗甲型流感病毒H5N1的实验
1.实验动物和前期准备
6周龄的C57BL/6雄鼠，购自北京维通利华实验技术有限公司。
与实例1条件一致。动物常规饲养于中国科学院微生物研究所P2和P3动物中心(Biosafety level 2and 3,P2/P3)，光照周期为12小时光照/12小时黑暗，动物自由采食，自由饮水。动物适应新环境1天后进行相关实验。中国农业大学和农业生物技 术国家重点实验室动物伦理委员会(SKLAB-2017-3-002)批准了动物实验。
将C57BL/6雄鼠随机分为2组，每组10只小鼠，从感染第4天起，每天腹腔注射给药1次，连续给药5天。1000pfu流感组用等量生理盐水注射。具体分组如下：
流感组(H5N1 1000pfu)：感染第4天开始注射等量生理盐水。
CBD组：H5N1 1000pfu感染第4天开始给药(大麻二酚，20mg/kg/d)。
2.实验方法
H5N1病毒的扩增方法和滴度检测方法与实施例1完全相同，只是病毒亚型是H5N1。
小鼠生理特征和发病症状的观察和记录与实例1完全相同。
H5N1感染小鼠当天(Day of post infection,DPI 0)至感染后11天(DPI 10)，每天进行称重，以当天体重减去DPI 0体重，再除以DPI 0体重，计算出体重变化百分数。
H5N1(以1000pfu剂量感染，监测体温的变化)感染小鼠每组10只，DPI 0至DPI 12，每天进行肛温测量，以当天肛温减去DPI0肛温，再除以DPI0肛温，计算出体温变化百分数。
在功毒的第9天(DPI)分别对小鼠的活动状况的进行了录像，检测和量化，小鼠行为的变化分为主动作和修饰动作(站立、坐，站起、行走、躺下、头转动、观望、闻、洗脸等)，详细情况参见实验材料和方法部分。取有代表性的1分钟内的行为活动，进行计数。
每天记录H5N1感染小鼠的死亡情况。
3.实验结果
(1)CBD组能显著的抑制H5N1引起的体重降低
如图13所示。
从图中可以看出，最开始小鼠体温波动，是由于小鼠购买后用于实验，适应新环境时出现的应激反应，但由于各组小鼠初期体重百分比曲线大致相当，可忽略该应激反应造成的变化。小鼠在感染流感病毒后第6天开始有明显体重下降，约第10天到达最低点，然后开始回升，但是第12天全部死亡。
(2)CBD组能显著的抑制H5N1引起的体温降低
如图14所示。
DPI 9小鼠体温变化具有显著性差异，与流感组相比，CBD组(20mg/kg/d)能 显著的抑制体温的降低。由于第9天小鼠死亡一半，不能进行统计学分析了，
(3)CBD组能显著的降低H5N1感染组的流感症状和行为不适
如图15所示。在感染DPI 9时(n＝5/组)，小鼠行为变化具有显著性差异(p<0.001)。与流感组(n＝5/组)相比，CBD组(20mg/kg/d)(n＝5/组)能显著的增加感染鼠的活动能力，减轻流感引起的不适度。
(4)CBD组能显著的延长H5N1感染组的生存时间
如图16所示。与流感组(10/10)相比，CBD组(10/10)死亡率没有显著性差异，但是，CBD组(20mg/kg/d)在每个时间点都能显著的延长H5N1感染后小鼠的生存时间为24小时(CBD组vs.流感组，P<0.05)。
另外，由于昆明鼠是封闭群，C57BL/6s是自交系，对药物的敏感度和反应程度不一样，同时又是两种不同的品系，这从另一方面说明CBD的广谱性。
实施例4：大麻二酚抑制甲型流感病毒H5N1的RNA聚合酶的体外实验
人非小细胞肺癌细胞系A549(
Figure PCTCN2017099956-appb-000006
CCL-185TM)，将其接种于6孔板，并分为如下4组：
对照组(高糖培养基DMEM，厂商是Sigma，货号是D5648-1L，在实验前用纯净水稀释成1升)、
H5N1感染对照组、
CBD组(1μM)和
达菲组(1μM)。
以上4组均在37℃过夜培养，18－24h细胞汇合度达100％且细胞间无间隙时感染H5N1。MOI＝0.01感染细胞1h。
将上清液吸弃，各组分别对应加入相同体积的含PBS、达菲和CBD的无血清的培养基，其中各无血清的培养基中含的PBS、达菲和CBD的浓度相同；在CO2培养箱中37℃下孵育24h。
提取RNA，反转录，qRT-PCR检测病毒mRNA的表达情况。检测的H5N1的NP、PA、PB1和PB2基因表达，RT-PCR引物如下：
Figure PCTCN2017099956-appb-000007
Figure PCTCN2017099956-appb-000008
实验结果分别如图17A、图17B、图17C和图17D所示。
结果显示，达菲组(1μM)和CBD组(1μM)能显著地降低H5N1的NP、PA、PB1和PB2基因的表达。结果表明，CBD能够有效地抑制流感病毒H5N1的RNA聚合酶的复制。
CBD抑制流感病毒RNA依赖的RNA聚合酶的表达，可能抑制IAV在宿主细胞中的复制，降低流感的感染。本发明提供了一个潜在的广谱抗流感病毒的药物。
参考文献
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Bernard,G.R.,Luce,J.M.,Sprung,C.L.,Rinaldo,J.E.,Tate,R.M.,Sibbald,W.J.,Kariman,K.,Higgins,S.,Bradley,R.,Metz,C.A.,et al.(1987).High-dose corticosteroids in patients with the adult respiratory distress syndrome.The New England journal of medicine 317,1565-1570.
Glezen,W.P.(2006).Influenza control.The New England journal of medicine 355,79-81.
Kolocouris,N.,Kolocouris,A.,Foscolos,G.B.,Fytas,G.,Neyts,J.,Padalko,E.,Balzarini,J.,Snoeck,R.,Andrei,G.,and De Clercq,E.(1996).Synthesis and antiviral activity evaluation of some new aminoadamantane derivatives.2.J Med Chem 39,3307-3318.
Ribeiro,A.,Almeida,V.I.,Costola-de-Souza,C.,Ferraz-de-Paula,V.,Pinheiro,M.L.,Vitoretti,L.B.,Gimenes-Junior,J.A.,Akamine,A.T.,Crippa,J.A.,Tavares-de-Lima,W.,et al.(2015).Cannabidiol improves lung function and inflammation in mice submitted to LPS-induced acute lung injury. Immunopharmacology and immunotoxicology 37,35-41.
尽管本发明的具体实施方式已经得到详细的描述，本领域技术人员将会理解。根据已经公开的所有教导，可以对那些细节进行各种修改和替换，这些改变均在本发明的保护范围之内。本发明的全部范围由所附权利要求及其任何等同物给出。

Claims (42)

  1. 选自如下的(1)－(3)中的任一项在制备治疗或预防流感的药物或者缓解流感症状的药物中的用途：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  2. 根据权利要求1所述的用途，其中，所述流感由选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种流感病毒引起；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
  3. 根据权利要求1所述的用途，其中，所述流感的患病对象为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
  4. 根据权利要求1所述的用途，其中，所述流感症状为流感引起的选自如下症状中的至少一种：
    发热、咳嗽、头痛、肌肉酸痛以及腹泻。
  5. 根据权利要求1所述的用途，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米 韦。
  6. 选自如下的(1)－(3)中的任一项在制备抗流感病毒(例如抑制流感病毒在宿主细胞中的复制)的药物中的用途：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  7. 根据权利要求6所述的用途，其中，所述流感病毒选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
  8. 根据权利要求6所述的用途，其中，所述宿主细胞为哺乳动物(例如人、猿、猴、猪、牛或羊)的细胞或者禽类(例如例如家禽如鸡、鸭或鹅，或者例如野生禽类)的细胞。
  9. 根据权利要求6所述的用途，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  10. 选自如下的(1)－(3)中的任一项在制备抑制流感病毒RNA聚合酶的复制的药物、抑制流感病毒RNA聚合酶的表达水平的药物或者抑制流感病毒RNA聚合酶的 活性的药物中的用途：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  11. 根据权利要求10所述的用途，其中，所述流感病毒RNA聚合酶选自甲型流感病毒RNA聚合酶、乙型流感病毒RNA聚合酶和丙型流感病毒RNA聚合酶中的一种或多种；优选地，所述甲型流感病毒RNA聚合酶为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒的RNA聚合酶。
  12. 根据权利要求10所述的用途，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  13. 一种药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料，并且所述的药物组合物，其还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  14. 一种组合产品，其包含独立包装的产品1和产品2，
    其中，
    所述产品1选自如下的(1)－(3)中的任意一种：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；和
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料；
    所述产品2包含有效量的选自如下的一种或者多种成分：肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  15. 选自如下的(1)－(3)中的任一项的产品，其用于治疗或预防流感或者缓解流感症状：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  16. 根据权利要求15所述的产品，其中，所述流感由选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种流感病毒引起；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病 毒。
  17. 根据权利要求15所述的产品，其中，所述流感的患病对象为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
  18. 根据权利要求15所述的产品，其中，所述流感症状为流感引起的选自如下症状中的至少一种：
    发热、咳嗽、头痛、肌肉酸痛以及腹泻。
  19. 根据权利要求15所述的产品，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  20. 选自如下的(1)－(3)中的任一项的产品，其用于抗流感病毒(例如抑制流感病毒在宿主细胞中的复制)：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  21. 根据权利要求20所述的产品，其中，所述流感病毒选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
  22. 根据权利要求20所述的产品，其中，所述宿主细胞为哺乳动物(例如人、猿、猴、猪、牛或羊)的细胞或者禽类(例如例如家禽如鸡、鸭或鹅，或者例如野生禽类)的细胞。
  23. 根据权利要求20所述的产品，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  24. 选自如下的(1)－(3)中的任一项的产品，其用于抑制流感病毒RNA聚合酶的复制、抑制流感病毒RNA聚合酶的表达水平或者抑制流感病毒RNA聚合酶的活性：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  25. 根据权利要求24所述的产品，其中，所述流感病毒RNA聚合酶选自甲型流感病毒RNA聚合酶、乙型流感病毒RNA聚合酶和丙型流感病毒RNA聚合酶中的一种或多种；优选地，所述甲型流感病毒RNA聚合酶为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒的RNA聚合酶。
  26. 根据权利要求24所述的产品，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  27. 一种治疗或预防流感或者缓解流感症状的方法，包括给予有需求的受试者以有效量的选自如下的(1)－(3)中的任一项的产品的步骤：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  28. 根据权利要求27所述的方法，其中，所述流感由选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种流感病毒引起；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
  29. 根据权利要求27所述的方法，其中，所述受试者为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
  30. 根据权利要求27所述的方法，其中，所述流感症状为流感引起的选自如下症状中的至少一种：
    发热、咳嗽、头痛、肌肉酸痛以及腹泻。
  31. 根据权利要求27所述的方法，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  32. 一种抗流感病毒(例如抑制流感病毒在宿主细胞中的复制)的方法，包括给予有需求的受试者以有效量的选自如下的(1)－(3)中的任一项的产品的步骤：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  33. 根据权利要求32所述的方法，其中，所述流感病毒选自甲型流感病毒、乙型流感病毒和丙型流感病毒中的一种或多种；优选地，所述甲型流感病毒为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒。
  34. 根据权利要求32所述的方法，其中，所述受试者为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
  35. 根据权利要求32所述的方法，其中，所述宿主细胞为哺乳动物(例如人、猿、猴、猪、牛或羊)的细胞或者禽类(例如例如家禽如鸡、鸭或鹅，或者例如野生禽类)的细胞。
  36. 根据权利要求32所述的方法，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶 抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  37. 一种抑制流感病毒RNA聚合酶的复制、抑制流感病毒RNA聚合酶的表达水平或者抑制流感病毒RNA聚合酶的活性的方法，包括给予有需求的受试者以有效量的选自如下的(1)－(3)中的任一项的产品的步骤：
    (1)大麻二酚或其药学上可接受的盐或酯；
    (2)含有大麻二酚的植物提取物；优选地，为含有大麻二酚的大麻提取物；优选地，为含有大麻二酚的工业大麻提取物；
    (3)药物组合物，其含有有效量的大麻二酚或其药学上可接受的盐或酯，以及一种或多种药学上可接受的辅料。
  38. 根据权利要求37所述的方法，其中，所述流感病毒RNA聚合酶选自甲型流感病毒RNA聚合酶、乙型流感病毒RNA聚合酶和丙型流感病毒RNA聚合酶中的一种或多种；优选地，所述甲型流感病毒RNA聚合酶为H1N1亚型、H2N2亚型、H3N2亚型、H5NI亚型、H7N9亚型或H9N2亚型的甲型流感病毒的RNA聚合酶。
  39. 根据权利要求37所述的方法，其中，所述受试者为哺乳动物(例如人、猿、猴、猪、牛或羊)或者禽类(例如家禽如鸡、鸭或鹅，或者例如野生禽类)。
  40. 根据权利要求37所述的方法，其中，所述药物组合物还包含有效量的选自如下的一种或者多种成分：
    肌苷单磷酸脱氢酶抑制药、干扰素诱导剂、M2离子通道蛋白抑制药和神经氨酸酶抑制药；
    优选地，所述肌苷单磷酸脱氢酶抑制药为利巴韦林；
    优选地，所述干扰素诱导剂为盐酸阿比朵尔；
    优选地，所述M2离子通道蛋白抑制药为盐酸金刚烷胺或盐酸金刚乙胺；
    优选地，所述神经氨酸酶抑制药为磷酸奥司他韦、奥塞米韦、扎那米韦或帕拉米韦。
  41. 根据权利要求27至40中任一权利要求所述的方法，其中，大麻二酚的剂量介于0.1－50mg/kg体重/天，更优选为0.5mg/kg－30mg/kg体重/天、0.5mg/kg－20mg/kg体重/天、5mg/kg－30mg/kg体重/天或者5mg/kg－20mg/kg体重/天，进一步优选为0.5mg/kg－10mg/kg体重/天，特别优选为0.5mg/kg－5mg/kg体重/天。
  42. 根据权利要求27至40中任一权利要求所述的方法，其中，通过口服给药。
PCT/CN2017/099956 2017-08-31 2017-08-31 大麻二酚在制备抗流感的药物中的用途 WO2019041239A1 (zh)

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