WO2021159570A1 - 黄芩素在制备预防和/或治疗新型冠状病毒感染疾病的药物中的应用 - Google Patents

黄芩素在制备预防和/或治疗新型冠状病毒感染疾病的药物中的应用 Download PDF

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WO2021159570A1
WO2021159570A1 PCT/CN2020/078252 CN2020078252W WO2021159570A1 WO 2021159570 A1 WO2021159570 A1 WO 2021159570A1 CN 2020078252 W CN2020078252 W CN 2020078252W WO 2021159570 A1 WO2021159570 A1 WO 2021159570A1
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baicalein
infection
new coronavirus
mice
coronavirus
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PCT/CN2020/078252
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English (en)
French (fr)
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杜冠华
吕扬
王珂
刘江宁
秦川
宋俊科
杨世颖
杨德智
张丽
张莉
王金华
张雯
袁天翊
邢逞
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中国医学科学院药物研究所
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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
    • A61K31/335—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
    • 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
    • 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
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

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  • the invention belongs to the technical field of medicine and relates to the application of baicalein in the preparation of drugs for preventing and/or treating novel coronavirus infections. Specifically, it relates to the application of baicalein and pharmaceutical compositions containing baicalein in the prevention and/or treatment of new coronavirus (SARS-CoV-2, hereinafter also referred to as "new coronavirus”) infectious diseases, including the use of baicalein Virus-induced mild, moderate, and severe infections, including but not limited to new coronary pneumonia.
  • SARS-CoV-2 new coronavirus
  • Coronavirus is a type of RNA virus with envelope. It is composed of a single ribonucleic acid RNA and belongs to single-stranded RNA virus. At present, about 15 different coronavirus strains have been discovered, which can infect humans, mice, pigs, cats, dogs, and avian vertebrates. Coronaviruses (CoVs) belong to the Coronavirus family (Coronaviridae), and are divided into 4 genera according to serotype and genome characteristics: Alpha Coronavirus ( ⁇ -CoV), Beta Coronavirus ( ⁇ -CoV), Gamma Coronavirus ( ⁇ -CoV), Delta Coronavirus ( ⁇ -CoV) can infect humans and a variety of animals, causing host respiratory, intestinal, liver and nervous system diseases.
  • Coronaviruses belong to the Coronavirus family (Coronaviridae), and are divided into 4 genera according to serotype and genome characteristics: Alpha Coronavirus ( ⁇ -CoV), Beta Coronavirus ( ⁇ -CoV), Gamma Coronavirus ( ⁇ -CoV), Delta Coronavirus ( ⁇ -CoV)
  • Beta Coronavirus is the most harmful to humans.
  • the International Committee on Taxonomy of Viruses ICTV
  • ICTV International Committee on Taxonomy of Viruses
  • the first 4 subgenres correspond to the original Beta.
  • the subgenus Hibecovirus consists of a class of ⁇ -CoVs that are isolated from Chinese bats and are phylogenetically related to the subgenus Sarbecovirus. Ascension comes.
  • 2019-nCoV 2019-nCoV
  • Wuhan City, Hubei province As the epidemic spreads, the disease has been included in the "Infectious Diseases of the People's Republic of China”.
  • Class B infectious diseases stipulated in the Prevention and Control Law, and measures to prevent and control Class A infectious diseases shall be adopted.
  • 2019-nCoV is a new type of coronavirus belonging to the beta genus. The virus can infect patients with pneumonia, and can also cause damage to the intestines, liver and nervous system and corresponding symptoms. The epidemic spread rapidly and spread quickly, but so far there is no specific medicine.
  • the pneumonia caused by the new type of coronavirus is uniformly referred to as "new coronavirus pneumonia", or “new coronary pneumonia” for short, and "Novel coronavirus pneumonia” in English, or “NCP” for short.
  • ICTV International Committee on Taxonomy of Viruses
  • SARS-CoV severe acute respiratory syndrome coronavirus 2
  • WHO World Health Organization
  • RNA viruses include HIV, hepatitis C virus, Japanese encephalitis virus, influenza virus, rhinovirus, polio virus, dengue virus, SARS virus, MERS virus, Ebola virus and so on.
  • Baicalein (chemical name: 5,6,7-trihydroxyflavone; English name: Baicalien), also known as baicalein and astragalin, is found in the dried roots of Scutellaria baicalensis Georgi, a plant in the Labiatase family.
  • the main flavonoids have a molecular formula of C 15 H 10 O 5 , a weight of 270.24, and a structure of formula (I).
  • Baicalein is a flavonoid compound with poor water solubility, and has the problem of low bioavailability in the body as a medicine.
  • the applicant invented a new ⁇ crystal form of baicalein in Chinese patent ZL200710177330.8.
  • the bioabsorption rate of ⁇ crystal form in vivo is significantly higher than that of ⁇ crystal form, which is about twice as much as that of ⁇ crystal form. .
  • the baicalein ⁇ crystal form can significantly improve the in vivo bioavailability of the compound, which provides the possibility for the development of baicalein-related solid oral pharmaceutical preparations.
  • Baicalein has a variety of pharmacological effects, including: anti-bacterial, anti-viral, anti-inflammatory, anti-allergic, anti-oxidation, scavenging free radicals, anti-tumor, anti-Parkinson, anti-coagulation, anti-thrombosis and protecting the liver, cardiovascular and cerebrovascular and The role of neurons and so on.
  • antiviral activity of baicalein has also been reported many times, such as anti-double-stranded DNA virus, herpes virus, a few negative-strand respiratory viruses, Japanese encephalitis virus, dengue fever virus and so on.
  • Chinese Patent Application Publication No. CN104203265A describes the "anti-flavivirus antiviral composition" invented by the University of Malaysia, which discloses an anti-viral compound that prevents or treats a flavivirus infection in humans or animals or diseases caused by it.
  • the active composition is characterized in that the composition contains baicalein, its analogues or its derivatives.
  • the composition may further comprise a pharmaceutically acceptable carrier.
  • the antiviral activity includes inhibition of virus attachment to host cells, inhibition of intracellular virus replication, and direct virus killing activity.
  • Flaviviruses include dengue virus type 1, dengue virus type 2, dengue virus type 3, dengue virus type 4, and Japanese encephalitis virus.
  • the number of dengue virus foci in Vero cells pretreated with 50 ⁇ g/ml baicalein was reduced by ⁇ 37% and the amount of dengue virus type 2 RNA production was reduced by 39.5% ⁇ 0.8.
  • the IC 50 of baicalein in the pretreated cells was 108.8 ⁇ g/ml.
  • 25 ⁇ g/ml of baicalein reduced the amount of dengue virus foci by 76.6% and the production of DENV-2RNA by 90.3% ⁇ 1.6, and the calculated IC 50 value was 7.14 ⁇ g/ml.
  • Chinese Patent Application Publication No. CN1925863A discloses the "Scutellaria extract for the treatment of SARS” invented by Vannova (UK) Ltd., which involves the total standardized extract of Scutellaria with anti-coronavirus activity, especially It has antiviral activity against those viruses that cause severe acute respiratory syndrome (SARS).
  • the Scutellaria extract PYN5C inhibits SARS-CoV infectivity by about 50% at the highest concentration (200 ⁇ g/ml), and the effect is dose-dependent Yes, the inhibitory effect at higher levels is greater than ribavirin (100 ⁇ g/ml).
  • baicalein and baicalin as Zika virus inhibitors (Archives of Virology, 164,585-593, 2019) records that baicalein and baicalin can down-regulate Zika virus (ZIKV) replication within 10 hours after infection. It has obvious preventive effect in pretreated cells. Baicalein exhibits the highest efficacy in the intracellular ZIKV replication process, and baicalin has the strongest inhibitory effect on virus entry. Predicted by silico internal interaction analysis, the two compounds show the strongest binding affinity to ZIKV NS5, and the viral envelope glycoprotein is the most unlikely target protein. These findings provide an important platform for further research on the potential anti-ZIKV mechanism of each compound.
  • baicalein has an inhibitory effect on influenza viruses in the body .
  • Oral administration of baicalein to BALB/c mice infected with influenza A virus (H1N1) has obvious effects of preventing death, increasing the average death time, inhibiting lung consolidation and reducing the titer of lung virus. These effects are believed to be due to baicalin, a metabolite of baicalein in the serum.
  • the covering medium when the concentration of baicalin was 2mg/ml, it showed a significant inhibitory effect in the plaque test.
  • the average IC 50 value of baicalin in the cytopathic test was 1.2mg/ml. The results show that baicalein as a potential anti-influenza virus drug is worthy of further study.
  • Baicalein can reduce the immediate early protein level of HCMV to close to the background level, while genistein does not.
  • the antiviral effect of genistein is completely reversible in cell culture. Adding flavonoids to the concentrated virus culture solution has no inhibitory effect on the replication of HCMV, indicating that baicalein cannot directly inactivate virus particles.
  • Baicalein can block epidermal growth factor receptor tyrosine kinase activity and HCMV nuclear translocation, while genistein cannot. 24 hours after infection, HCMV-infected cells treated with genistein continued to express immediate early protein and effectively phosphorylated IE1-72.
  • HCMV-induced increases in NF- ⁇ B and cell cycle regulatory protein levels are not related to immediate early protein function. It is suggested that the main mechanism of baicalein may be to block HCMV infection, and the main mechanism of genistein may be to block the immediate early protein function of HCMV.
  • One or more embodiments of the present application provide the use of baicalein in the preparation of medicines for preventing and/or treating diseases caused by novel coronavirus infection.
  • One or more embodiments of the present application provide a pharmaceutical composition for preventing and/or treating a novel coronavirus infection, which comprises baicalein and a pharmaceutically acceptable carrier, adjuvant or excipient, and/or a Or multiple additional antiviral agents or antibiotics.
  • the baicalein is ⁇ crystal form baicalein.
  • the disease caused by a novel coronavirus infection is a novel coronavirus pneumonia.
  • the new coronavirus is SARS-CoV-2.
  • the antiviral agent is remdesivir, chloroquine phosphate, ribavirin, nitazoxanide, nafamostat, penciclovir, favipiravir , Arbidol and/or Amantadine.
  • the antibiotics are lactams, macrolides, cephalosporins and/or sulfa antibiotics.
  • the content of baicalein in the pharmaceutical composition is 0.1 to 99.9% by weight.
  • the content of baicalein is 50 to 99.9% by weight.
  • the content of baicalein is 50, 60, 70, 80, 90, 99% by weight
  • the content of baicalein is 80 to 99.9% by weight.
  • the pharmaceutical composition is a solid preparation.
  • the solid preparations include tablets, capsules, pills, injection preparations, sustained-release or controlled-release preparations.
  • One or more embodiments of the present application provide the use of the pharmaceutical composition of the present application in the preparation of a medicament for preventing and/or treating diseases caused by a novel coronavirus infection.
  • the new coronavirus is SARS-CoV-2.
  • the novel coronavirus infection includes mild, moderate and severe infections.
  • the disease caused by the novel coronavirus infection includes pneumonia or enteritis caused by the novel coronavirus.
  • the baicalein inhibits the infection of the new coronavirus, inhibits the replication of the new coronavirus, or has protective and therapeutic effects on tissue damage caused by the new coronavirus.
  • the daily dose of baicalein is 50-1000 mg.
  • the daily dosage for the elderly and children is 50-800 mg.
  • the daily dose of baicalein is 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg.
  • baicalein is ⁇ crystal form baicalein.
  • One or more embodiments of the present application provide a method for treating diseases caused by a novel coronavirus infection, which comprises administering a therapeutically effective amount of baicalein or a composition containing baicalein to a subject in need thereof.
  • One or more embodiments of the present application provide a method of baicalein or a composition containing baicalein, which is used to prevent and/or treat diseases caused by a novel coronavirus infection.
  • baicalein Although the antiviral activity of baicalein against many common human viruses has been widely reported, its activity against the new coronavirus (SARS-CoV-2) has not been reported yet. In molecular screening, in vitro cells and experimental animal models, the inventors have used baicalein to achieve unexpected technical effects against the new coronavirus. Its half effective concentration EC 50 is equivalent to that of radcivir and significantly higher than that of riba.
  • Classical antiviral drugs such as virin and lopinavir-ritonavir have completed the present invention.
  • the technical problem to be solved in the first aspect of the present invention is to provide a new application of baicalein in pharmacy, that is, the application of baicalein in the preparation of drugs for preventing and/or treating diseases caused by novel coronavirus infection.
  • the prevention and/or treatment of new coronavirus (SARS-CoV-2) infection in the present invention includes mild, moderate and severe infections caused by the new coronavirus, including but not limited to new coronary pneumonia (NCP).
  • NCP new coronary pneumonia
  • baicalein can be used to prepare a pharmaceutical composition for the prevention and/or treatment of new coronavirus infections.
  • the pharmaceutical composition contains baicalein and a pharmaceutically acceptable carrier, preferably containing an effective dose of baicalein ⁇ crystal form .
  • the prevention and/or treatment of novel coronavirus infection refers to having antiviral activity after preventive infection, direct virus killing activity, or inhibition of virus attachment to host cells.
  • the daily dosage of baicalein is in the range of 50-1000 mg, such as 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 mg.
  • baicalein as the active ingredient.
  • the pharmaceutical composition can be prepared according to methods well known in the art.
  • the baicalein can be combined with one or more pharmaceutically acceptable solid or liquid excipients and/or adjuvants to prepare any dosage form suitable for human or animal use.
  • the content of baicalein of the present invention in its pharmaceutical composition is usually in the range of 0.1% to 100% by weight, such as 1%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% , 90%.
  • the baicalein of the present invention can be administered in a unit dosage form, and the route of administration can be enteral or parenteral, such as oral, intravenous, intramuscular, subcutaneous injection, nasal cavity, oral mucosa, eyes, lungs and respiratory tract, skin, Vagina, rectum, etc.
  • the dosage form of the present invention is preferably a solid dosage form.
  • the solid dosage form can be a tablet, including ordinary tablets, enteric-coated tablets, buccal tablets, dispersible tablets, chewable tablets, effervescent tablets, orally disintegrating tablets; capsules, including hard capsules, soft capsules, and enteric-coated capsules; granules; Powders; pellets; dripping pills; suppositories; films; patches, air (powder) sprays; sprays, etc.
  • the baicalein of the present invention can be made into ordinary preparations, and can also be made into slow-release preparations, controlled-release preparations, targeted preparations and various particle delivery systems.
  • the diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup, dextrin, syrup, honey, glucose solution, microcrystalline cellulose, acacia syrup, gelatin syrup, sodium carboxymethyl cellulose, methyl cellulose, hypromellose Base cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.
  • the disintegrant can be dry starch, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.
  • the humectant can be water, ethanol, iso Propanol, etc.
  • the binder can be starch syrup
  • the tablets can also be further made into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer tablets and multi-layer tablets.
  • the active ingredient can be mixed with a diluent or glidant, and the mixture can be directly placed in a hard capsule or a soft capsule.
  • the active ingredient can also be made into granules or pellets with diluents, binders, and disintegrants, and then placed in hard or soft capsules.
  • the various diluents, binders, wetting agents, disintegrants, and glidants used to prepare the baicalein tablets of the present invention can also be used to prepare the baicalein capsules of the present invention.
  • coloring agents can also be added to the pharmaceutical preparations.
  • the drug of the present invention can be administered by any known administration method.
  • the dose of baicalein and the pharmaceutical composition containing baicalein of the present invention can vary widely in accordance with the nature and severity of the disease to be prevented or treated, the individual conditions of the patient or animal, the route of administration, and the dosage form.
  • the above-mentioned dosage can be administered in one dosage unit or divided into several dosage units, depending on the doctor's clinical experience and the dosage regimen including the use of other treatment methods.
  • the baicalein or the composition of the present invention can be taken alone or combined with other therapeutic drugs or symptomatic drugs.
  • the baicalein of the present invention has a synergistic effect with other therapeutic drugs, its dosage should be adjusted according to the actual situation.
  • the present invention relates to the application of baicalein and a pharmaceutical composition containing baicalein in drugs for preventing and/treating new coronavirus infections, including but not limited to new coronary pneumonia.
  • the baicalein used in the present invention has a strong interaction with the key amino acid in the active site of the SARS-CoV-2 coronavirus key protein 3CL hydrolase (Mpro) in the computer virtual screening at the molecular level.
  • Mpro SARS-CoV-2 coronavirus key protein 3CL hydrolase
  • baicalein used in the present invention can interact strongly with key amino acids in the binding site of the viral Spike protein and ACE2 in the computer virtual screening at the molecular level.
  • baicalein of the present invention When the baicalein of the present invention is used to test the anti-coronavirus activity at the cellular level, a viral infection dose of 100TCID50 is used to determine the virus infectivity on the African green monkey kidney cell (VERO cell) model. The results show that baicalein Both the 0.1 ⁇ M and 1 ⁇ M doses have an unexpectedly significant inhibitory effect on the new coronavirus, and its inhibitory ability is equivalent to remdesivir.
  • the baicalein of the present invention is applied to the SARS-CoV-2 infected hACE2 transgenic mouse model.
  • the body weight of the mouse did not decrease due to the infection virus and maintained its increase.
  • the virus replication in the body was significantly inhibited, and the lung tissue lesions were slightly improved, which can be seen Baicalein has a certain viral inhibitory effect on SARS-CoV-2 infected hACE2 transgenic mice and improves lung inflammation.
  • baicalein of the present invention has an unexpected pharmacological effect on pneumonia in an in vitro cell model. Specifically, baicalein has a significant inhibitory effect on the nuclear translocation of NF- ⁇ B in Raw264.7 cells, and has a significant effect on intracellular NF- ⁇ B phosphorylation has a significant inhibitory effect.
  • baicalein of the present invention has a significant therapeutic effect on LPS-induced acute pneumonia injury at the overall animal level, which is specifically manifested in the improvement of the respiratory function of model mice, the improvement of histopathological changes, and the improvement of MPO activity in lung tissue.
  • Influence inflammatory cell concentration in bronchial perfusion fluid, cytokine concentration, serum cytokine concentration, spleen index, and inflammation-related gene mRNA expression level in lung tissue.
  • baicalein of the present invention has completed phase I clinical trial research, and the clinical trial results show that it has good safety.
  • FIG. 1 The interaction diagram between SARS-CoV-2 coronavirus 3CL hydrolase (Mpro) and baicalein (A: three-dimensional interaction diagram, B: two-dimensional interaction diagram);
  • Figure 4 The effect of baicalein on the end-expiratory pause time of mice with lung injury (A); the effect on the maximum expiratory flow per unit body weight of mice with lung injury (B); the effect on the degree of bronchoconstriction in mice with lung injury ( C); Effect on the respiratory rate of mice with lung injury (D);
  • Figure 7 The effect of baicalein on the concentration of inflammatory cells in mouse lung bronchial perfusion fluid
  • Figure 8 The effect of baicalein on cytokine concentration in mouse lung bronchial perfusion fluid
  • Figure 11 The effect of baicalein on the mRNA expression level of inflammation-related genes in lung tissue
  • Viral Spike Glycoprotein S uses ACE2 (D) to mediate the fusion of the viral membrane and the host membrane;
  • FIG. 13 Diagram of the interaction between baicalein and the binding site of virus S-protein-ACE2;
  • Figure 14 The weight loss rate of mice in the model group and the baicalein treatment group
  • FIG. 15 Viral load graphs of lung tissues in the model group and the baicalein treatment group
  • Figure 16 Pulmonary tissue lesions in the model group and the baicalein treatment group.
  • the new coronavirus (SARS-CoV-2) in the following examples are all preserved and provided by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences. Baicalein is provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences, with a purity of >98%, all ⁇ Crystal form.
  • Virus The new coronavirus (SARS-CoV-2), with a titer of 105TCID50/ml, stored at -80°C by the Pathogen Center of the Institute of Medical Laboratory Animals, Chinese Academy of Medical Sciences.
  • the virus titer used is 100TCID50.
  • test drug is diluted to 2 concentrations, each with 5 replicate holes, 100 ⁇ l per hole, and then an equal volume of 100 TCID 50 virus is added to each hole for 1 hour;
  • CPE cytopathic changes
  • baicalein can inhibit the damage of SARS-CoV-2 to cells at a concentration of 0.1 ⁇ M, suggesting that baicalein has anti-SARS-CoV-2 activity in vitro, and its strength of action is the same as that of Remdesivir ( The cell experiment works for more than 4 days after administration).
  • Baicalein (Baicalein, BC) was provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences, and its structure is shown in formula (I), with a purity of >98%; Caspase-1 small molecule inhibitor VX-765 was purchased from Shanghai Hanxiang Biotechnology Co., Ltd. Purity>98%; non-steroidal anti-inflammatory drug aspirin (Acetylsalicylic acid or Aspirin, ASP) and steroidal anti-inflammatory drug dexamethasone (Dexamethasone, DEX) were purchased from Sigma, purity>99%; the compounds were all DMSO Prepare a 100mM mother liquor and store it at -20°C for later use. LPS is formulated into 8mg/ml mother liquor with sterile normal saline, and stored in aliquots at -80°C for later use.
  • the mouse macrophage cell line Raw264.7 was cultured in RPMI-1640 medium containing 10% domestic FBS, 100U/ml penicillin and 100 ⁇ g/ml streptomycin; the culture conditions were 37°C, 5% CO 2 , and 80% humidity ; Passage once every 2 days and replace with fresh complete medium.
  • Normal human lung bronchial epithelial cells BEAS-2b were cultured in RPMI-1640 medium containing 10% imported FBS, 100U/ml penicillin and 100 ⁇ g/ml streptomycin; the culture conditions were 37°C, 5% CO 2 , and 80% humidity ; Passage once every 3 days, and replace with fresh complete medium during passaging.
  • the cells in the logarithmic growth phase were seeded in a 96-well culture plate, 1.0 ⁇ 10 4 cells per well, 37°C, 5% CO 2 , and humidity 80% for 24 hours; use different final concentrations of LPS or Treat cells with compound solution at 37°C for 24 hours; use MTT method to detect cell viability; use an inverted fluorescence microscope to photograph cells fixed with formaldehyde solution and record the cell morphology; use a high-content analyzer to analyze the nucleus morphology and count the average fluorescence of the nucleus strength.
  • the mouse macrophages Raw264.7 in the logarithmic growth phase were seeded on a 96-well culture plate with a black bottom, 2.0 ⁇ 10 4 cells per well, and the medium was containing 10% domestic FBS, 100U/ml penicillin and 100 ⁇ g /ml streptomycin RPMI-1640; culture for 24 hours at 37°C, 5% CO 2 , and humidity 80%; add LPS solution diluted in serum-free 1640 medium with a concentration of 2 times the final concentration, and shake gently Mix the plate so that the final concentrations of baicalein are 0.1, 1, and 10 ⁇ M, the final concentrations of dexamethasone, aspirin, and VX-765 are 10 ⁇ M, and the final concentration of LPS is 2 ⁇ g/ml; formaldehyde fixation, immunofluorescence
  • a high-content analyzer was used to analyze the degree of nuclear translocation of NF- ⁇ B p65 in cells.
  • Immunoblotting was used to analyze the phosphorylation of NF- ⁇ B p65 and TAK1 in Raw264.7 of mouse macrophages.
  • the specific operation steps are as follows: inoculate the mouse macrophages Raw264.7 in logarithmic growth phase in a 35mm culture dish, 1.0 ⁇ 10 6 cells per well, and the medium contains 10% domestic FBS, 100U/ml penicillin and 100 ⁇ g /ml streptomycin RPMI-1640, 2ml medium per dish; culture for 24 hours at 37°C, 5% CO 2 , and humidity 80%; add serum-free RPMI-1640 medium with a concentration of 2 times the final concentration The diluted LPS solution, gently shake and mix so that the final concentration of baicalein is 0.1, 1.0, 10 ⁇ M, the final concentration of dexamethasone, aspirin and VX-765 are 10 ⁇ M, and the final concentration of LPS is 1 ⁇ g/ ml, a total of 1ml/dish; after continuing to incubate
  • the experimental data is expressed by Mean ⁇ SD or Mean ⁇ SEM, using Origin 9.0 software effect statistics chart; using SPSS 22.0 software for statistical analysis, using single-factor variance Bonferroni analysis and Games-Howell analysis to analyze the differences between the two , P ⁇ 0.05 indicates a significant difference.
  • baicalein can also significantly inhibit the phosphorylation of NF- ⁇ B p65 in cells (P ⁇ 0.05); baicalein has no significant effect on the content of non-phosphorylated NF- ⁇ B p65. ,As shown in Figure 3.
  • baicalein also has a significant inhibitory effect on the nuclear translocation and NF- ⁇ B p65 phosphorylation of NF- ⁇ B p65 subunits induced by LPS in Raw264.7 cells of mice.
  • the final concentration of baicalein is 10 ⁇ M and below. It has no obvious effect on the proliferation activity of mouse macrophages Raw264.7, but it has a significant inhibitory effect on the proliferation activity of human normal lung epithelial cells BEAS-2b at a concentration above 10 ⁇ M.
  • the results of this part show that baicalein has obvious anti-inflammatory effects in vitro.
  • mice BALB/c mice (30 mice), male, weighing 18-22 g, SPF grade, purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., license number is SCXK (Beijing) 2012-0001. Feeding conditions: 5 animals/cage, room temperature 24 ⁇ 1°C, humidity 60%-65%. During the experiment, they were free to eat, drink, and illuminate for 12h/d. Before starting the experiment, adapt the culture for 3 days.
  • Baicalein is provided by the Institute of Materia Medica, Chinese Academy of Medical Sciences, with a purity of >98%.
  • the compound When administering to animals, the compound is formulated into a suspension of suitable concentration with an aqueous solution of carboxymethyl cellulose (CMC-Na) for intragastric administration (the baicalein in the suspension is tested to be in ⁇ crystal form); for LPS Sterile normal saline was prepared into a mother liquor of 8mg/ml, and stored in aliquots at -80°C for later use.
  • CMC-Na carboxymethyl cellulose
  • mice were randomly divided into eight groups, namely the normal control group, the LPS model group, the positive drug aspirin group, the positive drug VX-765, the positive drug dexamethasone group, and the baicalein low-dose group (50mg/kg) , Baicalein middle-dose group (100mg/kg) and baicalein high-dose group (200mg/kg), each group has 10 mice.
  • Acute lung injury mouse model modeling process first use a small amount of ether to lightly anesthetize the mice, let them inhale 400 ⁇ g of LPS through the nose, a total of 50 ⁇ l (concentration of 8mg/ml); normal control mice inhale the same through the nose Volume of saline. After half an hour and 12 hours after inhalation, the mice were given the compound to be evaluated and the positive drug according to the dose.
  • mice in each group were monitored for animal lung function and blood was taken. After the animals were sacrificed, lung bronchial lavage was performed and the lavage fluid was collected. Finally, tissues and organs such as lung, spleen, and thymus were taken for determination.
  • TI expiration time
  • PPF maximum inspiratory flow
  • PEF maximum expiratory flow
  • TV tidal volume
  • EV expiratory volume
  • RT expiration per minute Air volume
  • MV respiratory rate
  • F end-inspiratory pause
  • EIP end-expiratory pause
  • EEP degree of bronchoconstriction
  • the lung tracheal lavage After the lung tracheal lavage, the lungs, thymus, and spleen of the mice were carefully harvested, cleaned up, and their wet weight was recorded.
  • the left lung was put into a 5ml centrifuge tube and weighed again, and then baked at 80°C for 48 hours, and the dry weight was weighed to calculate the wet-to-dry weight ratio of the lung.
  • Part of the left lung tissue was fixed in 4% paraformaldehyde solution for 48 hours for pathological section analysis; the remaining lung tissue was stored in liquid nitrogen or -80°C refrigerator for later use. The pathology of lung tissue was analyzed by HE staining.
  • Enzyme-linked immunosorbent assay was used to determine the levels of inflammation-related cytokines IL-1 ⁇ , IL-6 and TNF- ⁇ in serum or lung bronchial perfusion fluid. Refer to the instructions of the cytokine determination kit for specific steps.
  • the myeloperoxidase activity assay kit was used to determine the activity of myeloperoxidase in lung tissue. Refer to the kit instructions for specific steps.
  • the experimental data is expressed by Mean ⁇ SEM, using the Origin 9.0 software effect statistical chart; using the SPSS 22.0 software for statistical analysis, using the Bonferroni analysis method of single-factor variance and the Games-Howell analysis method to analyze the difference between the two, P ⁇ 0.05 Indicates a significant difference.
  • baicalein at a dose of 50 mg/kg significantly shortened the end-tidal pause time in mice with lung injury (P ⁇ 0.05, Figure 4A); baicalein at doses of 50 and 100 mg/kg It significantly reduced the maximum expiratory flow per unit body weight of mice with lung injury (P ⁇ 0.05, P ⁇ 0.01, Figure 4B); doses of baicalein at 50, 100, and 200 mg/kg on the bronchus of mice with lung injury Intensified contraction has a significant reduction effect (P ⁇ 0.05, P ⁇ 0.05, P ⁇ 0.05, Figure 4C); doses of baicalein at 50, 100, and 200 mg/kg have no significant effect on the respiratory rate of mice with lung injury (Figure 4C). 4D).
  • the cell infiltration area of the LPS model group accounted for 71.83 ⁇ 1.61% of the total area, which was significantly higher than 29.38 ⁇ 2.38% of the normal group (P ⁇ 0.001); and the low, medium, and high-dose baicalein groups were respectively 44.51 ⁇ 2.23%, 42.25 ⁇ 2.79% and 41.55 ⁇ 2.75%, which were significantly lower than the LPS model group (P ⁇ 0.001, P ⁇ 0.001, P ⁇ 0.001), as shown in Figure 5B.
  • the MPO enzyme activity in the lung tissue of the LPS model mice was significantly increased (P ⁇ 0.001); compared with the LPS model group, the baicalein at the dose of 100 and 200 mg/kg could significantly reduce the lung Damage to the MPO activity in the lung tissue of mice (P ⁇ 0.05, P ⁇ 0.05), while baicalein at a dose of 50 mg/kg only had a tendency to reduce the MPO enzyme activity, and there was no statistical difference, as shown in Figure 6.
  • inhalation of 400 ⁇ g of LPS through the nose can significantly increase the total cells (P ⁇ 0.001), neutrophils (P ⁇ 0.001) and macrophages (P ⁇ 0.001) in the bronchial perfusion fluid of mice. 0.001) concentration.
  • baicalein at doses of 100 and 200 mg/kg can significantly reduce the concentration of total cells in the perfusate (P ⁇ 0.001, P ⁇ 0.001, Figure 7A), but the dose of 50mg/kg Baicalein has no significant effect on reducing the concentration of total cells; baicalein at doses of 50, 100, and 200 mg/kg can significantly reduce neutrophils in lung bronchial perfusate of mice with lung injury (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.001, Figure 7B) and macrophage concentration (P ⁇ 0.05, P ⁇ 0.001, P ⁇ 0.001, Figure 7C).
  • inhalation of 400 ⁇ g LPS through the nose can significantly increase the inflammatory cytokines IL-1 ⁇ (P ⁇ 0.001), IL-6 (P ⁇ 0.001) and TNF- in the bronchial perfusion fluid of mice.
  • the concentration of ⁇ (P ⁇ 0.001).
  • baicalein at a dose of 50 mg/kg can significantly reduce the concentration of IL-6 in the perfusate (P ⁇ 0.05, Figure 8).
  • baicalein at a dose of 50 and 200 mg/kg can significantly reduce the level of IL-1 ⁇ in serum (P ⁇ 0.05, P ⁇ 0.05, Figure 9A), while a dose of 100 mg/kg of baicalein There was only a tendency to suppress this, and there was no statistical difference; baicalein at doses of 50, 100, and 200 mg/kg had no significant inhibitory effect on the concentration of IL-6 in mouse serum ( Figure 9B); doses of 100 and 200 mg/kg 200mg/kg of baicalein also had a significant inhibitory effect on serum TNF- ⁇ (P ⁇ 0.01, P ⁇ 0.01, Figure 9C), but the dose of 50mg/kg of baicalein only reduced serum TNF- ⁇ The trend is not statistically different.
  • the spleen index of the mice in the LPS model group increased significantly (P ⁇ 0.001).
  • baicalein at a dose of 50 and 100 mg/kg can significantly reduce the spleen index of mice with lung injury (P ⁇ 0.05, P ⁇ 0.05), while baicalein at a dose of 200 mg/kg has a significant effect on the spleen.
  • the index only has a decreasing trend, and there is no statistical difference, as shown in Figure 10.
  • baicalein at doses of 50, 100, and 200 mg/kg had an effect on the inflammatory body genes NLRP3 (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.01), AIM2 (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.01) and inflammatory factor gene TNF- ⁇ (P ⁇ 0.01, P ⁇ 0.01, P ⁇ 0.05) mRNA expression levels have a significant inhibitory effect; baicalein at a dose of 50mg/kg has a significant effect on Caspase-1 (P ⁇ 0.05).
  • baicalein can significantly shorten the end-expiratory pause time of lung-injured mice, significantly reduce the maximum expiratory flow per unit weight, and significantly reduce the degree of bronchoconstriction; baicalein can significantly reduce lung injury in mice Lung cell infiltration and the activity of MPO enzyme in lung tissue; Baicalein can also significantly reduce the concentration of inflammatory cells in the lung bronchial perfusion fluid, and to a certain extent can reduce the concentration of cytokines in the perfusion fluid; Baicalein can Significantly reduce the concentration of IL-1 ⁇ and TNF- ⁇ in the serum; baicalein can significantly reduce the spleen index of mice with lung injury.
  • baicalein can reduce the serum inflammatory factor content in mice with lung injury, and has a certain degree of therapeutic effect on LPS-induced acute lung injury.
  • Baicalein can interfere with the binding of virus S protein to ACE2
  • Viral Spike Glycoprotein S ( Figure 12, A, B, C) binds to the host ACE2 ( Figure 12, D) receptor protein to mediate the fusion of the viral membrane and the host membrane to achieve the infection of the host by the virus . After the virus enters the host cell, it is cleaved by the host protease and multiplies in large numbers, leading to serious consequences.
  • FIG. 13 The binding site of baicalein binding virus Spike protein and ACE2 is shown in Figure 13: (A) 3D diagram of interaction, (B) 2D diagram of interaction.
  • the results of computer virtual screening show that the key amino acids of baicalein attacking the site where ACE2 binds to the receptor include ILE(B)405, VAL(B)404, PEH(B)483, LYS(B)390, TYR(B) 481, ASP(B)393.
  • Baicalein can interact with key amino acids in the binding site of virus S-protein-ACE2, indicating that it is a potential active compound that acts on this target.
  • the SARS-CoV-2 infection hACE2 transgenic mouse model was used to evaluate the therapeutic effect of baicalein.
  • Test substance name Baicalein, ready to use, 10mg/ml
  • each group has 6 rats, baicalein 200mg/kg, calculated according to 200 ⁇ l/10g per mouse. Gavage, 1 hour after challenge, once a day for 5 consecutive days, the model group was given 0.5% sodium carboxymethyl cellulose in an equal volume.
  • mice After the mice were challenged, the mice were observed for 5 consecutive days, the general symptoms were observed, and the weight changes were recorded. On the 3rd and 5th days after infection, 3 mice in each group were euthanized to detect the viral load of lung tissue, and on the 5th day, one lung tissue of 1 mouse was taken for histopathological examination.
  • mice in the model group were in general condition. Some animals showed upright hair and weight loss, with the highest average drop rate of 4.55%. Compared with the model group, the average weight of mice in the baicalein group increased slightly, with the highest rate of increase being 1.59%. See Figure 14 and Table 6.
  • mice in each group were euthanized. Three mice were taken to collect lung tissue for load detection, and half of the lung tissue of one mouse was used for pathological examination.
  • the lung tissue load of mice in the model group was 105.94copies/ml and 104.14copies/ml on the 3rd and 5th day after infection.
  • the lung tissue viral load of mice in the baicalein group was 104.45 copies/ml on the 3rd day after infection, which was significantly lower than that of the model group mice (p ⁇ 0.01); the lung tissue viral load on the 5th day was 103.36 copies/ml, which was significantly lower Yu and the model group (p ⁇ 0.05). See Figure 15 and Table 7.
  • the lung tissues of the hACE2 transgenic model group mice infected with SARS-CoV-2 for 5 days showed diffuse moderate interstitial pneumonia, with widened alveolar septum, inflammatory cell infiltration, and a small amount of inflammatory cell infiltration around the blood vessels.
  • the lung tissues of mice in the baicalein-treated group showed diffuse mild to moderate interstitial pneumonia, with widened alveolar septum, inflammatory cell infiltration, and a small amount of inflammatory cell infiltration around blood vessels.
  • the lung tissue lesions of the baicalein treatment group were slightly improved.
  • Figure 16 shows the lung tissue lesions of the model group and the baicalein treatment group.
  • mice in the baicalein treatment group Compared with mice in the model group, mice in the baicalein treatment group generally performed better, their body weight did not decrease due to the infection of the virus and maintained an increase, the virus replication in the body was significantly inhibited, and the lung tissue lesions were slightly improved, indicating that baicalein (e.g. The 200mg/kg/d dose of baicalein) can inhibit the virus and improve lung inflammation in SARS-CoV-2 infected hACE2 transgenic mice.
  • baicalein e.g. The 200mg/kg/d dose of baicalein

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Abstract

黄芩素在制备预防和/或治疗新型冠状病毒感染引起的疾病的药物中的应用。具体而言，涉及黄芩素及含有黄芩素的药物组合物在制备用于预防和/或治疗新型冠状病毒(SARS-CoV-2)感染所引起疾病的药物中的应用，所述感染疾病包括由新型冠状病毒引起的轻症、中症、重症感染，尤其是新型冠状病毒肺炎。

Description

黄芩素在制备预防和/或治疗新型冠状病毒感染疾病的药物中的应用 技术领域
本发明属于医药技术领域，涉及黄芩素在制备预防和/或治疗新型冠状病毒感染疾病药物中的应用。具体而言，涉及黄芩素及含有黄芩素的药物组合物在预防和/或治疗新型冠状病毒(SARS-CoV-2，此后亦简称“新冠病毒”)感染疾病的药物中的应用，包括由新冠病毒引起的轻症、中症、重症感染，包括但不限于新冠肺炎。
背景技术
冠状病毒是一类具有囊膜的RNA病毒，由单一的核糖核酸RNA构成，属于单链RNA病毒。目前大约有15种不同冠状病毒株被发现，能够感染人、鼠、猪、猫、犬、禽类脊椎动物。冠状病毒(Coronaviruses，CoVs)属冠状病毒科(Coronaviridae)，根据血清型和基因组特点分为4属：Alpha Coronavirus(α-CoV)、Beta Coronavirus(β-CoV)、Gamma Coronavirus(γ-CoV)、Delta Coronavirus(δ-CoV)，可感染人和多种动物，引起宿主呼吸道、肠道、肝和神经系统疾病，其中又以β-CoVs对人类危害最烈。2018年，国际病毒分类委员会(International Committee on Taxonomy of Viruses，ICTV)将Beta Coronavirus属进一步分为5个亚属，即Embecovirus、Sarbecovirus、Merbecovirus、Nobecovirus和Hibecovirus，其中前4个亚属分别对应原Beta Coronavirus属之下的A、B、C、D等4个进化簇(lineages或clusters)，Hibecovirus亚属则由分离于中国蝙蝠、且与Sarbecovirus亚属在系统发生上近缘的一类β-CoVs提升而来。
2019年12月以来，湖北省武汉市陆续发现了多例新型冠状病毒(2019 novel corona virus，2019-nCoV)感染的肺炎患者，随着疫情的蔓延，已将该病纳入《中华人民共和国传染病防治法》规定的乙类传染病，并采取甲类传染病的预防、控制措施。2019-nCoV为β属的一种新型冠状病毒，该病毒会使患者感染肺炎，还可引起肠道、肝脏和神经系统的损害和相应症状。疫情急剧蔓延，迅速播散，但截至目前尚无特效药物。根据国家卫健委的通知，新型冠状病毒感染的肺炎统一称谓为“新型冠状病毒肺炎”，简称“新冠肺炎”，英文名为“Novel coronavirus pneumonia”，简称为“NCP”。随后，国际病毒分类委员会(International Committee on Taxonomy of Viruses,ICTV)宣布，新型冠状病毒(2019-nCoV)的正式分类名为严重急性呼吸综合征冠状病毒2(severe acute respiratory syndrome coronavirus 2，SARS-CoV-2)。世界卫生组织(WHO)也同日宣布，由这一病毒导致的疾病的正式名称为COVID-19。
可感染人的冠状病毒，包括2019新型冠状病毒(SARS-CoV-2)在内共有7种，不同类型病毒的致病力不同，引起的临床表现也不尽相同。常见的RNA病毒还包括艾滋病病毒、丙型肝炎病毒、乙型脑炎病毒、流感病毒、鼻病毒、脊髓灰质炎病毒、登革热病毒、SARS病毒、MERS病毒、埃博拉病毒(Ebola virus)等。
黄芩素(化学名：5,6,7-三羟基黄酮；英文名：Baicalien)又名黄芩苷元、黄芪素，是存在于唇形科(Labiatase)植物黄芩(Scutellaria baicalensis Georgi)干燥根中的主要黄酮类化合物，分子式为C 15H 10O 5，分量为270.24，具有式(I)的结构。
Figure PCTCN2020078252-appb-000001
黄芩素为黄酮类化合物，水溶性较差，在生物体内存在生物利用度低的成药性难题。申请人通过前期研究，在中国专利ZL200710177330.8中发明了黄芩素的一种新β晶型物质，β晶型的体内生物吸收率明显高于α晶型，约为α晶型的2倍以上。黄芩素β晶型可以显著提升化合物的体内生物利用度，为研发黄芩素相关固体口服药物制剂提供了可能。
黄芩素具有多种药理作用，包括：抗菌、抗病毒、抗炎、抗变态反应、抗氧化、清除自由基、抗肿瘤、抗帕金森、抗凝、抗血栓形成和保护肝脏、心脑血管和神经元等作用。其中，黄芩素的抗病毒活性也被多次报道，如抗双链DNA病毒、疱疹病毒、少数负链呼吸道病毒、日本脑炎病毒、登革热病毒等。
中国专利申请公开号CN104203265A中记载了马来西亚大学发明的“抗黄病毒的抗病毒组合物”，其中公开了一种预防或治疗人类或动物中黄病毒感染或由其所产生的疾病的具有抗病毒活性的组合物，其特征在于该组合物中包含黄芩素、其类似物或其衍生物。该组合物可进一步包含医药上可接受的载剂。所述抗病毒活性包括对病毒附着于宿主细胞的抑制、对细胞内病毒复制的抑制、及直接杀病毒的活性。黄病毒包含登革热病毒1型、登革热病毒2型、登革热病毒3型、登革热病毒4型及日本脑炎病毒。相较于未处理的细胞，50μg/ml的黄芩素预处理Vero细胞的登革热病毒灶的数目减少～37％及登革热病毒第2型RNA产生量减少39.5％±0.8。预处理细胞中黄芩素的IC 50为108.8μg/ml。相较于未处理细胞，25μg/ml的黄芩素对登革热病毒灶的量减少76.6％及DENV-2RNA生产量降低90.3％±1.6，且计算的IC 50值为7.14μg/ml。
中国专利申请公开号CN1925863A中公开了由凡诺华(英国)有限公司发明的“用于治疗SARS的黄芩属提取物”，其中涉及了含有黄芩属中的总标准化提取物具有抗冠状病毒活性，特别是抗引起严重急性呼吸器官综合征(SARS)的那些病毒的抗病毒活性，该黄芩属提取物PYN5C在最高浓度(200μg/ml)抑制SARS-CoV感染性约50％，该效果是剂量依赖性的，在更高水平的抑制作用大于利巴韦林(100μg/ml)。
文献《Baicalein and baicalin as Zika virus inhibitors》(Archives of Virology,164,585-593，2019)，记载了黄芩素和黄芩苷能在感染后10小时内下调寨卡病毒(Zika virus，ZIKV)的复制，而在预处理的细胞中具有明显的预防作用。黄芩素在细胞内ZIKV复制过程中表现出最高的效力，而黄芩苷对病毒进入的抑制作用最强。通过silico内相互作用分析预测，两种化合物对ZIKV NS5表现出最强的结合亲和力，而病毒包膜糖蛋白是最不可能的靶蛋白。这些发现为进一步深入研究每种化合物的潜在抗ZIKV机制提供了重要平台。
文献《Inhibitory Effects of Baicalein on the Influenza Virus in Vivo Is Determined by Baicalin in the Serum》(Biol.Pharm.Bull.33(2)238-243,2010)，记载了黄芩素在体内对流感病毒有抑制作用。对感染甲型流感病毒(H1N1)的BALB/c小鼠口服黄芩素，具有明显的预防死亡、增加平均死亡时间、抑制肺实变和降低肺病毒滴度的作用。这些作用被认为是 由于血清中黄芩素的代谢产物黄芩苷。在覆盖培养基中，当黄芩苷浓度为2mg/ml时，在菌斑试验中表现出明显的抑制作用，细胞病变试验中黄芩苷的平均IC 50值为1.2mg/ml。结果表明，黄芩素作为一种潜在的抗流感病毒药物值得进一步研究。
文献《Human cytomegalovirus-inhibitory flavonoids:Studies on antiviral activity and mechanism of action.》(Antiviral Research,68(3),124-134，2005)，记载了饮食类黄酮对人巨细胞病毒(HCMV)的抗病毒作用及其可能的生化作用机制。十个评价的黄酮类化合物中的九个在浓度显著低于产生生长或静止期宿主细胞的细胞毒性的浓度下阻断了HCMV复制。黄芩素是该系列药物中最有效的抑制剂(IC 50＝0.4-1.2μM)，阳性对照更昔洛韦也不例外。以黄芩素和染料木素为模型化合物，两种黄酮均显著降低HCMV早期和晚期蛋白水平，并降低病毒DNA合成。黄芩素可将HCMV即刻早期蛋白水平降低到接近背景水平，而染料木素则没有。染料木素(genistein)的抗病毒作用在细胞培养中是完全可逆的。在浓缩的病毒培养液中加入黄酮类物质，对HCMV的复制没有抑制作用，说明黄芩素不能直接灭活病毒颗粒。黄芩素能阻断表皮生长因子受体酪氨酸激酶活性和HCMV核移位，而染料木素则不能。感染后24小时，染料木素处理的HCMV感染细胞继续表达即时早期蛋白，并有效磷酸化IE1-72。然而，HCMV诱导NF-κB和细胞周期调节蛋白水平的增加与即刻早期蛋白功能相关的事件是不存在的。提示黄芩素的主要作用机制可能是阻断HCMV的感染，而genistein的主要作用机制可能是阻断HCMV的即刻早期蛋白功能。
文献《Binding Aspects of Baicalein to HIV-1 Integrase》(Mol.Cells,12(1),127-130，2001)，记载了利用荧光光谱和圆二色谱(CD)研究了抑制剂黄芩素对HIV-1整合酶催化结构域In-(50-212/F185K)构象的影响。发现黄芩素与HIV-1整合酶催化核心区的疏水区结合，这种结合诱导了酶的构象变化。黄芩素与HIV-1整合酶催化结构域的结合率为2:1。
文献《Remdesivir and chloroquine effectively inhibit the recently emerged novel coronavirus(2019-nCoV)in vitro》(Cell Research,0:1–3,2020)，记载了瑞德西韦、磷酸氯喹、利巴韦林、硝唑尼特、萘莫司他等抗病毒药物在体外的抗SARS-CoV-2活性，结果显示5种药物对新冠状病毒的半数有效浓度(EC 50)分别为0.77μm、1.13μM、109.50μM、2.12μM、22.50μM，同时报道了喷昔洛韦、法匹拉韦的EC 50值分别为95.96μM，61.88μM。由实验结果可知，瑞德西韦体外抗SARS-CoV-2活性非常显著。
发明内容
本申请的一个或多个实施方式提供了黄芩素在制备用于预防和/或治疗由新型冠状病毒感染引起的疾病的药物中的用途。
本申请的一个或多个实施方式提供了用于预防和/或治疗新型冠状病毒感染的药物组合物，其包含黄芩素和药学上可接受的载体、辅料或赋形剂，和/或一种或多种另外的抗病毒剂或抗生素。
在本申请的一个或多个实施方式中，所述黄芩素为β晶型黄芩素。
在本申请的一个或多个实施方式中，所述由新型冠状病毒感染引起的疾病为新型冠状病毒肺炎。
在本申请的一个或多个实施方式中，所述新型冠状病毒为SARS-CoV-2。
在本申请的一个或多个实施方式中，所述抗病毒剂为瑞德西韦、磷酸氯喹、利巴韦林、 硝唑尼特、萘莫司他、喷昔洛韦、法匹拉韦、阿比多尔和/或金刚烷胺。
在本申请的一个或多个实施方式中，所述抗生素为内酰胺类、大环内酯类、头孢类和/或磺胺类抗生素。
在本申请的一个或多个实施方式中，所述药物组合物中的黄芩素的含量为0.1至99.9重量％。
在本申请的一个或多个实施方式中，所述黄芩素的含量为50至99.9重量％。
在本申请的一个或多个实施方式中，所述黄芩素的含量为50、60、70、80、90、99重量％
在本申请的一个或多个实施方式中，所述黄芩素的含量为80至99.9重量％。
在本申请的一个或多个实施方式中，所述药物组合物为固体制剂。
在本申请的一个或多个实施方式中，所述固体制剂包括片剂、胶囊、丸剂、注射用制剂、缓释或控释制剂。
本申请的一个或多个实施方式提供了本申请的药物组合物在制备用于预防和/或治疗由新型冠状病毒感染引起的疾病的药物中的用途。
在本申请的一个或多个实施方式中，所述新型冠状病毒为SARS-CoV-2。
在本申请的一个或多个实施方式中，所述新型冠状病毒感染包括轻症、中症和重症感染。
在本申请的一个或多个实施方式中，所述由新型冠状病毒感染引起的疾病包括由所述新型冠状病毒引起的肺炎或肠炎。
在本申请的一个或多个实施方式中，所述黄芩素抑制所述新型冠状病毒的感染、抑制所述新型冠状病毒的复制、或对新型冠状病毒引起的组织损伤具有保护和治疗作用。
在本申请的一个或多个实施方式中，其中所述黄芩素的每日给药剂量为50-1000mg。
在本申请的一个或多个实施方式中，其中针对老人和儿童的每日给药剂量为50-800mg。
在本申请的一个或多个实施方式中，其中所述黄芩素的每日给药剂量为50、100、200、300、400、500、600、700、800、900、1000mg。
在本申请的一个或多个实施方式中，其中所述黄芩素为β晶型黄芩素。
本申请的一个或多个实施方式提供了治疗由由新型冠状病毒感染引起的疾病的方法，其包括给予有此需要的对象治疗有效量的黄芩素或包含黄芩素的组合物。
本申请的一个或多个实施方式提供了黄芩素或包含黄芩素的组合物，其用于预防和/或治疗由新型冠状病毒感染引起的疾病的方法。
发明详述
尽管黄芩素对许多常见人类病毒的抗病毒活性已被广泛报道，但其对于新型冠状病毒(SARS-CoV-2)的活性尚无人报道。本发明人在分子筛选、体外细胞和实验动物模型上，使用黄芩素对抗新冠病毒的活性取得了预料不到的技术效果，其半数有效浓度EC 50与瑞德 西韦相当，显著高于利巴韦林、洛匹那韦-利托那韦等经典抗病毒药物，完成了本发明。
本发明的第一个方面要解决的技术问题是提供黄芩素在制药中的新用途，即黄芩素在制备预防和/或治疗新型冠状病毒感染引起的疾病的药物中的应用。
本发明中预防和/或治新冠病毒(SARS-CoV-2)感染，包括由新型冠状病毒引起的轻、中、重症感染，包括但不限于新冠肺炎(NCP)。
根据本发明，黄芩素可以制备用于预防和/或治疗新型冠状病毒感染的药物组合物，该药物组合物含有黄芩素和药学上可接受的载体，其中优选含有有效剂量的黄芩素β晶型。
与黄芩素β晶型相关的技术内容已经公开在专利文献第ZL200710177330.8中，在此声明将相关内容一并并入本发明专利文件中。
根据本发明的应用，其中预防和/或治疗新型冠状病毒感染是指具有预防性感染后抗病毒活性、直接杀病毒活性或者对病毒附着至宿主细胞的抑制。
在本发明的用途中，黄芩素的每日用药剂量在50-1000mg范围内，例如50、100、200、300、400、500、600、700、800、900、1000mg。
本发明的另一个方面还涉及以黄芩素作为活性成份的药物组合物。该药物组合物可根据本领域公知的方法制备。可通过将黄芩素与一种或多种药学上可接受的固体或液体赋形剂和/或辅剂结合，制成适于人或动物使用的任何剂型。本发明黄芩素在其药物组合物中的含量通常在0.1％～100％重量范围内，例如1％、10％、20％、30％、40％、50％、60％、70％、80％、90％。
本发明黄芩素可以单位剂量形式给药，给药途径可为经肠道或非肠道，如经口服、静脉注射、肌肉注射、皮下注射、鼻腔、口腔粘膜、眼、肺和呼吸道、皮肤、阴道、直肠等。
本发明的给药剂型优选是固体剂型。固体剂型可以是片剂，包括普通片、肠溶片、含片、分散片、咀嚼片、泡腾片、口腔崩解片；胶囊剂，包括硬胶囊、软胶囊、肠溶胶囊；颗粒剂；散剂；微丸；滴丸；栓剂；膜剂；贴片、气(粉)雾剂；喷雾剂等。
本发明黄芩素可以制成普通制剂、也可制成缓释制剂、控释制剂、靶向制剂及各种微粒给药系统。
为了将本发明黄芩素制成片剂，可以广泛使用本领域公知的各种赋形剂，包括稀释剂、黏合剂、润湿剂、崩解剂、润滑剂、助流剂。稀释剂可以是淀粉、糊精、蔗糖、葡萄糖、乳糖、甘露醇、山梨醇、木糖醇、微晶纤维素、硫酸钙、磷酸氢钙、碳酸钙等；湿润剂可以是水、乙醇、异丙醇等；粘合剂可以是淀粉浆、糊精、糖浆、蜂蜜、葡萄糖溶液、微晶纤维素、阿拉伯胶浆、明胶浆、羧甲基纤维素钠、甲基纤维素、羟丙基甲基纤维素、乙基纤维素、丙烯酸树脂、卡波姆、聚乙烯吡咯烷酮、聚乙二醇等；崩解剂可以是干淀粉、微晶纤维素、低取代羟丙基纤维素、交联聚乙烯吡咯烷酮、交联羧甲基纤维素钠、羧甲基淀粉钠、碳酸氢钠与枸橼酸、聚氧乙烯山梨糖醇脂肪酸酯、十二烷基磺酸钠等；润滑剂和助流剂可以是滑石粉、二氧化硅、硬脂酸盐、酒石酸、液体石蜡、聚乙二醇等。
还可以将片剂进一步制成包衣片，例如糖包衣片、薄膜包衣片、肠溶包衣片，或双层片和多层片。
为了将给药单元制成胶囊剂，可以将有效成分与稀释剂、助流剂混合，将混合物直接置于硬胶囊或软胶囊中。也可将有效成分先与稀释剂、黏合剂、崩解剂制成颗粒或微丸，再置于硬胶囊或软胶囊中。用于制备本发明黄芩素片剂的各种稀释剂、黏合剂、润湿剂、崩解剂、助流剂品种也可用于制备本发明黄芩素的胶囊剂。
此外，如需要，也可以向药物制剂中添加着色剂、防腐剂、香料、矫味剂或其它添加剂。
为达到用药目的，增强治疗效果，本发明的药物可用任何公知的给药方法给药。
本发明黄芩素及含有黄芩素的药物组合物，其给药剂量依照所要预防或治疗疾病的性质和严重程度，患者或动物的个体情况，给药途径和剂型等可以有大范围的变化。上述剂量可以一个剂量单位或分成几个剂量单位给药，这取决于医生的临床经验以及包括运用其它治疗手段的给药方案。
本发明黄芩素或组合物可单独服用，或与其他治疗药物或对症药物合并使用。当本发明黄芩素与其它治疗药物存在协同作用时，应根据实际情况调整它的剂量。
本发明涉及黄芩素及含有黄芩素的药物组合物在预防和/治疗新冠状病毒感染的药物中的应用，包括但不限于新冠肺炎。
本发明发现黄芩素在抗新冠状病毒方面的活性取得了预料不到的技术效果的至少一种：
1.本发明应用的黄芩素在分子水平的计算机虚拟筛选中，与SARS-CoV-2冠状病毒关键蛋白3CL水解酶(Mpro)活性位点中的关键的氨基酸产生较强相互作用。
2.本发明应用的黄芩素在分子水平的计算机虚拟筛选中，能够与病毒Spike蛋白与ACE2结合位点中的关键氨基酸产生较强相互作用。
3.本发明的黄芩素应用在细胞水平测试抗冠状病毒的活性时，采用100TCID50的病毒感染剂量，在非洲绿猴肾细胞(VERO细胞)模型上，进行病毒感染力的测定，结果显示黄芩素在0.1μM、1μM两个剂量下均对新冠病毒有预料不到的显著抑制作用，其抑制能力与瑞德西韦相当。
4.本发明的黄芩素应用在SARS-CoV-2感染hACE2转基因小鼠模型上，小鼠体重没有因为感染病毒下降且维持上升，体内病毒复制被显著抑制，肺组织病变有轻度改善，可见黄芩素对SARS-CoV-2感染hACE2转基因小鼠有一定的病毒抑制和改善肺部炎症的作用。
5.本发明的黄芩素应用在体外细胞模型上对肺炎具有预料不到的药理作用，具体而言，黄芩素对Raw264.7细胞内NF-κB核转位具有显著的抑制作用，对细胞内NF-κB磷酸化具有显著的抑制作用。
6.本发明的黄芩素应用在整体动物水平上对LPS诱导的急性肺炎损伤具有显著的治疗作用，具体表现在对模型小鼠呼吸功能的改善、组织病理变化的改善、肺组织中MPO活性的影响、支气管灌流液中炎性细胞浓度、细胞因子浓度、血清中细胞因子浓度的影响、脾脏指数的影响、以及肺组织中炎症相关基因mRNA表达水平的影响等方面。
7.本发明的黄芩素应用已完成I期临床试验研究，临床试验结果显示其具有良好的安全性。
附图说明
图1SARS-CoV-2冠状病毒3CL水解酶(Mpro)与黄芩素的相互作用图(A：三维相互作用图，B：二维相互作用图)；
图2黄芩素对Raw264.7细胞内NF-κB核转位的抑制作用；
图3黄芩素对细胞内NF-κB磷酸化的抑制作用；
图4黄芩素对肺损伤小鼠的呼气末期停顿时间的作用(A)；对肺损伤小鼠的单位体重最大呼气流量的作用(B)；对肺损伤小鼠的支气管收缩程度作用(C)；对肺损伤小鼠的呼吸频率的影响(D)；
图5黄芩素对急性肺损伤小鼠肺组织病理变化的改善作用；
图6黄芩素对急性肺损伤小鼠肺组织中MPO活性的影响；
图7黄芩素对小鼠肺支气管灌流液中炎性细胞浓度的影响；
图8黄芩素对小鼠肺支气管灌流液中细胞因子浓度的影响；
图9黄芩素对小鼠血清中细胞因子浓度的影响；
图10黄芩素对急性肺损伤小鼠脾脏指数的影响；
图11黄芩素对肺组织中炎症相关基因mRNA表达水平的影响；
图12病毒棘突糖蛋白S(Spike Glycoprotein，A，B，C)利用ACE2(D)介导病毒膜和宿主膜的融合；
图13黄芩素与病毒S-蛋白-ACE2结合位点的相互作用图；
图14模型组与黄芩素治疗组小鼠体重下降率图；
图15模型组与黄芩素治疗组肺组织病毒载量图；
图16模型组与黄芩素治疗组肺组织病变图。
具体实施方式
为示例说明本发明的技术方案，特给出以下实施例，但本发明并不仅限于此。
以下各实施例中的新冠病毒(SARS-CoV-2)均由中国医学科学院医学实验动物研究所病原中心保存并提供，黄芩素由中国医学科学院药物研究所提供，纯度>98％，均为β晶型。
实施例1
计算机虚拟对接预测黄芩素在分子水平抗冠状病毒的活性
利用SARS-CoV-2冠状病毒关键蛋白3CL水解酶(Mpro)的高分率晶体结构(注：晶体结构的坐标可由PDB蛋白质结构数据库(Protein Data Bank,PDB)下载(PDB ID:6LU7))，采用Discovery studio软件进行计算机虚拟对接，分子对接结果见表1所示。图1所示为Mpro与黄芩素的相互作用图。
表1黄芩素与Mpro的对接结果
黄芩素 对接能量
构象1 34.8954
构象2 34.4027
构象3 31.7587
构象4 31.6989
构象5 31.4707
构象6 31.3751
构象7 30.9768
构象8 30.9701
构象9 30.9509
构象10 30.8861
计算机虚拟筛选的结果表明：黄芩素结合SARS-CoV-2冠状病毒关键蛋白3CL水解酶(Mpro)的关键氨基酸包括HIS41、MET49、PHE140、CYS145、HIS163、MET165、GLU166、GLN189。黄芩素能够与活性位点中关键的氨基酸产生相互作用，因此具有作用于该靶点的潜在利用价值。
实施例2
黄芩素对SARS-CoV-2新冠病毒感染力的影响研究
【受试药物】：药物名称及浓度见表2。
【细胞】：VeroE6细胞，由中国医学科学院医学实验动物研究所病原中心保存并提供。
【病毒】：新冠病毒(SARS-CoV-2)，滴度为105TCID50/ml，由中国医学科学院医学实验动物研究所病原中心-80℃保存。使用病毒滴度为100TCID50。
【实验方法】
(1)无菌96孔培养板，每孔加入200μl浓度为5×10 4cell/ml Vero E6细胞，37℃下5％CO 2培养24小时；
(2)受试药物稀释成2个浓度，每个浓度5个复孔，每孔100μl，然后每孔再加入等体积100TCID 50病毒，作用1h；
(4)1h后，弃去96孔培养板中细胞培养液，加入上述混合液；
(5)同时设立细胞对照、空白对照(溶剂对照)和病毒对照(阴性对照)；
(6)细胞37℃，5％CO 2孵箱孵育4-5天；
(7)光学显微镜下观察细胞病变(CPE)，细胞完全病变记录为“++++”，75％病变记录为“+++”，50％病变记录为“++”，25％病变记录为“+”，未病变记录为“－”。
【实验条件】：以上实验操作均在BSL-3实验室内完成
【结果判断】：细胞不出现CPE为有效抑制病毒的浓度，出现CPE为无效。
【实验结果】
采用100TCID50的病毒感染剂量，在非洲绿猴肾细胞(VeroE6细胞)模型上，分别使用0.1μM、1μM两个剂量的黄芩素与瑞德西韦进行病毒感染力的测定研究，研究结果如表2所示。
表2不同剂量的黄芩素与瑞德西韦对SARS-CoV-2新型冠状病毒感染力数据
Figure PCTCN2020078252-appb-000002
【结论】
根据细胞水平的筛选结果，黄芩素在0.1μM浓度下，可抑制SARS-CoV-2对细胞的损伤，提示黄芩素具有体外抗SARS-CoV-2活性，其作用强度与瑞德西韦一致(细胞实验在给药后作用4天以上)。
表3黄芩素抗SARS-CoV-2作用分析
Figure PCTCN2020078252-appb-000003
细胞实验显示0.1μM的黄芩素对细胞具有100％的保护作用。
实施例3
黄芩素在细胞水平对肺炎的药理作用研究
选择炎性刺激物细菌脂多糖(LPS)和黄芩素，利用体外细胞模型，利用体外细胞模型，考察炎性刺激物及黄芩素的细胞毒性和对小鼠巨噬细胞Raw264.7内炎症相关信号途径的影响，以确证所选黄芩素的抗炎活性。此外，采用正常人肺支气管上皮细胞BEAS-2b，观察黄芩素对正常肺细胞增殖活性的作用特点和选择性。
【样品配制】
黄芩素(Baicalein，BC)由中国医学科学院药物研究所提供，其结构如式(I)所示，纯度>98％；Caspase-1小分子抑制剂VX-765购自上海瀚香生物科技有限公司纯度>98％；非甾体类抗炎药阿司匹林(Acetylsalicylic acid or Aspirin,ASP)和甾体类抗炎药地塞米松(Dexamethasone,DEX)购于Sigma公司，纯度>99％；化合物均用DMSO配制成100mM的母 液，于-20℃保存备用。LPS用无菌生理盐水配制成8mg/ml的母液，分装保存于-80℃备用。
【实验方法】
1.细胞的培养
小鼠巨噬细胞株Raw264.7培养于含有10％国产FBS、100U/ml青霉素和100μg/ml链霉素的RPMI-1640培养基中；培养条件为37℃，5％CO 2，80％湿度；2天传代一次，更换新鲜的完全培养基。正常人肺支气管上皮细胞BEAS-2b培养于含有10％进口FBS、100U/ml青霉素和100μg/ml链霉素的RPMI-1640培养基中；培养条件为37℃，5％CO 2，80％湿度；3天传代一次，传代时更换新鲜的完全培养基。
2.细胞增殖毒性实验
将处于对数生长期的细胞种于96孔培养板，1.0×10 4个细胞每孔，37℃，5％CO 2，湿度为80％的条件下培养24小时；用不同终浓度的LPS或化合物溶液在37℃条件下处理细胞24小时；使用MTT法检测细胞活力；使用倒置荧光显微镜拍摄经甲醛溶液固定的细胞，记录细胞的形态；用高内涵分析仪分析细胞核形态，统计细胞核的平均荧光强度。
3.细胞免疫荧光
将处于对数生长期的小鼠巨噬细胞Raw264.7种于黑色底透的96孔培养板，2.0×10 4个细胞每孔，培养基为含有10％国产FBS、100U/ml青霉素和100μg/ml链霉素的RPMI-1640；37℃，5％CO 2，湿度为80％的条件下培养24小时；加入浓度为最终浓度2倍的无血清1640培养基稀释的LPS溶液，轻轻振板混匀，使黄芩素最终的浓度分别为0.1、1、10μM，地塞米松、阿司匹林和VX-765的最终浓度分别为10μM，而LPS的终浓度为2μg/ml；甲醛固定，进行免疫荧光实验，用高内涵分析仪分析细胞内NF-κB p65的核转位程度。
4.免疫印迹分析
利用免疫印迹分析小鼠巨噬细胞Raw264.7中NF-κB p65和TAK1的磷酸化情况。具体操作步骤如下：将处于对数生长期的小鼠巨噬细胞Raw264.7接种于35mm培养皿，1.0×10 6个细胞每孔，培养基为含有10％国产FBS、100U/ml青霉素和100μg/ml链霉素的RPMI-1640，2ml培养基每皿；37℃，5％CO 2，湿度为80％的条件下培养24小时；加入浓度为最终浓度2倍的无血清RPMI-1640培养基稀释的LPS溶液，轻轻振摇混匀，使黄芩素最终的浓度分别为0.1、1.0、10μM，地塞米松、阿司匹林和VX-765的最终浓度分别为10μM，而LPS的终浓度为1μg/ml，共1ml/皿；37℃继续培养24小时后，进行蛋白提取，利用免疫印迹(western blot)，用FluorChem 5500分析软件对条带进行灰度扫描，并对其进行定量分析。
5.统计方法
实验数据用Mean±S.D.或Mean±S.E.M.表示，采用Origin 9.0软件作用统计图；采用SPSS 22.0软件进行统计分析，用单因素方差的Bonferroni分析法和Games-Howell分析法分析两两之间的差异性，P<0.05表示具有显著性差异。
【实验结果】
1.黄芩素对Raw264.7细胞内NF-κB核转位的抑制作用：
与LPS处理细胞相比，1.0和10μM的黄芩素对LPS诱导的NF-κB p65的核转位具有显著性抑制作用(P<0.05,P<0.01)，而0.1μM的黄芩素对LPS诱导的Raw264.7细胞内NF-κB p65的核转位没有抑制作用，如图2示。
2.黄芩素对细胞内NF-κB磷酸化的抑制作用：
与LPS处理组细胞相比，10μM的黄芩素也能够显著性的抑制细胞内NF-κB p65的磷酸化(P<0.05)；黄芩素对非磷酸化的NF-κB p65含量也无明显的影响，如图3所示。
【结论】
本实验表明，黄芩素对LPS诱导致小鼠Raw264.7细胞内NF-κB p65亚基发生的核转位和NF-κB p65磷酸化也具有明显的抑制作用，10μM及以下终浓度的黄芩素对小鼠巨噬细胞Raw264.7的增殖活性没有明显影响，但在10μM以上浓度对人正常肺上皮细胞BEAS-2b的增殖活性具有明显的抑制作用。该部分结果表明黄芩素在体外具有明显的抗炎作用。
实施例4
黄芩素在整体动物水平对LPS诱导急性肺炎损伤的治疗作用
【实验动物】
BALB/c小鼠(30只)，雄性，体重为18-22g，SPF级，购自北京维通利华实验动物技术有限公司，许可证号为SCXK(京)2012-0001。饲养条件：5只/笼，室温24±1℃，湿度60％～65％。实验期间自由摄食、饮水，光照12h/d。开始实验前，适应性培养3天。
【样品配制】
黄芩素由中国医学科学院药物研究所提供，纯度>98％。动物给药时，将化合物用羧甲基纤维素钠(CMC-Na)水溶液配制成浓度适合的混悬液灌胃给药(经检测混悬液中黄芩素为β晶型状态)；LPS用无菌生理盐水配制成8mg/ml的母液，分装保存于-80℃备用。
【实验方法】
1.动物分组、模型制备和给药
将BALB/c小鼠随机分为八组，分别为正常对照组、LPS模型组、阳性药阿司匹林组、阳性药VX-765、阳性药地塞米松组、黄芩素低剂量组(50mg/kg)、黄芩素中剂量组(100mg/kg)和黄芩素高剂量组(200mg/kg)，每组10只小鼠。
采用先造模后给药的方式考察黄芩素对LPS诱导急性肺损伤模型小鼠的治疗作用。急性肺损伤小鼠模型造模过程：先用少量乙醚对小鼠进行轻度麻醉，让其经鼻吸入400μg的LPS，共50μl(浓度为8mg/ml)；正常对照组小鼠经鼻吸入同体积的生理盐水。小鼠吸入后半小时和12小时，分别按照剂量给动物灌胃待评价的化合物及阳性药物。
各组小鼠在造模24小时后，进行动物肺功能监测，并取血。动物处死后进行肺支气管灌洗并收集灌洗液，最后取肺、脾、胸腺等组织器官进行测定。
2.动物肺功能监测
LPS诱导小鼠急性肺损伤造模24小时后，用EMKA清醒动物肺功能监测系统(Whole Body Plethysmography,WBP)监测各组小鼠在清醒无束缚状态下的肺呼吸情况，统计小鼠吸气时间(TI)、呼气时间(TE)、最大吸气流量(PIF)、最大呼气流量(PEF)、潮气量(TV)、呼气量(EV)、呼吸松弛时间(RT)、每分钟呼气量(MV)、呼吸频率(F)、吸气末期停顿(EIP)、呼 气末期停顿(EEP)、支气管收缩的程度(PENH)等12种呼吸功能相关的指标。
3.血清的收集
小鼠呼吸功能监测结束后，记录其体重；经皮下注射适量5％的水合氯醛溶液进行轻度麻醉，摘取眼球取血；室温静置60分钟后，4℃，5000rpm离心10分钟，收集的血清于-80℃保存备用。
4.肺气管灌流液的收集
取血后，暴露小鼠颈部气管，用0.3ml的预冷灭菌PBS进行肺气管插管灌洗，收集灌流液，每次0.3ml。三次混合后，4℃，1500rpm离心5分钟，上清保存于-80℃备用。沉淀用新的50μl预冷灭菌PBS重悬，保存4℃用于细胞分类统计。
5.组织器官的摘取
肺气管灌洗结束后，小心摘取小鼠的肺脏、胸腺和脾脏等器官，清理干净，记录其湿重。左肺装入5ml离心管再称湿重，然后80℃烘烤48小时，称干重以计算肺的湿干重比。部分左肺组织固定在4％的多聚甲醛溶液中48小时，用于病理切片分析；其余肺组织保存于液氮或-80℃冰箱中备用。用HE染色对肺组织病理进行分析。
6.一氧化氮含量的测定
采用Griess试剂法检测肺支气管灌流液中一氧化氮(Nitric oxide,NO)的含量。
7.炎症相关细胞因子的测定
采用酶联免疫吸附实验(ELISA)测定血清或肺支气管灌流液中的炎症相关细胞因子IL-1β、IL-6和TNF-α的含量。具体步骤参考细胞因子测定试剂盒说明书进行。
8.髓过氧化物酶活性的测定
采用髓过氧化物酶活性测定试剂盒测定肺组织中髓过氧化物酶的活性。具体步骤参考试剂盒说明书进行。
9.统计方法
实验数据用Mean±S.E.M.表示，采用Origin 9.0软件作用统计图；采用SPSS 22.0软件进行统计分析，用单因素方差的Bonferroni分析法和Games-Howell分析法分析两两之间的差异性，P<0.05表示具有显著性差异。
【实验结果】
1.黄芩素对急性肺损伤小鼠呼吸功能的影响
BALB/c小鼠呼吸功能指标的监测统计结果显示，与正常对照组相比，LPS模型组小鼠的呼气末期停顿时间显著性延长(P<0.001)、单位体重的最大呼气流量显著增大(P<0.01)、支气管收缩的程度明显加剧(P<0.001)、呼吸频率明显下降(P<0.001)。与LPS模型组相比，剂量为50mg/kg的黄芩素对肺损伤小鼠的呼气末期停顿时间有显著的缩短作用(P<0.05,图4A)；剂量为50、100mg/kg的黄芩素对肺损伤小鼠的单位体重最大呼气流量有显著性的降低作用(P<0.05,P<0.01,图4B)；剂量为50、100和200mg/kg的黄芩素对肺损伤小鼠的支气管收缩程度加剧有明显的降低作用(P<0.05,P<0.05,P<0.05,图4C)；剂量为50、100和200mg/kg的黄芩素对肺损伤小鼠的呼吸频率没有明显影响(图4D)。
2.黄芩素对急性肺损伤小鼠肺组织病理变化的改善作用
与正常对照组相比，经鼻腔吸入LPS的BALB/c小鼠肺组织中充斥着大量的细胞，肺泡空间被大量浸润的细胞所替代，肺泡结构发生明显变化。然而，与LPS模型组相比，剂量为50、100、200mg/kg的黄芩素均对肺部细胞浸润有改善作用(图5A)。经统计，LPS模型组的细胞浸润面积占总面积的71.83±1.61％，显著性地高于正常组的29.38±2.38％(P<0.001)；而黄芩素的低、中、高剂量组分别为44.51±2.23％、42.25±2.79％和41.55±2.75％，显著地低于LPS模型组(P<0.001,P<0.001,P<0.001)，如图5B所示。
3.黄芩素对急性肺损伤小鼠肺组织中MPO活性的影响
与正常对照组相比，LPS模型小鼠肺组织中MPO酶的活性显著升高(P<0.001)；与LPS模型组相比，剂量为100、200mg/kg的黄芩素均能够显著性降低肺损伤小鼠肺组织中的MPO活性(P<0.05,P<0.05)，而剂量为50mg/kg的黄芩素只有降低MPO酶活性的趋势，没有统计学差异，如图6所示。
4.黄芩素对小鼠肺支气管灌流液中炎性细胞浓度的影响
与正常对照组相比，经鼻腔吸入400μg的LPS能够显著性地增加小鼠肺支气管灌流液中的总细胞(P<0.001)、中性粒细胞(P<0.001)和巨噬细胞(P<0.001)的浓度。与LPS模型组相比，剂量为100、200mg/kg的黄芩素能够显著性地降低灌流液中的总细胞的浓度(P<0.001,P<0.001，图7A)，但剂量为50mg/kg的黄芩素对总细胞的浓度没有明显的降低作用；剂量为50、100、200mg/kg的黄芩素能够显著性地降低肺损伤小鼠肺支气管灌流液中的中性粒细胞(P<0.01,P<0.01,P<0.001，图7B)和巨噬细胞浓度(P<0.05,P<0.001,P<0.001，图7C)。
5.黄芩素对小鼠肺支气管灌流液中细胞因子浓度的影响
与正常对照组相比，经鼻腔吸入400μg的LPS能够显著性地升高小鼠肺支气管灌流液中的炎症细胞因子IL-1β(P<0.001)、IL-6(P<0.001)和TNF-α(P<0.001)的浓度。与LPS模型组相比，剂量为50mg/kg的黄芩素能够显著性地降低灌流液中IL-6的浓度(P<0.05，图8)。
6.黄芩素对小鼠血清中细胞因子浓度的影响
与正常对照组相比，LPS模型组小鼠的外周血清中炎症细胞因子IL-1β(P<0.001)、IL-6(P<0.001)和TNF-α(P<0.001)均显著性地升高。与LPS模型组相比，剂量为50、200mg/kg的黄芩素能够显著性地降低血清中IL-1β的含量(P<0.05,P<0.05,图9A)，而剂量为100mg/kg的黄芩素对此只有抑制的趋势，没有统计学差异；剂量为50、100、200mg/kg的黄芩素对小鼠血清中IL-6的浓度没有显著性的抑制作用(图9B)；剂量为100和200mg/kg的黄芩素对血清中TNF-α也具有显著性的抑制作用(P<0.01,P<0.01,图9C)，但剂量为50mg/kg的黄芩素对血清中TNF-α只具有降低的趋势，没有统计学差异。
7.黄芩素对急性肺损伤小鼠脾脏指数的影响
与正常对照组相比，LPS模型组小鼠的脾脏指数显著性地增加(P<0.001)。与LPS模型组相比，剂量为50、100mg/kg的黄芩素能够显著性地降低肺损伤小鼠的脾脏指数(P<0.05,P<0.05)，而剂量为200mg/kg的黄芩素对脾脏指数只有降低的趋势，没有统计学差异，如图10所示。
8.黄芩素对肺组织中炎症相关基因mRNA表达水平的影响
与LPS模型组相比，剂量为50、100、200mg/kg的黄芩素对炎性小体基因NLRP3(P<0.01, P<0.01,P<0.01)、AIM2(P<0.01,P<0.01,P<0.01)和炎症因子基因TNF-α(P<0.01,P<0.01,P<0.05)的mRNA表达水平有显著性地抑制作用；剂量为50mg/kg的黄芩素对Caspase-1(P<0.05)和IL-1β(P<0.01)的mRNA表达有明显地抑制作用，另两个剂量组的黄芩素降低作用不明显；剂量为100、200mg/kg的黄芩素对GAPDH(P<0.05,P<0.05)基因的mRNA表达水平有显著性地升高作用，但剂量为50mg/kg的黄芩素对此无明显的影响；与LPS模型组相比，剂量为50、100、200mg/kg的黄芩素对NLRP1、NLRC4、ASC和IL-6等4个炎症相关基因的mRNA表达水平没有明显地抑制作用，如图11所示。
【结论】
结果显示，黄芩素能够显著性地缩短肺损伤小鼠的呼气末期停顿时间、显著性地减小单位体重的最大呼气流量、明显减轻支气管收缩的程度；黄芩素能够明显减轻肺损伤小鼠肺部细胞浸润和肺组织中MPO酶的活性；黄芩素还能够显著性地降低肺支气管灌流液中炎性细胞的浓度，在一定程度上可以降低灌流液中的细胞因子的浓度；黄芩素可以显著降低血清中IL-1β和TNF-α的浓度；黄芩素能够显著性地降低肺损伤小鼠的脾脏指数。
上述结果表明，在建立的急性肺损伤模型中，黄芩素能够降低肺损伤小鼠血清中炎症因子的含量，对LPS诱导的急性肺损伤具有一定程度上的治疗作用。
实施例5
黄芩素可干扰病毒S蛋白与ACE2结合
病毒棘突糖蛋白S(Spike Glycoprotein，图12，A，B，C)通过与宿主ACE2(图12，D)受体蛋白结合，介导病毒膜和宿主膜的融合，实现病毒对宿主的感染。病毒进入宿主细胞后，被宿主蛋白酶裂解并大量繁殖，导致严重后果。
根据Scripps研究所报告的蛋白质结构数据(PDB code：6CS2)，病毒棘突糖蛋白S以三聚体形式与ACE2结合。因此，冠状病毒的抑制剂要么进攻ACE2与受体结合的位点，要么进攻糖蛋白S的三聚体结合面，或者进攻能导致任一种蛋白产生大的结构变化的结合口袋。
下载Scripps研究所报告的蛋白质结构数据(Protein Data Bank code：6CS2)，采用Discovery studio软件进行计算机虚拟对接，分子对接结果见表4所示。
黄芩素结合病毒Spike蛋白与ACE2的结合位点，详见图13：(A)相互作用的3D图，(B)相互作用的2D图。
表4黄芩素与ACE2-病毒蛋白S结合的位点的对接结果
黄芩素 对接能量
构象1 -5.10
构象2 -4.66
计算机虚拟筛选的结果表明：黄芩素进攻ACE2与受体结合的位点的关键氨基酸包括ILE(B)405、VAL(B)404、PEH(B)483、LYS(B)390、TYR(B)481、ASP(B)393。黄芩素能够与病毒S-蛋白-ACE2结合位点中的关键氨基酸产生相互作用，说明其为作用于该靶点的潜在活性化合物。
实施例6
黄芩素在小鼠模型上具有明显的抗新型冠状病毒作用
应用SARS-CoV-2感染hACE2转基因小鼠模型，评价黄芩素治疗效果。
1.材料与方法
1.1受试药物
受试物名称：黄芩素，现用现配，10mg/ml
1.2攻毒毒株
毒株名称：SARS-CoV-2
感染途径：滴鼻
感染剂量：105TCID50/只
感染体积：50微升
1.3实验动物
动物名称：hACE2转基因小鼠，SPF级，7周龄，体重16-24g
动物来源：来自中国医学科学院医学实验动物研究所
2.实验方法
2.1动物分组：
分为黄芩素组和模型组，每组6只，黄芩素200mg/kg，按照每只小鼠给予200μl/10g计算。灌胃给药，攻毒后1小时给药，每天1次，连续给药5天，模型组按照等体积给予0.5％羧甲基纤维素钠。
表5药物抗病毒效果实验动物分组
组别 hACE2小鼠(只) 给药剂量 感染
黄芩素组 6 200mg/kg/d 10 5TICD 50/只
模型组 6 / 10 5TICD 50/只
2.2观察指标
小鼠攻毒后连续观察5天，进行一般症状观察，记录体重变化。分别在感染后第3天和第5天，每组安乐死3只小鼠检测肺组织病毒载量，第5天取1只小鼠的1侧肺组织进行组织病理学检查。
2.3数据统计处理方法
本实验所产生的定量数据，应用统计处理软件SPSS(Version.12.0)进行方差分析。
3.实验结果
3.1一般状态观察
模型组小鼠感染后一般状态尚可，部分动物出现竖毛及体重下降，平均下降率最高为4.55％。与模型组比较，黄芩素组小鼠平均体重略有上升，上升率最高为1.59％。见图14和表6。
表6黄芩素给药后各组小鼠体重变化
Figure PCTCN2020078252-appb-000004
3.2病毒载量
各组小鼠感染后第3天安乐小鼠，分别取3只小鼠收集肺组织用于做载量检测，其中1只小鼠肺组织一半用于做病理检查。
模型组小鼠感染后第3天和5天肺组织载量检测结果为105.94copies/ml和104.14copies/ml。黄芩素组小鼠感染后第3天肺组织病毒载量为104.45copies/ml，显著低于模型组小鼠(p<0.01)；第5天肺组织病毒载量为103.36copies/ml，显著低于与模型组(p<0.05)。见图15和表7。
表7黄芩素对小鼠肺组织病毒载量影响
Figure PCTCN2020078252-appb-000005
各组受试物小鼠体重下降率与模型组比较，*p<0.05有显著性差异，**p<0.01有非常显著性差异
3.3病理诊断
SARS-CoV-2感染5天的hACE2转基因模型组小鼠肺组织呈弥漫性中度间质性肺炎改变，可见肺泡隔增宽，炎细胞浸润，血管周围少量炎细胞浸润。黄芩素治疗组小鼠肺组织呈弥漫性轻至中度间质性肺炎改变，可见肺泡隔增宽，炎细胞浸润，血管周围少量炎细胞浸润。与模型组相比，黄芩素治疗组的肺组织病变有轻微改善。图16所示为模型组与黄芩素治疗组的肺组织病变图。
表8肺组织病理改变程度简表
Figure PCTCN2020078252-appb-000006
注：—，未见病变；+，轻度病变；++，中度病变；+++，重度病变；++++，极重度病变。
4.实验结论
与模型组小鼠相比，黄芩素治疗组的小鼠一般表现较好，体重没有因为感染病毒下降且维持上升，体内病毒复制被显著抑制，肺组织病变有轻度改善，表明黄芩素(例如200mg/kg/d给药剂量的黄芩素)对SARS-CoV-2感染hACE2转基因小鼠有一定的抑制病毒和改善肺部炎症的作用。

Claims (13)

  1. 黄芩素在制备用于预防和/或治疗由新型冠状病毒感染引起的疾病的药物中的用途。
  2. 用于预防和/或治疗新型冠状病毒感染的药物组合物，其包含黄芩素和药学上可接受的载体、辅料或赋形剂，和/或一种或多种另外的抗病毒剂或抗生素，优选地所述黄芩素为β晶型黄芩素，优选地所述由新型冠状病毒感染引起的疾病为新型冠状病毒引起的肺炎和/或肠炎，优选地所述新型冠状病毒为SARS-CoV-2。
  3. 根据权利要求2所述的药物组合物，其中所述抗病毒剂为瑞德西韦、磷酸氯喹、利巴韦林、硝唑尼特、萘莫司他、喷昔洛韦、法匹拉韦、阿比多尔和/或金刚烷胺。
  4. 根据权利要求2所述的药物组合物，其中所述抗生素为内酰胺类、大环内酯类、头孢类和/或磺胺类抗生素。
  5. 根据权利要求2所述的药物组合物，其中所述药物组合物中的黄芩素的含量为0.1至99.9重量％，优选地所述黄芩素的含量为50至99.9重量％，更优选地所述黄芩素的含量为80至99.9重量％。
  6. 根据权利要求2所述的药物组合物，其为固体制剂，优选地所述固体制剂包括片剂、胶囊、丸剂、注射用制剂、缓释或控释制剂。
  7. 权利要求2-6中任一项所述的药物组合物在制备用于预防和/或治疗由新型冠状病毒感染引起的疾病的药物中的用途。
  8. 根据权利要求1或7所述的用途，所述新型冠状病毒为SARS-CoV-2。
  9. 根据权利要求1或7所述的用途，所述新型冠状病毒感染包括轻症、中症和重症感染。
  10. 根据权利要求1或7所述的用途，所述由新型冠状病毒感染引起的疾病包括由所述新型冠状病毒引起的肺炎或肠炎。
  11. 根据权利要求1或7所述的用途，所述黄芩素抑制所述新型冠状病毒的感染、抑制所述新型冠状病毒的复制、或对新型冠状病毒引起的组织损伤具有保护和治疗作用。
  12. 根据权利要求1或7所述的用途，其中所述黄芩素的每日给药剂量为50-1000mg，其中针对老人和儿童的每日给药剂量为50-800mg。
  13. 根据权利要求1或7所述的用途，其中所述黄芩素为β晶型黄芩素。
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