WO2020114444A1 - 双醋瑞因在制备抗病毒药物及治疗病毒感染中的用途 - Google Patents

双醋瑞因在制备抗病毒药物及治疗病毒感染中的用途 Download PDF

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WO2020114444A1
WO2020114444A1 PCT/CN2019/123209 CN2019123209W WO2020114444A1 WO 2020114444 A1 WO2020114444 A1 WO 2020114444A1 CN 2019123209 W CN2019123209 W CN 2019123209W WO 2020114444 A1 WO2020114444 A1 WO 2020114444A1
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virus
refers
infection
enterovirus
diacerein
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French (fr)
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朱水芳
丛浩龙
王晨光
田志清
姜帆
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中检科医药科技(北京)集团有限公司
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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
    • A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
    • A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
    • A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
    • A61K31/222—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin with compounds having aromatic groups, e.g. dipivefrine, ibopamine
    • 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
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • 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
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • 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
    • A61P31/18—Antivirals for RNA viruses for HIV
    • 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
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes 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

Definitions

  • the present invention relates to the new use of diacerein, in particular to the use of diacerein in the preparation of antiviral drugs and the treatment of viral infections.
  • Viruses have different characteristics due to their transmission routes, characteristics, and mechanisms.
  • the mechanism of action of antiviral drugs is also divided into various types, such as direct inhibition or killing of viruses, interference with virus adsorption, prevention of virus penetration into cells, inhibition of virus biosynthesis, Inhibit the release of viruses or enhance the host's anti-virus capabilities.
  • Some viruses such as hepadnaviruses, retroviruses, enteroviruses, and orthomyxoviruses, etc., due to their special transmission routes, lack of effective preventive vaccines, virus mutations, and drug resistance, make the treatment of viral diseases face many difficulties .
  • Enteroviruses for example, belong to the family of small RNA viruses. They usually break out in summer and autumn. Their infections are widely distributed and their clinical manifestations are complex and diverse. Currently, there is a lack of effective therapeutic drugs for enterovirus infections. Among enteroviruses, in addition to individual preventive vaccines, there are still more than 110 enteroviruses that pose a huge threat to human health. Enterovirus 71 (EV-A71) is the main pathogen of hand-foot-mouth disease. Over the past 30 years, hand-foot-mouth disease has been widespread in the Asia-Pacific region, with increasing morbidity and mortality, and epidemic strains also changing [1] .
  • the clinically used anti-hepatitis B virus drugs are mainly nucleoside drugs, mainly including lamivudine, entecavir, tenofovir, mainly targeting reverse transcriptase, blocking hepatitis B virus replication [4] .
  • nucleoside analogues The disadvantage of nucleoside analogues is that the treatment course is long and requires lifelong treatment. Taking such drugs for a long time, the drug-resistant side effects that are prone to appear will also increase.
  • the use of lamivudine is susceptible to drug resistance, and some patients become more ill after the mutation of drug-resistant strains occurs. Entecavir and tenofovir are new hepatitis B drugs, but the price of these two drugs is too high, and the side effects are large.
  • the human immunodeficiency virus HIV is the most infected and most widely affected. It mainly attacks human CD4-positive T cells, impairs the body's cellular immune function, and eventually causes tumors or infections, resulting in death.
  • the number of HIV-infected people in our country is showing a trend of becoming younger and expanding, with 100,000 to 100,000 new infections each year.
  • anti-AIDS drugs there are six categories of anti-AIDS drugs, which are nucleoside (acid) reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors ( NNRTIs), protease inhibitors (PIs), integrase inhibitors (INSTIs), fusion inhibitors, co-receptor antagonists.
  • NRTIs nucleoside (acid) reverse transcriptase inhibitors
  • NNRTIs non-nucleoside reverse transcriptase inhibitors
  • PIs protease inhibitors
  • INSTIs integrase inhibitors
  • fusion inhibitors co-receptor antagonists
  • co-receptor antagonists co-receptor antagonists.
  • compound preparations composed of different types of drugs [6] .
  • the above drugs have gradually shown their shortcomings. For example, although the treatment can achieve good results, it cannot completely remove the virus infection. The drug resistance problem gradually reduces the effect of the treatment; the above drugs usually also bring many toxic
  • Influenza is an acute respiratory infection caused by influenza virus. Influenza viruses have caused multiple pandemics in history, such as the influenza A(H1N1) in 1918, the influenza A(H2N2) in 1957, and the influenza A(H1N1) in 2009, which caused tens of millions of deaths. At present, hundreds of thousands of people worldwide still die from influenza virus infection every year. In recent years, reports of human infection with highly pathogenic avian influenza have also increased. Due to the characteristics of influenza virus transmission, high variability and wide host range, the prevention and control of influenza virus is facing a severe form. Currently, vaccination and the use of anti-influenza drugs are the main ways to prevent and treat influenza viruses.
  • Influenza virus-specific drugs can serve the purpose of preventing and treating influenza virus infections.
  • the first-line anti-influenza drugs currently in clinical use are mainly divided into two categories.
  • One is the M2 particle channel inhibitor amantadine and amantadine.
  • the drugs are only effective against influenza A virus and have no inhibitory effect on influenza B virus. Because most current influenza A virus strains have strong resistance to amantadine drugs, the World Health Organization has not recommended the use of amantadine drugs for the prevention and treatment of influenza viruses.
  • Another class of drugs against influenza viruses are neuraminidase (NA) inhibitors.
  • Diacerein chemical name 4,5-diacetyl-9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid, has the structure of formula (I). Diacerein exerts its medicinal function in the form of rhein (such as the structure of formula II) in the body.
  • Diacerein and its analogues are mainly used clinically for the treatment of osteoarthritis for anti-inflammatory. It has been reported in some literatures that it also has anticancer activity, catharsis/diuretic effect, treatment of psoriasis, treatment of hyperuricemia and its related metabolic disorders, immunosuppression, etc. The related reports mainly focus on the treatment of arthritis-related disorders .
  • a medicament for preventing or treating viral infection characterized in that the medicinal components of the medicament are selected from or contain diacerein, rhein, chrysophanol, monoacetyl rhein, or an academically acceptable salt, Ester or prodrug.
  • the viral infection refers to hepadnavirus, retrovirus, enterovirus, orthomyxovirus, fibrovirus, reovirus, sand virus, hepatitis E virus, astrovirus, circovirus, parvovirus Virus, adenovirus, polyoma virus, herpes virus, pox virus, human papilloma virus, paramyxovirus, flavivirus, herpes virus, alphavirus, rhabdovirus; rubella virus. ;
  • the hepadnavirus refers to human hepatitis B virus and duck hepatitis B virus;
  • the enterovirus refers to human enterovirus, enterovirus A71 type, Coxsackie virus, Coxsackie virus A16, and poliovirus;
  • the orthomyxovirus refers to influenza A and influenza B;
  • the retrovirus refers to AIDS virus
  • the paramyxovirus refers to respiratory syncytial virus
  • the herpes virus refers to herpes simplex type 1 or herpes simplex type 2;
  • the flavivirus refers to West Nile virus, dengue virus, Zika virus and/or Japanese encephalitis virus infection;
  • the rhabdovirus refers to rabies virus
  • the human ring-shaped virus refers to Norovirus
  • the herpes virus refers to human cytomegalovirus.
  • the viral infection is a retrovirus combined with hepadnavirus infection;
  • the retrovirus combined with enterovirus infection
  • the retrovirus is combined with hepadnavirus and enterovirus infection
  • Retroviruses with orthomyxoviruses, enterovirus infections are Retroviruses with orthomyxoviruses, enterovirus infections
  • the viral infection refers to hepadnavirus, retrovirus, enterovirus, orthomyxovirus, fibrovirus, reovirus, sand virus, hepatitis E virus, astrovirus, circovirus, parvovirus Viruses, adenoviruses, polyomaviruses, herpesviruses, poxviruses, human papillomaviruses, paramyxoviruses, flaviviruses, herpesviruses, alphaviruses, rhabdoviruses; rubella virus infection with any two or more viruses.
  • the medicament further comprises a pharmaceutically acceptable carrier, the medicinal oral preparation, injection preparation, tablet, capsule, granule, suspension or pill.
  • Another aspect of the present invention provides a method for treating a virus-induced disease, which comprises administering to a subject a medicine containing a therapeutically effective dose of a compound selected from diacerein, rhein, Chrysophanol, monoacetyl rhein or its academically acceptable salts, esters or prodrugs.
  • the subject is a patient suffering from a disease caused by one or more of the following viral infections: the virus refers to hepadnavirus, retrovirus, enterovirus, orthomyxovirus, fibrovirus, reovirus Virus, sand virus, hepatitis E virus, astrovirus, circovirus, parvovirus, adenovirus, polyoma virus, herpes virus, pox virus, human papilloma virus, paramyxovirus, flavivirus, herpes virus, Alphavirus, rhabdovirus; rubella virus, respiratory syncytial virus, herpes simplex virus type 1, herpes simplex virus type 2, norovirus, West Nile virus, rabies virus and/or human rhinovirus;
  • the hepadnavirus refers to human hepatitis B virus and duck hepatitis B virus;
  • the enterovirus refers to human enterovirus, enterovirus A71 type, Coxsackie virus, Coxsackie virus A16, and poliovirus;
  • the orthomyxovirus refers to influenza A and influenza B;
  • the retrovirus refers to AIDS virus
  • the paramyxovirus refers to respiratory syncytial virus
  • the herpes virus refers to herpes simplex type 1 or herpes simplex type 2;
  • the flavivirus refers to West Nile virus, dengue virus, Zika virus and/or Japanese encephalitis virus infection;
  • the rhabdovirus refers to rabies virus
  • the human ring-shaped virus refers to Norovirus
  • the herpes virus refers to human cytomegalovirus.
  • the subject is a patient suffering from a disease caused by one or more of the following viral infections:
  • the retrovirus combined with enterovirus infection
  • the retrovirus is combined with hepadnavirus and enterovirus infection
  • Retroviruses with orthomyxoviruses, enterovirus infections are Retroviruses with orthomyxoviruses, enterovirus infections
  • the subject is a patient suffering from a disease caused by one or more of the following viral infections:
  • the medicine used in the method further comprises a pharmaceutically acceptable carrier
  • the drugs preferably used in the method are oral preparations, injection preparations, tablets, capsules, granules, suspensions or pills.
  • the inventor discovered the antiviral activity of diacerein and its analogs.
  • diacerein can effectively inhibit the replication of various viruses.
  • viruses refer to hepadnavirus, retrovirus, enterovirus, orthomyxovirus, fibrovirus, Reovirus, Sand Virus, Hepatitis E virus, Astrovirus, Circovirus, Parvovirus, Adenovirus, Polyoma virus, Herpes virus, Pox virus, Human papilloma virus, Paramyxovirus, Flavivirus, Herpes virus, alphavirus, rhabdovirus; rubella virus.
  • the hepadnavirus refers to human hepatitis B virus and duck hepatitis B virus;
  • the enterovirus refers to human enterovirus, enterovirus A71, Coxsackie virus, Coxsackie virus A16, and polio virus;
  • the orthomyxovirus refers to influenza A and B; the retrovirus refers to HIV; the paramyxovirus refers to respiratory syncytial virus; the herpes virus refers to herpes simplex type 1 or herpes simplex type 2;
  • the flavivirus refers to West Nile virus, dengue virus, Zika virus and/or Japanese encephalitis virus infection; the rhabdovirus refers to rabies virus; the human circovirus refers to norovirus; the herpes virus refers to Human cytomegalovirus.
  • Experimental results show that, for example, diacerein effectively reduces the level of mature virions of human hand, foot and mouth disease virus, and significantly inhibits virus replication.
  • diacerein can significantly inhibit the replication of hepatitis B virus, reduce the level of S antigen, and its inhibition level reaches more than 50%.
  • diacerein can also effectively inhibit the replication of human HIV, with an inhibition efficiency of more than 70%.
  • diacerein has significant activity in inhibiting orthomyxovirus.
  • diacerein has an inhibitory effect of more than 90% on the 2018 new influenza strains of influenza virus types A and B at a concentration of 25 There is no significant cytotoxicity below the micromolar concentration.
  • diacerein can effectively protect mice from death caused by influenza virus infection, and its inhibitory efficiency is comparable to oseltamivir.
  • Figure 1A Indirect immunofluorescence detection of diacerein for the replication of EV-A71 in RD cells. It can be seen that as the concentration of diacerein increases, the fluorescent signal representing EV-A71 virus protein decreases; it shows that diacerein can be effective Inhibit the replication of EV-A71 in RD cells;
  • INC003 is a diacerein-treated group
  • Mock is uninfected RD cells
  • DMSO is DMSO-treated cells.
  • Figure 1B Western blot to detect the replication of diacerein on EV-A71 in RD cells
  • Mock is uninfected RD cells
  • INC003 is diacerein treatment group.
  • EV-A71 VP1 represents the amount of viral VP1 protein, and RD cells were treated with 25 ⁇ M. It can be seen that diacerein has a significant inhibitory effect on EV-A71.
  • ribavirin 250 ⁇ M was set as a positive control.
  • GAPDH was used as an internal reference.
  • Figure 1C Virus titer detection determines the titer of EV-A71 virus particles in the cell supernatant.
  • INC003 is a diacerein treatment group. It can be seen that the number of mature virions in the diacerein treatment group remained at a low level, and the 48-hour virus titer Log value was only 2.3, the DMSO treatment group was 5.4, and the ribavirin (250 ⁇ M) treatment group was 3.94. It can be seen that diacerein can significantly reduce the number of mature virions compared to the ribavirin control group.
  • Figure 1D Indirect immunofluorescence analysis of the number of virus-infected cells to determine the half-inhibitory concentration of diacerein exerting anti-EV-A71.
  • INC003 is a diacerein treatment group. After incubating the virus with the cells, diacerein or ribavirin was added, and 24 hours after infection, the number of virus-infected cells was analyzed by immunofluorescence. The results showed that the half-inhibitory concentration of diacerein on EV-A71 was about 10 ⁇ M. Significantly lower than ribavirin.
  • Figure 2A Elisa test detects hepatitis B virus S antigen in the supernatant of HepG-2-2215 cell model of Hepatitis B.
  • INC.3 is the diacerein treatment group
  • Positive Control is the positive control group
  • Negative Control is the S antigen negative control group. It can be seen that the concentration of diacerein at 50 ⁇ M and 25 ⁇ M can effectively inhibit the production of hepatitis B virus S antigen.
  • Figure 2B Fluorescence quantitative PCR to detect the copy number of mature HBV DNA in the supernatant of HepG-2-2215 cell model.
  • Mock is a negative control group.
  • Control is the DMSO processing group.
  • INC.3 is the diacerein treatment group. It can be seen that the concentration of diacerein is above 3.125 ⁇ M, and the viral DNA copy number in the supernatant decreases significantly. Its degree of decline exceeds 50%. It is suggested that diacerein can inhibit the replication of hepatitis B virus in HepG-2-2215 cells.
  • FIG. 2C CCK-8 method was used to detect the cytotoxicity of diacerein on HepG-2-2215 cells.
  • Cells were treated with diacerein at different dilutions, and 48 hours later, CCK-8 was added and assayed by enzyme labeling. The results showed that diacerein was below 25 ⁇ M concentration and did not cause significant cytotoxicity.
  • FIG. 3A Dual luciferase reporter system detects the effect of diacerein on the replication of HIV-1 virus.
  • HeLa cells were infected with HIV-1 and treated with diacerein; luciferase activity was detected 48 hours after infection.
  • the results show that diacerein can significantly inhibit HIV-1 replication at 25 ⁇ M and 12.5 ⁇ M concentrations.
  • FIG. 3B CCK-8 detects the cytotoxicity of diacerein on HeLa cells.
  • FIG. 4A Western blot detection of the inhibitory effect of diacerein on the HIN1 influenza virus-PR8 strain.
  • DMSO is the DMSO treatment group
  • INC.3 is the diacerein treatment group
  • INC.1 is the compound control group
  • Figure 4B Comparison of the effect of diacerein on the inhibition of HIN1 type A influenza virus-PR8 strain and oseltamivir phosphate (Oseltamivir). After the influenza virus was inoculated into MDCK cells, the cells were treated with diacerein and oseltamivir phosphate at different concentrations. After 24 hours, the cells were lysed and the NP protein signal was detected by Western blot. DMSO is the DMSO treatment group, and Mock is the uninfected group. The results showed that, at the same molar concentration, diacerein had better inhibitory effect on HIN1 than oseltamivir.
  • Figure 4C Comparison of the effect of diacerein in inhibiting the 2018 HIN1 influenza pandemic virus strain and oseltamivir phosphate. After the influenza virus was inoculated into MDCK cells, the cells were treated with diacerein and oseltamivir phosphate at a concentration of 50 ⁇ M. After 24 hours, the cells were lysed and the NP protein signal was detected by Western blot. DMSO is the DMSO treatment group, and Mock is the uninfected group. The results showed that at the same molar concentration, diacerein had a better inhibitory effect on the 2018 HIN1 epidemic strain than oseltamivir.
  • Figure 4D Comparison of the effect of diacerein in inhibiting the 2018 B-type HIN influenza epidemic strains BY and BV and oseltamivir phosphate.
  • the influenza virus was inoculated into MDCK cells, the cells were treated with diacerein and oseltamivir phosphate at a concentration of 50 ⁇ M. After 24 hours, the cells were lysed and the NP protein signal was detected by Western blot.
  • DMSO is the DMSO treatment group, and Mock is the uninfected group. The results showed that at the same molar concentration, diacerein had a better inhibitory effect on the 2018 type B epidemic strain than oseltamivir.
  • Figure 4E Indirect immunofluorescence analysis of the number of cells infected with influenza virus to determine the half-inhibitory concentration of diacerein exerting anti-influenza virus effects.
  • INC003 is a diacerein treatment group. After incubating the virus with the cells, diacerein or oseltamivir phosphate (the initial concentration is 50 ⁇ M) was added. After 24 hours of infection, the number of virus-infected cells was analyzed by NP protein immunofluorescence. The results showed that the half-inhibitory concentration of diacerein on EV-A71 was about 4.46 ⁇ M. The half inhibitory concentration of oseltamivir phosphate is 5.2 micromolar.
  • Figure 4F Mouse experiment to detect the inhibitory effect of diacerein on influenza virus.
  • DMSO is the DMSO treatment group
  • Oseltamivir is the oseltamivir phosphate treatment group
  • INC. 3 is the diacerein treatment group.
  • Mock is the uninfected group.
  • 50 ⁇ l of diacerein and oseltamivir phosphate were intragastrically administered at 10 mg/kg body weight per day, and 50 ⁇ l of DMSO was administered intragastrically in the DMSO group.
  • mice were weighed regularly every day, and the results showed that the weight of the mice in the oseltamivir phosphate group and diacerein group decreased significantly on the fourth day and reached the lowest value on the sixth day. Then gradually recover and increase. In the DMSO group, the decline continued to increase after the fourth day, and all died on the ninth day. The Mock group has been rising. It shows that diacerein has a protective effect on mice infected with influenza virus.
  • Figure 4G Effect of diacerein on mortality of mice infected with influenza virus.
  • DMSO is the DMSO treatment group
  • Oseltamivir is the oseltamivir phosphate treatment group
  • INC. 3 is the diacerein treatment group.
  • Mock is the uninfected group.
  • 50 ⁇ l of diacerein and oseltamivir phosphate were intragastrically administered at 10 mg/kg body weight per day, and 50 ⁇ l of DMSO was administered intragastrically in the DMSO group. Observe the mortality of mice.
  • FIG. 4H CCK-8 method was used to detect the cytotoxicity of diacerein on MDCK cells.
  • Cells were treated with diacerein at different dilutions, and 48 hours later, CCK-8 was added and assayed by enzyme labeling. The results showed that diacerein was below 50 ⁇ M concentration, and did not cause significant cytotoxicity in MDCK cells.
  • INC1 and INC16 are compound controls.
  • Oseltamivir is oseltamivir phosphate treatment group
  • INC3 is diacerein group.
  • FIG. 4I CCK-8 method was used to detect the cytotoxicity of diacerein on A549 cells. Cells were treated with diacerein at different dilutions, and 48 hours later, CCK-8 was added and assayed by enzyme labeling. The results showed that diacerein was below 25 ⁇ M concentration and did not cause significant cytotoxicity.
  • INC1 and INC16 are compound controls. Oseltamivir is oseltamivir phosphate treatment group, INC3 is diacerein group.
  • Figure 4J Indirect immunofluorescence detection of the effect of diacerein on the replication of influenza A virus in MDCK and A549 cells. The results showed that diacerein was below the concentration of 25 ⁇ M and 2.5 ⁇ M, significantly reducing the level of influenza virus NP protein signal. It shows that diacerein can significantly inhibit the replication of influenza A virus. Mock is uninfected cells. DMSO is DMSO-treated cells.
  • RD cells were cultured in DMEM medium containing 10% FBS. When the number of cells is sufficient, divide them into 10cm culture dishes with a density of 70%.
  • the RD cells were digested with 2.5% trypsin, counted, and the cells were placed in each well of a 96-well plate at 3000 cells per well. 10% FBS DMEM medium was cultured for 24 hours.
  • ribavirin is a positive control, and the concentration is 250 ⁇ M.
  • the RD cells were digested with trypsin, counted, and the cells were plated to a 12-well plate at 70% confluence per well.
  • diacerein was added at final concentrations of 25 ⁇ M, 12.5 ⁇ M and 6.25 ⁇ M.
  • RD cells were digested with trypsin, and 12-well culture plates were plated at 70% confluence.
  • Elisa test detects hepatitis B virus S antigen in HepG2-2215 cell model supernatant of hepatitis B.
  • the supernatant of HepG2-2215 cells treated with diacerein was drawn, and the DMSO group was used as a control.
  • the pre-experiment detects the different dilution ratios of the cell supernatant, with a view to satisfying them within the range of the instrument with different dilutions.
  • the specific detection methods are as follows:
  • washing solution wash three times with washing solution, patting the washing solution dry each time.
  • Fluorescence quantitative PCR absolute quantification was used to detect the copy number of HepG2-2215 cell model supernatant HBV DNA.
  • HepG2-2215 cells were treated with diacerein at different concentrations. After 24 and 48 hours of treatment, 100 ⁇ l of cell supernatant was taken, treated with lysate and proteinase K, and incubated at 37 degrees for 45 minutes.
  • the supernatant was drawn into the DNA binding column, centrifuged at 12,000 rpm for 1 minute, and the liquid in the collection tube was discarded. Wash the binding column twice with Washing Buffer and centrifuge at 12000 for 10 minutes. Place on a clean bench to dry for 20 minutes, and dissolve with 50 ⁇ l of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0).
  • Nanodrop measures DNA concentration and purity. Using pCMV-HB X plasmid as the standard, calculate the molecular weight and the plasmid moles based on the plasmid concentration. Dilute the plasmid at a 10-fold ratio, and set a total of 10 2 -10 6 copies/microliter of five concentration standards.
  • the sample is mixed with 2 ⁇ SYBgreen Mix, primers and sterilized water to prepare quantitative PCR reaction solution.
  • HB X -R 5'-CCCAACTCCTCCCAGTCCTTAA.
  • diacerein can inhibit the replication of hepatitis B virus in HepG-2-2215 cells.
  • HepG2-2215 cells were cultured in 10% FBS DMEM medium.
  • Cells were digested with 2.5% trypsin, and 100 ⁇ l of 5000 cells were added to each well to divide into 96-well plates. After cultivating for 24 hours, diacerein was diluted twice with 10% FBS DMEM medium to make the concentration from 200 micromolar to 0.9 micromolar, and incubated at 37 degrees for 48 hours.
  • the wells with the corresponding amounts of cell culture fluid and CCK-8 solution but no cells were used as blank control.
  • the incubation in the cell incubator was continued for 0.5 hours, and the absorbance was measured at 450 nm.
  • Diacerein is below 25 ⁇ M and does not cause significant cytotoxicity.
  • Exemplary Experimental Example III Diacerein on human immunodeficiency virus (HIV-1) inhibitory effect cell detecting the level of pharmacodynamic
  • 293T cells were cultured in 10% FBS DMEM medium.
  • the plasmids of pNL4-3Luc-R-E and VSVG were extracted in large quantities, and Nanodrop measured the plasmid concentration and purity.
  • the cells in better condition are digested and divided into 10cm petri dishes. After culturing for 24 hours, the medium was discarded, rinsed twice with DMEM, and 5 ml of DMEM was added.
  • HeLa cells were cultured in 10% FBS DMEM medium.
  • the cells were trypsinized and divided into 6-well plates. Count and plate cells to a six-well plate at 50% confluence. Incubate at 37 degrees for 24 hours, remove the medium, rinse with ice PBS three times, infect HIV-1 pseudovirus at the appropriate virus concentration, discard the supernatant, rinse three times with ice PBS, add 10% FBS DMEM medium, and add double concentrations of different concentrations Control of vinegarin and DMOS. Set up an uninfected control group.
  • CCK-8 was used to detect the cytotoxicity of diacerein on HeLa cells. As shown in Figure 3B, cells were treated with diacerein at different dilutions. After 48 hours, CCK-8 was added and the enzyme labeling was performed. The results showed that diacerein did not cause significant cytotoxicity at 25 ⁇ M and 12.5 ⁇ M concentrations.
  • HIN1-PR8, HIN1-2018, H3N2-2018, BY-2018 and BV-2018 influenza viruses are all amplified using the chicken embryo amniotic cavity method.
  • the specific method is: take out the chicken embryo, look at the chicken embryo with a flashlight, irradiate the egg device, draw a pencil on the edge of the airbag and draw a horizontal line. Mark the injection location. Each dilution is 3-4 pieces, half starting from -1 dilution.
  • Ultra clean table preparation alcohol lamp, needle, large needle. First spray the egg with alcohol, then burn it with a large needle, then punch the top of the air bag, and then punch the hole at the marked position. Use a 1ml syringe to inject the diluted venom.
  • the depth is not too deep, about 0.3-0.5cm, right hand injection, no bubbles. After the wax has melted, apply it. Place the 37 degree incubator for 72 hours to collect the poison. Put in 4 degree refrigerator overnight. Spray alcohol to sterilize and smash the top air chamber with tweezers.
  • MDCK cells were cultured in 8% FBS DMEM medium, and the cells were divided into 96-well plates with trypsin digestion, 2000 cells per well. 10% FBS DMEM medium was cultured for 24 hours. Pipette 100 ⁇ l of virus solution or virus culture supernatant, and dilute with DMEM 10 times dilution.
  • Trypsin digests MDCK cells, counts and divides the cells into 12-well culture plates. After 24 hours, inoculate influenza virus with MOI 1, add diacerein or other controls, and add TPCK pancreatin at a volume ratio of 1:2000. After 45 minutes of adsorption, discard the supernatant and add 5% FBS DMEM medium TPCK trypsin was added in a volume ratio of 1:2000; after 24 or 48 hours of culture, the supernatant was discarded, washed three times with ice PBS, and 100 ⁇ l of lysis solution was added. After the cells are completely lysed, they are drawn into a 1.5ml centrifuge tube, 12,000 rpm, and centrifuged at 4°C for 10 minutes.
  • TBST Tris-HCI buffer (0.5M pH7.6) 100ml NaCl 8.5-9g (0.15mol/L), 1ml/L Triton-20) was washed 3 times, 5% skimmed milk powder was blocked overnight, TBST was washed 3 After the second time, add rabbit anti-influenza virus NP protein monoclonal antibody or rabbit anti-influenza virus NP protein polyclonal antibody (1% skimmed milk powder, antibody 1/1000 dilution), incubate at 37°C for 1 hour, and wash 3 times with TBST ( After 10 minutes each time, add a horseradish peroxidase-labeled secondary antibody (purchased from Zhongshan Jinqiao) (1% skimmed milk powder, antibody 1/3000 dilution), incubate at 37°C for 45 minutes, and wash three times with TBST (per 10 minutes), then use super-sensitive luminescent liquid to develop color and take a picture.
  • Use Image J to analyze the gray value of WB band After the color development is complete, TBST wash the membrane, add TBST diluted anti-GAPDH mouse monoclonal antibody, incubate for 1 hour at room temperature, wash three times with TBST, add the horseradish peroxidase labeled secondary antibody (purchased from Zhongshan Jinqiao) (1% skimmed milk powder) , Diluted 1/3000 of antibody), incubate at 37°C for 45 minutes, wash three times with TBST (10 minutes each time), and then develop the color with super-sensitive luminescent solution and take a picture.
  • Use Image J to analyze the gray value of WB band.
  • Diacerein can significantly inhibit the replication of HIN1 influenza virus in cells, which is mainly manifested in the large reduction of influenza virus NP protein. The efficiency of inhibiting HIN1 influenza virus reached more than 90%.
  • Figure 4B shows that after influenza virus was inoculated into MDCK cells, the cells were treated with diacerein or oseltamivir phosphate at different concentrations. After 24 hours, the cells were lysed and the NP protein signal was detected by Western blot.
  • DMSO is the DMSO treatment group. Mock is the uninfected group. The results showed that, at the same molar concentration, diacerein had better inhibitory effect on HIN1 than oseltamivir.
  • Figure 4C shows that after influenza virus was inoculated into MDCK cells, the cells were treated with diacerein or oseltamivir phosphate at a concentration of 50 ⁇ M. After 24 hours, the cells were lysed and the NP protein signal was detected by Western blot.
  • DMSO is the DMSO treatment group. Mock is the uninfected group. The results showed that at the same molar concentration, diacerein had a better inhibitory effect on the 2018 HIN1 epidemic strain than oseltamivir.
  • Figure 4D shows that after influenza virus inoculates MDCK cells, the cells are treated with 50 ⁇ M diacerein or oseltamivir phosphate. After 24 hours, the cells are lysed and the NP protein signal is detected by Western blot.
  • DMSO is the DMSO treatment group. Mock is the uninfected group. The results showed that at the same molar concentration, diacerein had a better inhibitory effect on the 2018 type B epidemic strain than oseltamivir.
  • MDCK cells were digested with trypsin, counted, and cells were plated to a 24-well plate at 50% confluence per well.
  • diacerein below 25 ⁇ M and 2.5 ⁇ M concentrations significantly reduces the level of influenza virus NP protein signal. Shows that diacerein can significantly inhibit the replication of influenza A virus
  • mice SPF grade 18-21 g male and female BALB/c mice were divided into cages. Five mice per cage were set up in the uninfected group, DMSO group, oseltamivir group, diacerein group, and 8 mice in each group. Place the SPF feeding room for a week. The mice were anesthetized with isoflurane. After nasal inoculation of 90 ⁇ l of influenza virus (10 5 TCID50/ml), mice in the diacerein group were given 10 mg/kg of body weight every day, and dioserein in the oseltamivir group was given 50 ⁇ L/kg of body weight. Oseltamivir phosphate, DMSO group was given DMSO at 50 ⁇ L/kg body weight. Weigh the mice regularly every day, continue to observe, and count the number of dead mice.
  • DMSO is the DMSO treatment group
  • Oseltamivir is the oseltamivir phosphate treatment group
  • INC. 3 is the diacerein treatment group
  • Mock is the uninfected group.
  • mice were vaccinated with influenza virus (105TCID50/ml) in the nasal cavity, the diacerein group was gavaged with 10 mg/kg body weight, and the oseltamivir phosphate-treated group was gavaged with 50 ⁇ L/kg body weight.
  • DMSO group was given DMSO at 50 ⁇ L/kg body weight. Observe the mortality of mice.
  • CCK-8 method was used to detect the cytotoxicity of diacerein on MDCK cells.
  • Oseltamivir is an oseltamivir phosphate treatment group.
  • INC3 is the diacerein group.
  • the CCK-8 method was used to detect the cytotoxicity of diacerein on A549 cells.
  • NC1 and INC16 are compound controls.
  • Oseltamivir is an oseltamivir phosphate treatment group.
  • INC3 is the diacerein group.

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Abstract

双醋瑞因的一种制药用途，实验证明，双醋瑞因能够有效抑制嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、冠状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、甲病毒、弹状病毒。

Description

双醋瑞因在制备抗病毒药物及治疗病毒感染中的用途 技术领域
本发明涉及双醋瑞因新用途，特别涉及双醋瑞因在制备抗病毒药物及治疗病毒感染中的用途。
背景技术
病毒由于其传播感染途径、特性和机制各有特点，抗病毒药的作用机制也分为多种类型，比如直接抑制或杀灭病毒、干扰病毒吸附、阻止病毒穿入细胞、抑制病毒生物合成、抑制病毒释放或增强宿主抗病毒能力等。一些病毒例如嗜肝DNA病毒、逆转录病毒、肠道病毒及正黏病毒等，由于其传播途径特殊，缺乏有效的预防性疫苗、病毒变异及耐药等原因，使得病毒病的治疗面临诸多困难。
例如肠道病毒，属于小RNA病毒科，通常在夏秋季节爆发流行，其感染分布广泛，临床表现复杂多样。目前，对肠道病毒的感染缺乏有效的治疗性药物。在肠道病毒中，除个别病毒有有效的预防性疫苗外，仍有超过110种肠道病毒对人类健康造成巨大威胁。肠道病毒71型(EV-A71)是手足口病的主要病原体。近30年来，手足口病在亚太地区广泛流行，发病率与死亡率不断提高，流行毒株也在不断变迁 [1]。我国每年的丙型传染病病例中，有将近200万人的手足口病病毒感染者，防护及治疗形势严峻。目前手足口疫情的控制仍以常规卫生及消毒等预防为主，临床尚未有特异性治疗药物。临床资料表明，EV-A71灭活疫苗可预防手足口病毒的感染，并已经获批上市。然而，该疫苗尚未列入国家免疫规划，且保护效率和安全性仍有待长期广泛验证 [2,3]。
嗜肝DNA病毒中，感染人群最多的当属乙肝病毒。目前，临床上使用较多 的抗乙肝病毒药物主要为核苷类药物，主要包括，拉米夫定，恩替卡韦，替诺福韦，主要靶向逆转录酶，阻断乙肝病毒复制 [4]。核苷类似物的缺点是疗程漫长，需要终身治疗，长期服用此类药物，容易出现药物的耐药性副作用也随之增加。拉米夫定的使用易产生耐药性，有些病人在发生耐药毒株变异后出现病情加重。恩替卡韦和替诺福韦属于新型乙肝药物，但这两种药的价格过高，毒副作用较大。
逆转录病毒中，感染人数多且影响最广泛的当属人免疫缺陷病毒HIV。其主要攻击人的CD4阳性T细胞，使人体的细胞免疫功能受损，最终发生肿瘤或感染等，从而引起死亡。当前，世界范围内有7200万HIV感染者。有将近100万人的感染者，每年有1.5万至2万人死于HIV感染 [5]。当前，我国的HIV感染人数呈现年轻化，扩大化的趋势，每年新增10-15万的感染者。当前，针对HIV的感染，有40多种各类药物，抗艾滋药物共有六大类，为核苷(酸)类反转录酶抑制剂(NRTIs)、非核苷类反转录酶抑制剂(NNRTIs)、蛋白酶抑制剂(PIs)、整合酶抑制剂(INSTIs)、融合抑制剂、辅助受体拮抗剂。除此之外，还有不同类别药品混合组成的复合制剂 [6]。但上述药物已经逐渐显现出其缺陷，如治疗虽然能够取得很好的效果但并不能完全去除病毒的感染，耐药性问题使治疗的效果逐渐降低；上述药物通常也会带来很多毒副作用 [7]。因此，为满足抗艾滋病药物的新需求，克服上述一种或几种缺点，有必要进一步开发新型的抗HIV药物。
流行性感冒是由流感病毒引起的急性呼吸道传染病。流感病毒在历史上曾造成多次大流行，如1918年甲型H1N1大流感，1957年的甲型H2N2流感，2009年的甲型H1N1流感疫情，共造成数千万人的死亡。目前，全球每年仍有几十万人死于流感病毒感染。近年来，人感染高致病性禽流感的报道也在不断增加。 由于流感病毒的传播途径、高变异性及宿主范围广等特点，使得流感病毒的防控面临严峻的形式。当前，疫苗接种及抗流感药物的使用是预防和治疗流感病毒的主要途径。但由于流感病毒的变异速度较快，接种一种流感疫苗往往只能得到暂时的保护，起不到长期有效的预防作用 [8]。流感病毒的特异性药物能够起到预防和治疗流感病毒感染的目的，目前临床上使用的一线抗流感药物主要分为两类，一类是M2粒子通道抑制剂金刚烷胺和金刚乙胺，该类药物只对甲型流感病毒有效，对乙型流感病毒无抑制作用。由于当前的大部分甲型流感病毒毒株对金刚烷胺类药物都存在较强的耐药性，世界卫生组织已经不推荐使用金刚烷胺类药物用于流感病毒的预防和治疗的首选药物。抗流感病毒的另一类药物是神经氨酸酶(NA)抑制剂。其对当前流行的大多数甲型及乙型流感均有较好的抗病毒效果，是WHO推荐的流感预防和治疗流感病毒治疗的首选药物。随着NA抑制剂的广泛使用，已经出现了耐药株 [9]。2008-2009的季节性H1N1流感对大部分的NA抑制剂耐药。在欧洲的一些国家，奥司他韦耐药株的发生率在逐年增加。因此，可以预见，在不久的将来，奥司他韦的持续使用必将导致大规模耐药株的出现。
可以看出，不同病毒的感染传播途径、核酸构成特点、自身复制增殖特性、难点都不同，因此针对不同病毒采用的药物、治疗方案都不一样。广谱型抗病毒药非常少。
双醋瑞因，化学名4，5-二乙酰-9，10-二氢-9，10-二氧-2-蒽羧酸，具有式(I)的结构。双醋瑞因在体内以大黄酸(如式II结构)的形式发挥药效功能。
Figure PCTCN2019123209-appb-000001
双醋瑞因及其类似物(大黄酸、大黄酚、单乙酰大黄酸或其要学上可接受的盐、酯或前药)在临床主要用于治疗骨关节炎中用于消炎。一些文献中报道，其也具有抗癌活性、导泻/利尿作用、治疗牛皮癣、可治疗高尿酸血症及其相关的代谢性病症、免疫抑制等，相关报道主要还是集中在治疗关节炎相关病症。
但未见到关于双醋瑞因抗病毒的报道，尤其是针对当前一些如手足口病、HIV等主要病毒的报道。
参考文献：
1.Pallansch,M.A.and M.S.Oberste,Enterovirus 71 encephalitis:a new vaccine on the horizon？ Lancet,2013.381(9871):p.976-977.
2.Li,R.C.,et al.,An Inactivated Enterovirus 71 Vaccine in Healthy Children.New England Journal of Medicine,2014.370(9):p.829-837.
3.Zhu,F.C.,et al.,Efficacy,Safety,and Immunogenicity of an Enterovirus 71 Vaccine in China.New England Journal of Medicine,2014.370(9):p.818-828.
4.Dienstag,J.L.,Drug therapy-Hepatitis B virus infection.New England Journal of Medicine,2008.359(14):p.1486-1500.
5.Zhang,B.,et al.,Tracking HIV infection and networks of drugs users in China:a national series,cross-sectional study.Lancet,2018.392:p.48-48.
6.Harter,Z.J.,et al.,Drug abuse and HIV-related pulmonary hypertension:double hit injury.Aids,2018.32(18):p.2651-2667.
7.Bertagnolio,S.,M.R.Jordan,and M.Doherty,HIV Drug Resistance.New England Journal of Medicine,2018.378(9):p.874-874.
8.Watanabe,T.,et al.,Influenza Virus-Host Interactome Screen as a Platform for Antiviral Drug Development.Cell Host&Microbe,2014.16(6):p.795-805.
9.Kelso,A.and A.C.Hurt,Drug-resistant influenza viruses:why fitness matters.Nature Medicine,2012.18(10):p.1470-1471.
发明内容
本发明的发明人在研发中抗病毒药研发中，以病毒沉默抑制子进行了基于 结构生物学的小分子药物筛选，通过大量的筛选工作及体外体内实验，发现双醋瑞因能够依赖其与新靶点的结合，表现出对多种重要病毒的强有效的抑制效果。基于此发现及实验验证数据，本发明请求保护以下技术方案：
一种预防或治疗病毒感染的药物，其特征在于：所述药物的药效成分选自或包含双醋瑞因、大黄酸、大黄酚、单乙酰大黄酸或其要学上可接受的盐、酯或前药。
所述病毒感染是指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒；风疹病毒。；
优选地，
所述嗜肝DNA病毒指人乙型肝炎病毒、鸭乙型肝炎病毒；
所述肠道病毒指人肠道病毒、肠道病毒A71型、柯萨奇病毒、柯萨奇病毒A16、脊髓灰质炎病毒；
所述正黏病毒指A型流感、B型流感；
所述逆转录病毒指艾滋病病毒；
所述副黏病毒指呼吸道合胞病毒；
所述疱疹病毒指单纯疱疹1型或单纯疱疹2型；
所述黄病毒指西尼罗河病毒、登革病毒、寨卡病毒和/或乙型脑炎病毒感染；
所述弹状病毒指狂犬病毒；
所述人环装病毒指诺如病毒；
所述疱疹病毒指人巨细胞病毒。所述病毒感染是逆转录病毒合并嗜肝 DNA病毒感染；
所述逆转录病毒合并肠道病毒感染；
所述逆转录病毒合并嗜肝DNA病毒、肠道病毒感染；
逆转录病毒合并正黏病毒、肠道病毒感染；
逆转录病毒并正黏病毒感染；
肠道病毒感染合并正黏病毒感染；或
肠道病毒感染合并嗜肝DNA病毒感染。
所述病毒感染是指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒；风疹病毒任意两种或多种病毒合并感染。
所述的药物还包含药学上可接受的载体，所述的药物口服制剂、注射制剂、片剂、胶囊剂、颗粒剂、混悬剂或丸剂。
本发明的另一方面，提供一种治疗病毒所致疾病的方法，其特征在于，包括向受试者施用含治疗有效剂量的化合物的药物，所述化合物选自双醋瑞因、大黄酸、大黄酚、单乙酰大黄酸或其要学上可接受的盐、酯或前药。
所述受试者是患有以下一种或多种病毒感染所致疾病的患者：所述病毒指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒；风疹病毒、呼吸道合胞病毒、单纯疱疹病毒1型、单纯疱疹病毒2型、诺如病毒、西尼罗河病毒、狂犬病毒和/或人鼻病毒；
优选地，
所述嗜肝DNA病毒指人乙型肝炎病毒、鸭乙型肝炎病毒；
所述肠道病毒指人肠道病毒、肠道病毒A71型、柯萨奇病毒、柯萨奇病毒A16、脊髓灰质炎病毒；
所述正黏病毒指A型流感、B型流感；
所述逆转录病毒指艾滋病病毒；
所述副黏病毒指呼吸道合胞病毒；
所述疱疹病毒指单纯疱疹1型或单纯疱疹2型；
所述黄病毒指西尼罗河病毒、登革病毒、寨卡病毒和/或乙型脑炎病毒感染；
所述弹状病毒指狂犬病毒；
所述人环装病毒指诺如病毒；
所述疱疹病毒指人巨细胞病毒。所述受试者是患有以下一种或多种病毒感染所致疾病的患者：
逆转录病毒合并嗜肝DNA病毒感染；
所述逆转录病毒合并肠道病毒感染；
所述逆转录病毒合并嗜肝DNA病毒、肠道病毒感染；
逆转录病毒合并正黏病毒、肠道病毒感染；
逆转录病毒并正黏病毒感染；
肠道病毒感染合并正黏病毒感染；或
肠道病毒感染合并嗜肝DNA病毒感染。
所述受试者是患有以下一种或多种病毒感染所致疾病的患者：
嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多 瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒；风疹病毒中的任意两种或多种病毒合并感染。
用于所述方法中的药物还包含药学上可接受的载体；
优选地用于所述方法中的药物是口服制剂、注射制剂、片剂、胶囊剂、颗粒剂、混悬剂或丸剂。
本发明中，发明人发现了双醋瑞因及其类似物的抗病毒活性。在体内病毒复制影响程度的实验中，显示双醋瑞因可强有效地抑制多种病毒的复制，这些病毒指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒；风疹病毒。
所述嗜肝DNA病毒指人乙型肝炎病毒、鸭乙型肝炎病毒；所述肠道病毒指人肠道病毒、肠道病毒A71型、柯萨奇病毒、柯萨奇病毒A16、脊髓灰质炎病毒；
所述正黏病毒指A型流感、B型流感；所述逆转录病毒指艾滋病病毒；所述副黏病毒指呼吸道合胞病毒；所述疱疹病毒指单纯疱疹1型或单纯疱疹2型；所述黄病毒指西尼罗河病毒、登革病毒、寨卡病毒和/或乙型脑炎病毒感染；所述弹状病毒指狂犬病毒；所述人环装病毒指诺如病毒；所述疱疹病毒指人巨细胞病毒。实验结果显示，例如，双醋瑞因有效降低人手足口病病毒成熟病毒粒子水平，显著抑制病毒的复制。在25微摩尔浓度下，其抑制效果达到百分之七十以上，且不引起显著的细胞毒性。在乙肝病毒模型中，双醋瑞因能够显著抑制乙肝病毒的复制，降低S抗原的水平，其抑制水平达到50％以上。此外，双醋瑞因也能够有效抑制人艾滋病病毒的复制，其抑制效率达到70％以上。同样，双醋瑞因具有显著的抑制正粘病毒的活性，在细胞水平，双醋瑞 因对2018年流感病毒A型和B型的新型流行毒株抑制效果达到90％以上，在浓度为25微摩尔浓度以下无显著细胞毒性。在实验动物水平上，双醋瑞因能够有效保护小鼠因感染流感病毒引起的死亡，其抑制效率与奥司他韦相当。
附图说明
图1.双醋瑞因对人手足口病病毒EV-A71的抑制效果
图1A：间接免疫荧光检测双醋瑞因对EV-A71在RD细胞中的复制，可见随双醋瑞因浓度的增加，代表EV-A71病毒蛋白的荧光信号减弱；显示双醋瑞因能够有效抑制EV-A71在RD细胞内的复制；
INC003为双醋瑞因处理组，Mock为未感染RD细胞，DMSO为DMSO处理细胞。
图1B：Western blot检测双醋瑞因对EV-A71在RD细胞中的复制
Mock为未感染RD细胞，INC003为双醋瑞因处理组。EV-A71 VP1代表病毒VP1蛋白量，RD细胞以25μM处理。可见双醋瑞因对EV-A71有显著抑制作用。为评价双醋瑞因对病毒的抑制效果，设置利巴韦林(250μM)为阳性对照。为衡量蛋白上样量，以GAPDH为内参照。
图1C:病毒滴度检测确定细胞上清中EV-A71病毒粒子的滴度。
INC003为双醋瑞因处理组。可见双醋瑞因处理组成熟病毒粒子数维持较低水平，其48小时病毒滴度Log值仅为2.3，DMSO处理组为5.4，利巴韦林(250μM)处理组为3.94。可见双醋瑞因较利巴韦林对照组，能够非常显著的降低成熟病毒粒子的数目。
图1D：以间接免疫荧光分析病毒感染细胞数测定双醋瑞因发挥抗EV-A71的半数抑制浓度。INC003为双醋瑞因处理组。病毒与细胞孵育后，加入双醋瑞 因或利巴韦林，感染24小时后，免疫荧光分析病毒感染细胞数目。结果显示，双醋瑞因对EV-A71的半数抑制浓度为10μM左右。显著低于利巴韦林。
图2.双醋瑞因对乙肝病毒病毒的抑制效果。
图2A:Elisa实验检测乙肝HepG-2-2215细胞模型上清乙肝病毒S抗原。
INC.3为双醋瑞因处理组、Positive Control为阳性对照组、Negative Control为S抗原阴性对照组。可见双醋瑞因浓度为50μM及25μM时，能够有效抑制乙肝病毒S抗原的产生。
图2B：荧光定量PCR检测HepG-2-2215细胞模型上清中成熟乙肝病毒DNA拷贝数。Mock为阴性对照组。Control为DMSO处理组。INC.3为双醋瑞因处理组。可见双醋瑞因浓度在3.125μM以上，上清中病毒DNA拷贝数显著下降。其下降程度超过50％。提示双醋瑞因能够抑制HepG-2-2215细胞中乙肝病毒的复制。
图2C:CCK-8法检测双醋瑞因对HepG-2-2215细胞的细胞毒性。以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标测定。结果显示，双醋瑞因在25μM浓度以下，不引起显著的细胞毒性。
图3.双醋瑞因对逆转录病毒HIV-1的抑制效果
图3A:双荧光素酶报告系统检测双醋瑞因对HIV-1病毒的复制的影响。
HeLa细胞感染HIV-1并以双醋瑞因处理；感染48小时后检测荧光素酶活性。结果显示，在25μM及12.5μM浓度下，双醋瑞因能够显著抑制HIV-1的复制。
图3B:CCK-8检测双醋瑞因对HeLa细胞的细胞毒性。
以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标测定。结果显示，双醋瑞因在25μM及12.5μM浓度下不引起显著的细胞毒性。图4.双醋瑞因对流感病毒的抑制效果
图4A：Western blot检测双醋瑞因对A型HIN1流感病毒-PR8株的抑制效果。DMSO为DMSO处理组、INC.3为双醋瑞因处理组、INC.1为化合物对照组，
所用浓度均为50μM。结果显示，双醋瑞因能够显著抑制HIN1流感病毒在细胞内的复制，主要表现在流感病毒NP蛋白的大量减少。抑制HIN1流感病毒的效率达到90％以上。
图4B:双醋瑞因抑制A型HIN1流感病毒-PR8株与磷酸奥司他韦(Oseltamivir)的效果比较。流感病毒接种MDCK细胞后，以不同浓度双醋瑞因与磷酸奥司他韦处理细胞，24小时后，细胞裂解并以Western blot检测NP蛋白信号。DMSO为DMSO处理组、Mock为未感染组。结果显示,在相同摩尔浓度下，双醋瑞因对HIN1的抑制效果要好于奥司他韦。
图4C：双醋瑞因抑制2018年A型HIN1流感流行病毒株与磷酸奥司他韦(Oseltamivir)的效果比较。流感病毒接种MDCK细胞后，以50μM浓度双醋瑞因与磷酸奥司他韦处理细胞，24小时后，细胞裂解并以Western blot检测NP蛋白信号。DMSO为DMSO处理组、Mock为未感染组。结果显示,在相同摩尔浓度下，双醋瑞因对2018年HIN1流行毒株的抑制效果要好于奥司他韦。
图4D：双醋瑞因抑制2018年B型HIN流感流行病毒株BY及BV与磷酸奥司他韦(Oseltamivir)的效果比较。流感病毒接种MDCK细胞后，以50μM 浓度双醋瑞因与磷酸奥司他韦处理细胞，24小时后，细胞裂解并以Western blot检测NP蛋白信号。DMSO为DMSO处理组、Mock为未感染组。结果显示,在相同摩尔浓度下，双醋瑞因对2018年B型流行毒株的抑制效果要好于奥司他韦。
图4E：以间接免疫荧光分析流感病毒感染细胞数测定双醋瑞因发挥抗流感病毒效果的半数抑制浓度。INC003为双醋瑞因处理组。病毒与细胞孵育后，加入梯度稀释的双醋瑞因或磷酸奥司他韦(初始浓度为50μM)，感染24小时后，NP蛋白免疫荧光分析病毒感染细胞数目。结果显示，双醋瑞因对EV-A71的半数抑制浓度为4.46μM左右。磷酸奥司他韦的半数抑制浓度为5.2微摩尔。
图4F:小鼠实验检测双醋瑞因对流感病毒的抑制效果。DMSO为DMSO处理组，Oseltamivir为磷酸奥司他韦处理组，INC.3为双醋瑞因处理组。Mock为未感染组。小鼠鼻腔接种流感病毒(10 5TCID50/ml)后，以每天，10mg/kg体重灌胃小鼠双醋瑞因及磷酸奥司他韦50微升，DMSO组灌胃50微升DMSO。每日定时称量小鼠体重，结果显示，磷酸奥司他韦组及双醋瑞因组的小鼠体重在第四天出现显著下降，在第6天达到最低值。之后逐渐恢复并增高。DMSO组则在第四天后一直加剧下降，于第九天，全部死亡。Mock组则一直在升高。显示出双醋瑞因对流感病毒感染的小鼠具有保护效果。
图4G:双醋瑞因对流感病毒感染小鼠死亡率的影响。DMSO为DMSO处理组，Oseltamivir为磷酸奥司他韦处理组，INC.3为双醋瑞因处理组。Mock为未感染组。小鼠鼻腔接种流感病毒(10 5TCID50/ml)后，以每天，10mg/kg体重灌胃小鼠双醋瑞因及磷酸奥司他韦50微升，DMSO组灌胃50微升DMSO。观察小鼠死亡率。结果显示，在每组8只实验小鼠中，奥司他韦组出现一只死亡，双醋瑞因组出现一只死亡。DMSO组则在病毒感染后的第8天出现死亡，第10天全部死亡。上述结果说明，双醋瑞因及磷酸奥司他韦均能有效降低流感病毒 感染引起的小鼠死亡。
图4H：CCK-8法检测双醋瑞因对MDCK细胞的细胞毒性。以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标测定。结果显示，双醋瑞因在50μM浓度以下，MDCK细胞中不引起显著的细胞毒性。INC1及INC16为化合物对照。Oseltamivir为磷酸奥司他韦处理组、INC3为双醋瑞因组。
图4I:CCK-8法检测双醋瑞因对A549细胞的细胞毒性。以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标测定。结果显示，双醋瑞因在25μM浓度以下，不引起显著的细胞毒性。INC1及INC16为化合物对照。Oseltamivir为磷酸奥司他韦处理组、INC3为双醋瑞因组。
图4J:间接免疫荧光检测双醋瑞因对A型流感病毒在MDCK及A549细胞中的复制的影响。结果显示，双醋瑞因在25μM及2.5μM浓度以下，显著降低流感病毒NP蛋白信号水平。表明双醋瑞因能够显著抑制A型流感病毒的复制。Mock为未感染细胞。DMSO为DMSO处理细胞。
具体实施方式
本发明通过下述实施例进行举例说明，所述实施例不限制在权利要求中描述的本发明的范围。
下述实施例中的方法，如无特殊说明，均为常规方法(分子克隆实验指南第四版，2017年，J.萨姆布鲁克，E.F弗里奇，科学出版社)，或依据试剂说明书进行操作。
示例性实验例一、双醋瑞因对人肠道病毒的抑制效果细胞水平药效学检测
Figure PCTCN2019123209-appb-000002
1、细胞培养、EV-A71病毒的扩增及纯化
用含10％FBS的DMEM培养基培养RD细胞。待细胞数目足够，将其分至10cm培养皿，密度70％。
放37度二氧化碳培养箱培养。24小时后，用DMEM稀释病毒并弃掉培养基，每皿加入4ml病毒稀释液，按MOI＝1接种EV-A71病毒。37度吸附1小时后，弃上清，加入5％FBS培养基10ml。37度培养48小时，观察细胞是否达到50％脱落。收集上清，2000转离心10分钟。取上清。
配制5×PEG8000 NaCl溶液。配制称取NaCl 8.766g；PEG8000 50g溶解在200ml纯水中。高压灭菌30分钟。室温冷却后与病毒上清液均匀混合。置4度过夜。12000离心一小时。沥干液体，将沉淀用PBS重悬。病毒液分装置-70度保存。
2、EV-A71病毒滴度测定
2.5％胰酶消化RD细胞，计数，以每孔3000个细胞将细胞置于96孔板的每个小孔中。10％FBS DMEM培养基培养24小时。
吸取病毒液或病毒培养上清100μl，以DMEM 10倍稀释度倍比稀释。
取出96孔培养板，去除每孔培养基，按每孔100微升加入稀释的病毒液，37度吸附1小时，补加100微升10％FBS DMEM培养基。
37度培养48-72小时，观察每个稀释度病变孔，以Reed-Muench法计算病毒滴度；利巴韦林为阳性对照，使用浓度为250μM。
结果图1C所示可见双醋瑞因处理组成熟病毒粒子数维持较低水平，其48小时病毒滴度Log值仅为2.3，DMSO处理组为5.4，利巴韦林(250μM)处理组为3.94。可见双醋瑞因较利巴韦林对照组，能够非常显著的降低成熟病毒粒子的数目。Mock为未感染组。
3、双醋瑞因对EV-A71抑制效果的间接免疫荧光检测
以胰酶消化RD细胞，计数，每孔70％汇合度铺细胞至12孔板。
10％FBS DMEM培养24小时，弃上清，冰DMEM洗三次，以MOI＝1用DMEM稀释EV-A71病毒，加入12孔板中，每孔500微升；并设置未感染对照孔。
4度孵育1小时，弃上清，以冰DMEM漂洗三次，加入10％FBS培养基。
同时以终浓度25μM、12.5μM和6.25μM加入双醋瑞因。
置37度培养24小时，以4％多聚甲醛固定10分钟。
用0.2％Triton-X100冰上破膜10分钟，加入3％BSA，室温封闭1小时。
加入1μg/ml的EV-A71鼠单抗(密理博)，室温孵育1小时，PBS洗三次，每次10分钟。加入0.5μg/ml的FITC标记山羊抗小鼠二抗。室温孵育45分钟。PBS洗涤三次，每次10分钟。
以488波长，在荧光显微镜下观察并拍照。以Image J计算绿色信号细胞 数。以DAPI计算细胞总数，计算感染效率或半数抑制浓度。
结果如图1A所示，随双醋瑞因浓度的增加，代表EV-A71病毒蛋白的荧光信号减弱，显示双醋瑞因能够有效抑制EV-A71在RD细胞内的复制。
结果如图D所示，双醋瑞因对EV-A71的半数抑制浓度为10μM左右，显著低于利巴韦林。
4、双醋瑞因对EV-A71抑制效果的WB检测
以胰酶消化RD细胞，以70％汇合度铺12孔培养板。
10％FBS DMEM培养24小时。弃上清，冰DMEM洗三次，以MOI＝1用DMEM稀释EV-A71病毒，加入12孔板中，每孔500微升。设置未感染对照孔。
4度孵育1小时，弃上清，以冰DMEM漂洗三次，加入10％FBS培养基。同时以终浓度50μM加入双醋瑞因；置37度培养24小时；24小时后，吸掉上清，冰浴,用PBS轻轻洗涤3次，加入100微升细胞裂解液(Sigma)，待至细胞裂解完全后，吸入1.5ml离心管中，12000转/分，4℃离心10分钟。
将上清转移到离心管中，用Nanodrop测量蛋白浓度，以等量总蛋白上样，12％SDS-PAGE，80V电泳2小时，将所述蛋白通过湿转的方法转移至PVDF膜(GE Health)上(200mA、90min)，考马斯亮蓝染色确定转膜效率，脱色后TBST(Tris-HCI缓冲液(0.5M pH7.6)100ml NaCl 8.5～9g(0.15mol/L)，1ml/L的Triton-20)洗涤3次，5％脱脂奶粉封闭过夜，TBST洗涤3次后，加入EV-A71鼠单克隆抗体(1％脱脂奶粉，抗体1/1000稀释)，37℃孵育1小时，TBST洗涤3次(每次10分钟)后，加入辣根过氧化酶标记的二抗(1％脱脂奶粉，抗体1/3000稀释)，37℃孵育45分钟，TBST洗涤3次后(每次10分钟)，再用超敏发光液显色，拍照。
以Image J分析WB条带灰度值,显色完成后，TBST洗膜，加入TBST稀释的抗GAPDH鼠单抗，室温孵育1小时，TBST洗三次，加入辣根过氧化酶标记的二抗(1％脱脂奶粉，抗体1/3000稀释)，37℃孵育45分钟，TBST洗涤3次后(每次10分钟)，再用超敏发光液显色，拍照。以Image J分析WB条带灰度值。
结果如图1B所示，可见双醋瑞因对EV-A71有显著抑制作用
示例性实验例二.双醋瑞因对乙肝病毒的抑制效果细胞水平药效学检测
Figure PCTCN2019123209-appb-000003
1.Elisa实验检测乙肝HepG2-2215细胞模型上清乙肝病毒S抗原。
吸取双醋瑞因处理的HepG2-2215细胞上清，以DMSO组为对照。依据检测试剂盒说明书，预实验检测细胞上清的不同稀释比例，以期以不同的稀释度满足其在仪器量程内。
具体检测方法如下：
将稀释的细胞上清加入酶标孔，每孔100微升。
37度孵育1小时，使S抗原与抗体(试剂盒中酶标板上的抗体)结合。
以洗涤液洗涤三次，每次将洗涤液拍干。加入50微升显色液A，50微升显色液B。37度孵育20分钟，加入终止液，读取450nm光吸收值，以630nm光吸收为参比波长。
2.荧光定量PCR(绝对定量)检测HepG2-2215细胞模型上清乙肝病毒DNA拷贝数。
以不同浓度双醋瑞因处理HepG2-2215细胞，处理24和48小时后，取细胞上清100微升，以裂解液及蛋白酶K处理上清，37度孵育45分钟。
将上清液吸入DNA结合柱上，12000转离心1分钟，弃去收集管中液体。以Washing Buffer洗涤结合柱两次，12000离心10分钟。置超净台干燥20分钟，以50微升TE缓冲液(10mM Tris-HCl,1mM EDTA,pH 8.0)溶解。
Nanodrop测量DNA浓度及纯度。以pCMV-HB X质粒为标准品，计算分子量并以质粒浓度计算质粒摩尔数。按10倍比例稀释质粒，共设置10 2-10 6拷贝数/微升五个浓度的标准品。
样品经适当稀释后与2×SYBgreen Mix、引物及灭菌水混合，配制定量PCR反应液，
HB X-F:5’-ACGTCCTTTGTTTACGTCCCGT；
HB X-R:5’-CCCAACTCCTCCCAGTCCTTAA。
将其在REAL-TIME PCR仪(Bio-Rad)上进行检测，绘制标准曲线，通过Ct值计算DNA拷贝数。
结果如图2A所示，双醋瑞因浓度为50μM及25μM时，能够有效抑制乙肝病毒S抗原的产生。
结果如图2B所示，可见双醋瑞因浓度在3.125μM以上，上清中病毒DNA拷贝数显著下降，其下降程度超过50％。
提示双醋瑞因能够抑制HepG-2-2215细胞中乙肝病毒的复制。
3.CCK-8法检测双醋瑞因对HepG2-2215细胞的毒性
以10％FBS DMEM培养基培养HepG2-2215细胞。
以2.5％胰酶消化细胞，以每孔加入100微升5000个细胞分细胞至96孔板。培养24小时，以10％FBS DMEM培养基2倍倍比稀释双醋瑞因，使其浓度从200微摩尔至0.9微摩尔,置37度培养48小时。
每孔加入10微升CCK-8溶液(碧云天生物科技)。
以用加相应量细胞培养液和CCK-8溶液但没有加入细胞的孔作为空白对照。细胞培养箱内继续孵育0.5小时，在450nm测定吸光度。
结果如图2C所示，双醋瑞因在25μM浓度以下，不引起显著的细胞毒性。 示例 性实验例三.双醋瑞因对人免疫缺陷病毒(HIV-1)的抑制效果细胞水平药效学 检测
Figure PCTCN2019123209-appb-000004
1.HIV-1假病毒的包装
以10％FBS DMEM培养基培养293T细胞。将pNL4-3 Luc-R-E及VSVG的质粒进行大量提取，Nanodrop测量质粒浓度及纯度。将状态较好的细胞消化并分至10cm培养皿。培养24小时后，弃培养基，以DMEM漂洗两次，加入5毫升DMEM。将pNL4-3 Luc-R-E及VSVG培养基按摩尔数1:1混合(总质量为6微克)，加入60微升PEI转染试剂。转染293T细胞。37度孵育2小时后，补加4毫升DMEM。继续培养46小时。吸取上清，1200转离心5分钟。保留上清。配制5×PEG8000 NaCl溶液。配制称取NaCl 8.766g；PEG8000  50g溶解在200ml纯水中。高压灭菌30分钟。室温冷却后与病毒上清液均匀混合。置4度过夜。12000离心一小时。沥干液体，用PBS重悬沉淀。
感染HeLa细胞，分析感染效率。
2.双荧光素酶检测双醋瑞因对HIV-1的抑制
以10％FBS DMEM培养基培养HeLa细胞。
将细胞胰酶消化并分至6孔板。计数，并按50％汇合度铺细胞至六孔板。37度培养24小时，去除培养基，冰PBS漂洗三次，按适当病毒浓度感染HIV-1假病毒，弃上清，以冰PBS冲洗三次，加入10％FBS DMEM培养基，同时加入不同浓度的双醋瑞因及DMOS对照。设置未感染对照组。
培养48小时后，弃上清，以冰PBS漂洗三次。每孔加入500微升PLB裂解液，室温摇床孵育15分钟，12000转离心5分钟，取上清。
取一96孔板，按照每个样品三个重复加入20微升蛋白裂解液。
每孔加入100微升LAR II荧光底物，测量萤火虫荧光素酶活力。之后迅速每孔加入100微升终止液，测量海肾荧光素酶活性。
计算荧光素酶活性与海肾荧光素酶活性的比值。
结果如图3A所示，在25μM及12.5μM浓度下，双醋瑞因能够显著抑制HIV-1的复制。
CCK-8检测双醋瑞因对HeLa细胞的细胞毒性，如图3B以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标测定。结果显示，双醋瑞因在25μM及12.5μM浓度下不引起显著的细胞毒性。
示例性实验例四、双醋瑞因对流感病毒的细胞及动物水平药效学检测
Figure PCTCN2019123209-appb-000005
1.流感病毒的扩增及纯化
HIN1-PR8、HIN1-2018、H3N2-2018、BY-2018及BV-2018流感病毒均采用鸡胚羊膜腔方法扩增。具体方法为：取出鸡胚，用手电筒，照蛋器照射看鸡胚，在气囊边缘拿铅笔划一横线，下方找到无血管位置划一叉。标记出注射位置。每个稀释度3-4枚，一半从-1的稀释度开始。超净台准备，酒精灯，针头，大针头。先将蛋喷酒精，然后拿大针烧一烧后先在气囊顶端打孔，再在标记位置打孔。用1ml注射器将稀释好的毒液注射。深度不易过深，大概0.3-0.5cm，右手注射，不能有气泡。蜡块儿融化后涂抹。放置37度培养箱培养72h收毒。放到4度冰箱过夜。喷酒精消毒，用镊子捣碎顶端气室。
烧镊子，撕破顶端膜结构。
用注射器吸取尿囊液，鸡胚需灭菌处理。分装冻存。取100微升测量血凝实验。
取适量鸡红细胞用PBS洗一次，不要超过3000转，短暂离心。PBS重悬，放到排枪吸盒中，上面套个一次性手套，用尖底一次性96皿，设置隐形对照，按1:1 25微升：25微升比例加入红细胞，加入十倍倍比稀释的病毒悬液，放置 20分钟，室温观察。病毒液2000转，4度离心10分钟，病毒液分装冻存。
以8％FBS DMEM培养基培养MDCK细胞，胰酶消化分细胞至96孔板，每孔2000个细胞。10％FBS DMEM培养基培养24小时。吸取病毒液或病毒培养上清100μl，以DMEM 10倍稀释度倍比稀释。
取出96孔培养板，去除每孔培养基，按每孔100微升加入稀释的病毒液(按1:2000加入TPCK胰酶)，4度吸附1小时，加200微升10％FBS DMEM培养基，按1:2000体积比加入TPCK胰酶。37度培养48-72小时，观察每个稀释度病变孔，以Reed-Muench法计算病毒滴度。
2.WB检测双醋瑞因对流感病毒的抑制效果
胰酶消化MDCK细胞，细胞计数并分细胞至12孔培养板。24小时后，以MOI＝1接种流感病毒，并加入双醋瑞因或其它对照，同时按1:2000体积比加入TPCK胰酶，吸附45分钟后，弃上清，加入5％FBS DMEM培养基，按1:2000体积比加入TPCK胰酶；培养24或48小时后，弃上清，冰PBS洗三次，加入裂解液100微升。待至细胞裂解完全后，吸入1.5ml离心管中，12000转/分，4℃离心10分钟。
将上清转移到离心管中，用Nanodrop测量蛋白浓度，以等量总蛋白上样，12％SDS-PAGE，80V电泳2小时，将所述蛋白通过湿转的方法转移至PVDF膜(GE Health)上(200mA、90min)，考马斯亮蓝染色确定转膜效率；
脱色后TBST(Tris-HCI缓冲液(0.5M pH7.6)100ml NaCl 8.5～9g(0.15mol/L)，1ml/L的Triton-20)洗涤3次，5％脱脂奶粉封闭过夜，TBST洗涤3次后，加入兔抗A型流感病毒NP蛋白单克隆抗体或兔抗B型流感NP蛋白多克隆抗体(1％脱脂奶粉，抗体1/1000稀释)，37℃孵育1小时，TBST洗涤3次(每次 10分钟)后，加入辣根过氧化酶标记的二抗(购买自中衫金桥)(1％脱脂奶粉，抗体1/3000稀释)，37℃孵育45分钟，TBST洗涤3次后(每次10分钟)，再用超敏发光液显色，拍照。
以Image J分析WB条带灰度值。显色完成后，TBST洗膜，加入TBST稀释的抗GAPDH鼠单抗，室温孵育1小时，TBST洗三次，加入辣根过氧化酶标记的二抗(购买自中衫金桥)(1％脱脂奶粉，抗体1/3000稀释)，37℃孵育45分钟，TBST洗涤3次后(每次10分钟)，再用超敏发光液显色，拍照。以Image J分析WB条带灰度值。
结果如图4A显示，双醋瑞因能够显著抑制HIN1流感病毒在细胞内的复制，主要表现在流感病毒NP蛋白的大量减少。抑制HIN1流感病毒的效率达到90％以上。
图4B显示，流感病毒接种MDCK细胞后，以不同浓度双醋瑞因或磷酸奥司他韦处理细胞，24小时后，细胞裂解并以Western blot检测NP蛋白信号。DMSO为DMSO处理组。Mock为未感染组。结果显示,在相同摩尔浓度下，双醋瑞因对HIN1的抑制效果要好于奥司他韦。
图4C显示，流感病毒接种MDCK细胞后，以50μM浓度双醋瑞因或磷酸奥司他韦处理细胞，24小时后，细胞裂解并以Western blot检测NP蛋白信号。DMSO为DMSO处理组。Mock为未感染组。结果显示,在相同摩尔浓度下，双醋瑞因对2018年HIN1流行毒株的抑制效果要好于奥司他韦。
图4D显示，流感病毒接种MDCK细胞后，以50μM浓度双醋瑞因或磷酸奥司他韦处理细胞，24小时后，细胞裂解并以Western blot检测NP蛋白信号。DMSO为DMSO处理组。Mock为未感染组。结果显示,在相同摩尔浓度下，双醋瑞因对2018年B型流行毒株的抑制效果要好于奥司他韦。
3.双醋瑞因对流感病毒抑制效果的间接免疫荧光检测
以胰酶消化MDCK细胞，计数，每孔50％汇合度铺细胞至24孔板。
8％FBS DMEM培养24小时。弃上清，冰PBS洗三次，以MOI＝1用DMEM稀释病毒(按1:2000加入TPCK胰酶)，加入24孔板中，每孔250微升。设置未感染对照孔。37度孵育1小时，弃上清，以冰DMEM漂洗三次，加入10％FBS培养基(按1:2000加入TPCK胰酶)，同时加入双醋瑞因或其它对照药物。
置37度培养48小时，以4％多聚甲醛固定10分钟。0.2％Triton-X100冰上破膜10分钟，加入3％BSA，室温封闭1小时。加入1μg/ml的流感病毒NP蛋白抗体，室温孵育1小时，PBS洗三次，每次10分钟。加入0.5μg/ml的FITC标记山羊抗小鼠二抗。
室温孵育45分钟。PBS洗涤三次，每次10分钟。以488波长，在荧光显微镜下观察并拍照。以Image J计算绿色信号细胞数。以DAPI计算细胞总数。计算感染效率或半数抑制浓度。
如图4E显示：在病毒与细胞孵育后，加入梯度稀释的双醋瑞因或磷酸奥司他韦(初始浓度为50μM)，感染24小时后，NP蛋白免疫荧光分析病毒感染细胞数目,结果显示，双醋瑞因对流感病毒的半数抑制浓度为4.46μM左右。磷酸奥司他韦的半数抑制浓度为5.2微摩尔。
如图4J显示，双醋瑞因在25μM及2.5μM浓度以下，显著降低流感病毒NP蛋白信号水平。表明双醋瑞因能够显著抑制A型流感病毒的复制
4.双醋瑞因对小鼠感染流感病毒的保护效果评价
SPF级18-21克雌雄BALB/c小鼠分笼。每笼5只，设置未感染组，DMSO 组，奥司他韦组，双醋瑞因组，每组8只小鼠。放置SPF饲养室适应一周。小鼠以异氟烷麻醉。鼻腔接种流感病毒(10 5TCID50/ml)90微升后，以每天，双醋瑞因组以10mg/kg体重灌胃小鼠双醋瑞因，奥司他韦组以50μL/kg体重灌胃磷酸奥司他韦，DMSO组以50μL/kg体重灌胃DMSO。每日定时称量小鼠体重，持续观察，统计小鼠死亡数目。
结果如图4F显示，磷酸奥司他韦组及双醋瑞因组的小鼠体重在第四天出现显著下降，在第6天达到最低值。之后逐渐恢复并增高。DMSO组则在第四天后一直加剧下降，于第九天，全部死亡。Mock组则一直在升高。显示出双醋瑞因对流感病毒感染的小鼠具有保护效果。
5.双醋瑞因对流感病毒感染小鼠死亡率的影响。
DMSO为DMSO处理组，Oseltamivir为磷酸奥司他韦处理组，INC.3为双醋瑞因处理组。Mock为未感染组。
小鼠鼻腔接种流感病毒(105TCID50/ml)后，双醋瑞因组以10mg/kg体重灌胃小鼠双醋瑞因，磷酸奥司他韦处理组以50μL/kg体重灌胃磷酸奥司他韦，DMSO组以50μL/kg体重灌胃DMSO。观察小鼠死亡率。
结果如图4G显示，在每组8只实验小鼠中，奥司他韦组出现一只死亡，双醋瑞因组出现一只死亡。DMSO组则在病毒感染后的第8天出现死亡，第10天全部死亡。上述结果说明，双醋瑞因及磷酸奥司他韦均能有效降低流感病毒感染引起的小鼠死亡。
6.CCK-8法检测双醋瑞因对MDCK细胞的细胞毒性。
以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标 测定。INC1及INC16为化合物对照。Oseltamivir为磷酸奥司他韦处理组。INC3为双醋瑞因组。
结果显示如图4H，双醋瑞因在50μM浓度以下，MDCK细胞中不引起显著的细胞毒性。
7.CCK-8法检测双醋瑞因对A549细胞的细胞毒性。
以不同稀释浓度的双醋瑞因处理细胞，48小时后，加入CCK-8并进行酶标测定。NC1及INC16为化合物对照。Oseltamivir为磷酸奥司他韦处理组。INC3为双醋瑞因组。
结果显示如图4I:，双醋瑞因在25μM浓度以下，不引起显著的细胞毒性。

Claims (13)

  1. 一种预防或治疗病毒感染的药物，其特征在于：所述药物的药效成分选自双醋瑞因、大黄酸、大黄酚、单乙酰大黄酸或其要学上可接受的盐、酯或前药。
  2. 一种预防或治疗病毒感染的药物，其特征在于：所述药物的药效成分包含双醋瑞因、大黄酸、大黄酚、单乙酰大黄酸或其要学上可接受的盐、酯或前药。
  3. 根据权利要求1或2所述的药物，其特征在于：所述病毒感染是指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、冠状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒。
  4. 根据权利要求3所述的药物，其特征在于：
    所述嗜肝DNA病毒指人乙型肝炎病毒、鸭乙型肝炎病毒；
    所述肠道病毒指人肠道病毒、肠道病毒A71型、柯萨奇病毒、柯萨奇病毒A16、脊髓灰质炎病毒；
    所述正黏病毒指A型流感、B型流感；
    所述逆转录病毒指艾滋病病毒；
    所述副黏病毒指呼吸道合胞病毒；
    所述疱疹病毒指单纯疱疹1型或单纯疱疹2型；
    所述黄病毒指西尼罗河病毒、登革病毒、寨卡病毒和/或乙型脑炎病毒感染；
    所述弹状病毒指狂犬病毒；
    所述人环装病毒指诺如病毒；
    所述疱疹病毒指人巨细胞病毒。
  5. 根据权利要求4所述的药物，其特征在于：所述病毒感染是指
    逆转录病毒合并嗜肝DNA病毒感染；
    所述逆转录病毒合并肠道病毒感染；
    所述逆转录病毒合并嗜肝DNA病毒、肠道病毒感染；
    逆转录病毒合并正黏病毒、肠道病毒感染；
    逆转录病毒并正黏病毒感染；
    肠道病毒感染合并正黏病毒感染；或
    肠道病毒感染合并嗜肝DNA病毒感染。
  6. 根据权利要求3所述的药物，其特征在于：所述病毒感染是指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒中的任意两种或多种病毒合并感染。
  7. 根据权利要求1或2所述的药物，其特征在于：还包含药学上可接受的载体。
  8. 根据权利要求1或2所述的药物，其特征在于：所述药物为口服制剂、注射制剂、片剂、胶囊剂、颗粒剂、混悬剂或丸剂。
  9. 一种治疗病毒所致疾病的方法，其特征在于，包括向受试者施用含治疗有效剂量的化合物的药物，所述化合物选自双醋瑞因、大黄酸、大黄酚、单乙酰大黄酸或其要学上可接受的盐、酯或前药；
  10. 根据权利要求9所述的方法，其特征在于，所述受试者是患有以下一种或多种病毒感染所致疾病的患者：嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒。
  11. 根据权利要求10所述的方法，其特征在于，
    所述嗜肝DNA病毒指人乙型肝炎病毒、鸭乙型肝炎病毒；
    所述肠道病毒指人肠道病毒、肠道病毒A71型、柯萨奇病毒、柯萨奇病毒A16、脊髓灰质炎病毒；
    所述正黏病毒指A型流感、B型流感；
    所述逆转录病毒指艾滋病病毒；
    所述副黏病毒指呼吸道合胞病毒；
    所述疱疹病毒指单纯疱疹1型或单纯疱疹2型；
    所述黄病毒指西尼罗河病毒、登革病毒、寨卡病毒和/或乙型脑炎病毒感染；
    所述弹状病毒指狂犬病毒；
    所述人环装病毒指诺如病毒；
    所述疱疹病毒指人巨细胞病毒。
  12. 根据权利要求9所述的方法，其特征在于，所述受试者是患有以下一种或多种病毒感染所致疾病的患者：
    逆转录病毒合并嗜肝DNA病毒感染；
    所述逆转录病毒合并肠道病毒感染；
    所述逆转录病毒合并嗜肝DNA病毒、肠道病毒感染；
    逆转录病毒合并正黏病毒、肠道病毒感染；
    逆转录病毒并正黏病毒感染；
    肠道病毒感染合并正黏病毒感染；或
    肠道病毒感染合并嗜肝DNA病毒感染。
  13. 根据权利要求9所述的方法，其特征在于，所述受试者是患有以下一种 或多种病毒感染所致疾病的患者：
    所述病毒感染是指嗜肝DNA病毒、逆转录病毒、肠道病毒、正黏病毒、纤丝病毒、呼肠孤病毒、沙粒病毒、戊型肝炎病毒、星状病毒、环状病毒、细小病毒、腺病毒、多瘤病毒、疱疹病毒、痘病毒、人乳头瘤病毒、副黏病毒、黄病毒、疱疹病毒、甲病毒、弹状病毒中的任意两种或多种病毒合并感染。
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