WO2022007713A1 - 牛磺罗定在抗病毒中的应用 - Google Patents

牛磺罗定在抗病毒中的应用 Download PDF

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WO2022007713A1
WO2022007713A1 PCT/CN2021/104197 CN2021104197W WO2022007713A1 WO 2022007713 A1 WO2022007713 A1 WO 2022007713A1 CN 2021104197 W CN2021104197 W CN 2021104197W WO 2022007713 A1 WO2022007713 A1 WO 2022007713A1
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virus
taurolidine
infection
pharmaceutically acceptable
cell
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PCT/CN2021/104197
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English (en)
French (fr)
Chinese (zh)
Inventor
高玉伟
李元果
吕朝相
苏忠
李松
王铁成
钟武
向海洋
周彤
孙伟洋
王雪峰
周博
李恩涛
冯娜
赵永坤
杨松涛
夏咸柱
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Academy Of Military Sciences Pla China Academy Of Military Medical Sciences Institute Of Military Veterinary Medicine
Changchun Mailing Biological Engineering Co Ltd
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Academy Of Military Sciences Pla China Academy Of Military Medical Sciences Institute Of Military Veterinary Medicine
Changchun Mailing Biological Engineering Co Ltd
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Priority claimed from CN202010639186.0A external-priority patent/CN111773227A/zh
Priority claimed from CN202010638926.9A external-priority patent/CN111658652A/zh
Priority claimed from CN202010994928.1A external-priority patent/CN112641789A/zh
Application filed by Academy Of Military Sciences Pla China Academy Of Military Medical Sciences Institute Of Military Veterinary Medicine, Changchun Mailing Biological Engineering Co Ltd filed Critical Academy Of Military Sciences Pla China Academy Of Military Medical Sciences Institute Of Military Veterinary Medicine
Priority to JP2023501231A priority Critical patent/JP7752675B2/ja
Priority to BR112023000308A priority patent/BR112023000308A2/pt
Publication of WO2022007713A1 publication Critical patent/WO2022007713A1/zh
Anticipated expiration legal-status Critical
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/549—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame having two or more nitrogen atoms in the same ring, e.g. hydrochlorothiazide
    • 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
    • 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

Definitions

  • This application is based on the CN application number of 202010638926.9, the application date of July 6, 2020, the CN application number of 202010639186.0, the application date of July 6, 2020, and the CN application number of 202010994928.1, and the application date of September 2020.
  • the disclosure content of this CN application is hereby incorporated into this application in its entirety.
  • the invention relates to the field of drug therapy, in particular to the application of taurolidine in antiviral, especially in the treatment and/or prevention of novel coronavirus and influenza virus infection.
  • Coronaviruses are a large group of viruses that exist widely in nature. In biological classification, coronaviruses belong to the order Nidovirales, the family Coronaviridae, and the genus Coronavirus. The diameter is 80-120nm, and the nucleic acid is non-segmented single-stranded (+) RNA, 27-31kb in length. Coronaviruses are viruses with the longest RNA nucleic acid strands among RNA viruses, and contain important structural features unique to positive-strand RNAs: that is, the 5' end of the RNA chain has a methylated "cap" and the 3' end has a PolyA "tail” structure. This structure is very similar to eukaryotic mRNA, and it is also an important structural basis for its genomic RNA itself to function as a translation template.
  • Coronavirus can be excreted through respiratory secretions, transmitted through oral fluid, sneezing, contact, and spread through air droplets, thereby infecting vertebrates and humans, such as humans, mice, horses, pigs, cats, dogs, poultry, etc. Cause respiratory infection and acute gastroenteritis and other diseases.
  • Influenza virus is the main virus that causes acute respiratory infectious diseases, leading to influenza.
  • Influenza virus belongs to the family Orthomyxoviridae and is an RNA virus that mainly includes influenza A virus, influenza B virus and influenza C virus.
  • Influenza A virus has high variability, transmissibility and pathogenicity, and can easily cause seasonal epidemics. Human infection with influenza virus can cause severe pneumonia, acute respiratory distress syndrome, sepsis with shock, etc. The mortality rate is very high, posing a huge threat to social public health security.
  • Currently commonly used drugs for anti-influenza virus mainly include alkylamine drugs and neuraminidase inhibitor drugs.
  • alkylamine drugs are only effective against influenza A virus, and it has been found that influenza virus can rapidly develop resistance to such antiviral drugs through genetic mutation or drug response.
  • Neuraminidase inhibitor drugs can inhibit viral replication by preventing the release of progeny viruses.
  • the side effects of such drugs have been a difficult problem in clinical application, including hallucinations, abnormal behavior, hearing and visual disturbances. Therefore, it is of great significance to find a variety of subtypes of influenza virus and develop a class of drugs with universal applicability.
  • the new coronavirus (Severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2) is the seventh known coronavirus that can infect humans.
  • SARS-CoV-2 severe Acute Respiratory Syndrome Coronavirus 2, SARS-CoV-2
  • SARS-CoV-2 severe Acute Respiratory Syndrome Coronavirus 2
  • SARS-CoV-2 severe Acute Respiratory Syndrome Coronavirus 2
  • Taurolidine (English name Taurolidine, chemical name is 4,4'-methylenebis[tetrahydro-2H-1,2,4-thiadiazine] 1,1,1',1'-tetraoxide , the molecular formula is C 7 H 16 N 4 O 4 S 2 ) is a derivative of the amino acid taurine, and its structure is as follows:
  • Taurolidine has anti-endotoxin, anti-bacterial and anti-adherent properties.
  • taurolidine can chemically react with cell walls, endotoxins and exotoxins to inhibit microbial adhesion and play an antibacterial role.
  • taurolidine can induce cytotoxicity of tumor cells by inducing apoptosis, autophagy and necrosis. The extent to which these processes are involved may vary with the type of tumor cell. Until July 2020, there were more than 260 foreign literature searches on taurolidine research reports, most of which focused on the exploration of the effect of taurolidine on tumor-related signaling pathways, while the application of taurolidine in antiviral activity was not yet available. See research reports.
  • taurolidine can significantly inhibit influenza virus (for example, H1N1, H3N2 or H5N1) and coronavirus (for example, new coronavirus SARS-CoV-2, canine coronavirus CCV or murine hepatitis virus MHV).
  • influenza virus for example, H1N1, H3N2 or H5N1
  • coronavirus for example, new coronavirus SARS-CoV-2, canine coronavirus CCV or murine hepatitis virus MHV.
  • taurolidine has a significant protective effect on the lungs and can prolong the survival time of mice infected with influenza virus or coronavirus.
  • taurolidine can be used for the prevention and treatment of pulmonary diseases caused by influenza virus or coronavirus. Therefore, one purpose of the present application is to broaden the efficacy range of taurolidine and provide its application in antiviral.
  • the application provides taurolidine or derivatives, prodrugs, solvates or pharmaceutically acceptable salts thereof, or containing taurolidine or derivatives, prodrugs, solvates or pharmaceutically acceptable salts thereof Use of a composition of acceptable salts in the manufacture of an antiviral medicament.
  • the application provides taurolidine or derivatives, prodrugs, solvates or pharmaceutically acceptable salts thereof, or taurolidine or derivatives, prodrugs, solvates or pharmaceutically acceptable salts thereof Use of a composition of an acceptable salt of the above in the manufacture of a medicament for inhibiting the replication and/or multiplication of a virus in a cell.
  • the application provides taurolidine or derivatives, prodrugs, solvates or pharmaceutically acceptable salts thereof, or taurolidine or derivatives, prodrugs, solvates or pharmaceutically acceptable salts thereof Use of a composition of the above acceptable salt in the preparation of a medicament for preventing and/or treating a disease or infection caused by a virus.
  • the application provides a method of inhibiting viral infection, comprising administering to a cell infected with the virus, a cell susceptible to infection with the virus, or a subject in need thereof an effective amount of taurolidine or its Derivatives, prodrugs, solvates, or pharmaceutically acceptable salts, or compositions containing taurolidine or derivatives, prodrugs, solvates, or pharmaceutically acceptable salts thereof.
  • the method further comprises the step of administering to the cell or subject an effective amount of an antibiotic, such as erythromycin, acetylspiramycin, azithromycin, and the like.
  • an antibiotic such as erythromycin, acetylspiramycin, azithromycin, and the like.
  • the antibiotic is not vancomycin.
  • the antibiotic is administered concurrently, separately or sequentially with the taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt, or composition thereof.
  • the present application provides a method of inhibiting the replication and/or propagation of a virus in a cell, comprising combining the cell with an effective amount of taurolidine or a derivative, prodrug, solvate or pharmaceutical thereof A step of contacting an acceptable salt above, or a composition containing taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof.
  • the method further comprises the step of contacting the cells with an effective amount of an antibiotic, such as erythromycin, acetylspiramycin, azithromycin, and the like.
  • an antibiotic such as erythromycin, acetylspiramycin, azithromycin, and the like.
  • the antibiotic is not vancomycin.
  • the antibiotic is administered to the cells simultaneously, separately, or sequentially with the taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt, or composition thereof.
  • the present application provides a method of preventing and/or treating a disease or infection caused by a virus, comprising administering to a subject in need thereof an effective amount of taurolidine or a derivative, prodrug, The step of solvating or pharmaceutically acceptable salt, or a composition comprising taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof.
  • the method further comprises the step of administering to the subject an effective amount of an antibiotic, such as erythromycin, acetylspiramycin, azithromycin, and the like.
  • an antibiotic such as erythromycin, acetylspiramycin, azithromycin, and the like.
  • the antibiotic is not vancomycin.
  • the antibiotic is administered to the subject simultaneously, separately or sequentially with the taurolidine, or a derivative, prodrug, solvate or pharmaceutically acceptable salt, or composition thereof .
  • any one of the above described herein the virus is an RNA virus, such as influenza, corona virus, hepatitis virus or HIV.
  • influenza virus is influenza A, B, or C, eg, H1N1, H2N2, H3N2, H5N1, H7N7, or H9N2.
  • the coronavirus is novel coronavirus SARS-CoV-2, canine coronavirus CCV, or murine hepatitis virus MHV.
  • the coronavirus is CCV.
  • the coronavirus is SARS-CoV-2.
  • the coronavirus is MHV.
  • any one of the above-described herein in said cell is a mammalian cell.
  • the mammal is selected from the group consisting of bovine, equine, ovine, porcine, canine, feline, rodent, and primate.
  • the mammal is a human, cat, chicken, pig, or dog.
  • the cells are human-derived cells or chicken embryo cells.
  • any one of the above-described herein is a disease or infection in the lung disease.
  • any one of the above-described herein is selected from influenza illness or infection, simple infections, including severe pneumonia, pneumonia, severe acute respiratory infections or acute, hypoxic respiratory failure, acute respiratory distress syndrome, septic sepsis, septic shock or severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and novel coronavirus pneumonia (COVID-19).
  • the uncomplicated infection is fever, cough, or sore throat.
  • the disease or infection is COVID-19.
  • any one of the above-described compositions described herein further comprises an antibiotic, such as erythromycin, acetylspiramycin, azithromycin.
  • the antibiotic is not vancomycin.
  • the composition may also contain pharmaceutically acceptable carriers such as diluents, absorbents, wetting agents, excipients, fillers, binders, disintegrants, surfactants and any combination thereof.
  • PVP-KF-17 is included in the composition.
  • the composition is an injection, infusion, tablet, capsule, spray, aerosol, or douche. In some embodiments, the composition is presented in unit dosage form.
  • the composition contains 1-1000 mg (eg, 1-800 mg, 1-500 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-20 mg, or 1-10 mg) of taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof.
  • the composition formula can be: 2g of taurolidine and 5g of PVP-KF-17 per 100ml of solution.
  • the preparation process is as follows: first dissolve PVP-KF-17 in water at room temperature, and after it is completely dissolved, add taurolidine at a temperature of 45°C to 50°C, stir until it is completely dissolved, and add an appropriate amount of 4% sodium hydroxide. The pH of the solution was adjusted, an appropriate amount of activated carbon was added, the temperature was maintained for 30 minutes, and the solution was filtered while hot. The filtrate is then filtered through a 0.25mm membrane, bottled and capped.
  • TRD refers to taurolidine
  • the term "pharmaceutically acceptable salts” includes inorganic or organic acid salts of taurolidine, as well as inorganic or organic base salts such as sodium, potassium, calcium, lithium, meglumine salts , Hydrochloride, Hydrogen Oxalate, Hydrogenate, Nitrate, Sulfate, Hydrogen Sulfate, Phosphate, Hydrogen Phosphate, Acetate, Propionate, Butyrate, Oxalate, Trimethyl Ethyl acetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate Sugar salt, benzoate, mesylate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate or pamoate, etc.
  • derivatives refer to sulfonamides derived from the substitution of hydrogen atoms or other atomic groups in taurolidine, which have a biological activity equivalent to more than 10% of taurolidine.
  • similar compounds are formed by simple substitution of two thiol groups in taurolidine molecule or structural changes brought about by adding or subtracting groups -NH- and -CH 2 -.
  • prodrug refers to a derivative with new physical, chemical and biological properties obtained by modifying the chemical structure of taurolidine, which itself does not show the pharmacology of the original drug (that is, taurolidine). It can be converted into the original drug in the body and play a role.
  • Taurolidine may exist in the form of a solvate (preferably a hydrate) comprising a polar solvent, in particular water, methanol or ethanol, as a structural element of taurolidine crystals.
  • a polar solvent in particular water, methanol or ethanol
  • the amount of polar solvent, especially water may be present in stoichiometric or non-stoichiometric ratios. It should be understood that any solvate of taurolidine used in the treatment of a disease or infection described herein, while likely to provide different properties (including pharmacokinetic properties), once absorbed into a subject. , taurolidine will be obtained, such that the use of taurolidine covers the use of any solvate of taurolidine, respectively.
  • the term "therapeutically effective amount” or “prophylactically effective amount” refers to an amount sufficient to treat or prevent a patient's disease but low enough to avoid serious side effects (at a reasonable benefit/risk ratio) within the scope of sound medical judgment.
  • a therapeutically effective amount of a compound will depend on the particular compound selected (eg, taking into account the potency, effectiveness and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, the severity of the patient being treated. Factors such as age, size, weight and physical ailment, medical history of the patient being treated, duration of treatment, nature of concurrent therapy, desired therapeutic effect, and the like vary, but can still be routinely determined by those skilled in the art.
  • the specific dosage and method of use for different patients will depend on many factors, including the patient's age, weight, sex, natural health, nutritional status, active strength of the drug, time of administration, metabolic rate, severity of the condition, and diagnosis and treatment. Physician's subjective judgment. It is preferred here to use doses between 0.001 and 1000 mg/kg body weight/day.
  • the present application provides the application of taurolidine in antiviral.
  • Vero-E6, MDCK, A549, Huh7 and other cells were used as cell models for the antiviral effect of taurolidine.
  • the virus liquid was harvested according to the cytopathic state, and the inhibitory effect of taurolidine on the virus was determined.
  • the results show that taurolidine can significantly inhibit influenza viruses (eg, H1N1, H3N2, or H5N1) and coronaviruses (eg, novel coronavirus SARS-CoV-2, canine coronavirus CCV, or murine hepatitis virus HMV) at the cellular level. .
  • influenza viruses eg, H1N1, H3N2, or H5N1
  • coronaviruses eg, novel coronavirus SARS-CoV-2, canine coronavirus CCV, or murine hepatitis virus HMV
  • taurolidine has a significant protective effect on the lungs and can prolong the survival time of mice infected with influenza virus or SARS-CoV-2 virus. It shows that taurolidine can be used for the prevention and treatment of pulmonary diseases caused by influenza virus or coronavirus.
  • the scope of efficacy of taurolidine is expanded, which provides a research basis and opens up new directions for the research and development of drugs for the prevention or treatment of pulmonary infections caused by viruses.
  • Figure 1 Sensitivity test results of taurolidine to MDCK, Vero-E6, and F81 cells.
  • Figure 2 Inhibitory effect of 14 ⁇ M taurolidine on H3N2 subtype influenza virus.
  • Figure 3 Inhibitory effect of TRD on H1N1 subtype influenza virus.
  • Figure 3B After converting the concentration of TRD to logarithm and virus titer to percentage, semi-logarithmic fit curve to calculate EC50 value.
  • Figure 3C Sensitivity of A549 cells measured by MTT after treatment with the solvent DMSO and different TRD concentrations for 24 hours.
  • Figure 3D After infection with H1N1-UI182 at the indicated MOIs, cells were treated with the solvents DMSO and TRD for 12 hours, and oseltamivir (OSTA) was used as a control. Nuclei were stained with DAPI and infected cells were detected by nuclear vNP staining (scale bar 10 ⁇ m).
  • Figure 3E Quantification of the number of NP-positive nuclei.
  • Figure 3F Total protein expression levels of NPs in A549 cells. Whole cell extracts were analyzed by western blot using ⁇ -actin as a control.
  • Figure 4 The inhibitory effect of TRD on different influenza virus subtypes.
  • Figure 4A-D After 12 hours of infection of A549 cells with influenza virus strains H1N1-UI182, H1N1-PR8, H3N2 and H5N1 strains, cells were treated with DMSO and TRD for 24 hours, and virus titers were determined.
  • Figure 4E-H After infection of MDCK cells with influenza strains H1N1-UI182, H1N1-PR8, H3N2 and H5N1 strains, cells were treated with DMSO and TRD for 24 hours and virus titers were determined. OSTA was used as a positive control drug.
  • the viral titers of the strains were expressed as Log 10 TCID 50 . Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • Figure 5 Inhibitory effect of 15 ⁇ M and 30 ⁇ M taurolidine on pseudorabies virus.
  • Figure 6 Inhibitory effect of 15 [mu]M and 30 [mu]M taurolidine on parvovirus.
  • Figure 7 TRD inhibits SARS-CoV-2 replication in cells.
  • Figure 7A Detection of virus pathogenicity.
  • Figure 7C Sensitivity of Vero-E6 cells measured by MTT after treatment with the solvent DMSO and different TRD concentrations for 24 hours.
  • Figure 7D After infection of Vero-E6 cells with novel coronavirus SARS-CoV-2 (100TCID 50 ) and (200 TCID 50 ), respectively, cells were treated with DMSO and TRD for 24 hours, and viral titers were determined.
  • Figure 7E Total protein expression levels of NPs in Vero-E6 cells. Whole cell extracts were analyzed by western blot using ⁇ -actin as a control.
  • Figure 8 Anti-SARS-CoV-2 activity of TRD.
  • Figure 8A,C Vero-E6 and Huh7 cells were imaged with light microscopy to detect typical SARS-CoV-2-induced cytolysis.
  • FIG. 9 Anti-SARS-CoV-2 activity of TRD.
  • Fig. 9A,C After infection with SARS-CoV-2 at the indicated MOI, Vero-E6 (left) and Huh7 (right) cells were treated with the solvent DMSO and different doses of TRD for 24 h, respectively, and the nuclei were Infected cells were stained with DAPI and detected by viral nuclear NP staining (scale bar 10 ⁇ m).
  • Figure 10 Progression of influenza virus infection is limited after TRD treatment in vivo.
  • Figure 10A the mice were divided into blank control groups (Control, ), the virus control group (Virus, ) and the TRD treatment group (TRD, ). Cumulative body weight change plotted against days after infection with H1N1-UI182.
  • Figure 10B Survival curves plot changes in mouse survival after infection with H1N1-UI182.
  • Figure 10C,D Virus titers in mouse lung tissue were measured on days 3 and 5 after infection with H1N1-UI182.
  • Figure 10E Total protein expression levels of NPs in mouse lung tissue at 5 days. Whole cell extracts were analyzed by western blot using ⁇ -actin as a control.
  • Figure 10F Quantification of NP expression levels. Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • FIG 11 TRD treatment limits the course of infection of mice with different influenza virus subtypes.
  • Figure 11A the mice were divided into blank control groups (Control, ), the virus control group (Virus, ) and the TRD treatment group (TRD, ). Cumulative weight changes were plotted against days post-infection with H3N2.
  • Figure 11B Survival curves plot changes in mouse survival after infection with H3N2.
  • Figure 11C,D Virus titers in mouse lung tissue were measured on days 3 and 5 after infection with H3N2.
  • Figure 11E Cumulative change in body weight plotted against days after infection with H5N1.
  • Figure 11F Survival curves plot changes in mouse survival after infection with H5N1.
  • Figure 11G,H Virus titers in mouse lung tissue were measured on days 3 and 5 after infection with H5N1. Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • Figure 12 The protective effect of TRD on the lungs.
  • Figure 12A, B After the 3rd and 5th day of H1N1-UI182 virus infection, the protective effect of the drug on the lung tissue of BALB-C mice was detected.
  • Figure 12C, D Drug protection on BALB-C mouse lung tissue was measured on days 3 and 5 after H3N2 virus infection.
  • Figure 12E, F Drug protection on BALB-C mouse lung tissue was measured on days 3 and 5 after H5N1 virus infection. Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • FIG. 13 TRD improves the extent of lung injury.
  • HE staining was performed on the lung tissues of mice in the control group, virus group and TRD-treated group, respectively, and representative photos of different groups are shown, left (HIN1-UI182), middle (H3N2), right (H5N1).
  • Figure 14 TRD improves body weight and survival in SARS-CoV-2 infected mice.
  • Figure 14A BALB-C female mice were divided into control groups (Control, ), the virus control group (Virus, ) and the TRD treatment group (TRD, ). Cumulative weight change is plotted against days after infection with SARS-CoV-2.
  • Figure 14B Survival curve of virus-infected BALB-C mice.
  • Figure 14C C57BL mice were divided into control groups (Control, ), the virus control group (Virus, ) and the TRD treatment group (TRD, ). Cumulative weight change is plotted against days after virus infection.
  • Figure 15 TRD improves prognosis in SARS-CoV-2 infected mice.
  • Figure 15A Viral RNA production in lung tissues of different groups of BALB-C mice was measured on days 3 and 5 after infection with SARS-CoV-2.
  • Figure 15B Drug protection on BALB-C mouse lung tissue was measured on days 3 and 5 post virus infection.
  • Figure 15C Viral titers were measured in lung tissue of C57BL mice on days 3 and 5 post virus infection.
  • FIG. 16 Effects of TRD on leukocytes following SARS-CoV-2 infection.
  • Fig. 16A, B The percentage of immune cells in the serum of BALB-C mice was determined on days 3 and 5 after virus infection.
  • Figure 16C,D The percentage of immune cells in the serum of C57BL mice was determined on days 3 and 5 post virus infection. Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • FIG. 17 Effects of TRD on host platelets following SARS-CoV-2 infection.
  • Fig. 17A, B Platelet numbers in serum of BALB-C mice were measured on days 3 and 5 after virus infection.
  • Figure 17C,D Platelet numbers in serum of C57BL mice were measured on days 3 and 5 post virus infection. Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • Figure 18 TRD ameliorates the malignancy of SARS-CoV-2 in mouse lung tissue lesions.
  • Figure 18A Pathological analysis flow.
  • Figure 18B Different groups of mouse lung tissue were sampled and photographed separately. Black arrows ( ⁇ ) indicate severe tissue lesions.
  • FIG. 19 TRD ameliorates the malignancy of SARS-CoV-2 in mouse lung, liver, spleen and kidney tissue lesions.
  • HE staining was performed on the lung, liver, spleen, and kidney tissues of mice in the control group, virus group, and TRD-treated group, respectively, and representative photos of different groups are shown.
  • Black arrows ( ⁇ ) indicate severe tissue hemorrhage.
  • FIG. 20 TRD inhibits histopathological expression of ACE2 following viral infection. After infection with SARS-CoV-2 virus, immunohistochemical detection was performed on the lung tissues of mice (BALB-C and C57BL) in the control group, virus group and TRD treatment group, respectively, and representative photos of different groups are shown (left), And the percentage of NP-positive expressing cells was calculated by quantitative analysis (right). Red arrows ( ⁇ ) indicate NP-positive cells. Data represent mean ⁇ SD and statistical significance was assessed by ANOVA analysis, *p ⁇ 0.05, **p ⁇ 0.01 and ***p ⁇ 0.001.
  • the BALB/c and C57BL/6N mice and taurolidine used in the examples are all commercially available products, wherein taurolidine is provided by Changchun Mailing Bioengineering Co., Ltd., Vero-E6, MDCK, A549, Huh7 cells and influenza
  • the virus originated from the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Academy of Military Sciences, and the SARS-CoV-2 virus was derived from Beijing isolates, which were stored in the Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Academy of Military Sciences.
  • MDCK, Vero-E6, and F81 cells were stored in liquid nitrogen, taken out and recovered, and passed on for three consecutive generations. After the cells grew well, they were used for experimental research. MDCK, Vero-E6, and F81 cells were seeded in 96-well plates, and the number of cells in each well was 1 ⁇ 10 5 , and cultured overnight in a 37° C., 5% CO 2 constant temperature cell incubator. When the cell density is 60-70%, add 2% FBS DMEM containing different concentrations of taurolidine (initial concentration 4mg/ml, with 10-fold gradient to dilute the drug to be tested, a total of 10 concentrations) in 2% FBS DMEM. 100 ⁇ L/well of solution, and 4 replicate wells were measured for each concentration.
  • a blank cell control and a PBS control were also set up, and 4 repetitions were performed, and the culture was continued, and the cell status was observed under a microscope every day.
  • 10 ⁇ L of MTT solution (5 mg/ml) was added, incubated at 37° C. and 5% CO 2 for 1 hour, and the OD 570 value was determined.
  • the data were analyzed using Graphpad Prism 8.0 software, and the median inhibitory concentration (IC 50 ) of the drugs on the above three cells was calculated.
  • Embodiment 2 Taurolidine inhibits the detection result of influenza virus infectivity
  • the MDCK cells were stored in liquid nitrogen, taken out and recovered, and passed on for three consecutive generations. After the cells grew well, they were used for experimental research.
  • the preserved H3N2 influenza virus strain was slowly thawed on ice, inoculated into monolayer MDCK cells (less than 24 hours), and cultured for 72-96 hours, and the virus liquid was harvested according to the cytopathic state. And measure its content, take TCID 50 /100 ⁇ l as the calculation unit.
  • the drug was mixed with the H3N2 subtype influenza virus solution obtained above and then inserted into the cells.
  • the well-growing MDCK cells were digested with trypsin to calculate the cell content, and seeded in a 96-well plate, and the number of cells in each well was 1 ⁇ 10 5 . Drug effect studies were performed within 12 hours of seeding and when the cells were in a monolayer state.
  • the H3N2 subtype influenza virus with a virus content of 100 TCID 50 was mixed with taurolidine (14 ⁇ M), and then inoculated into a plated 96-well plate after exposure to room temperature for 10 minutes. A blank cell control and a virus control were also set up for 3 replicates. Cell plates inoculated placed in 37 °C, 5% CO 2 incubator after 72 hours and cultured in Cytopathic, cell proliferation rate is calculated.
  • H1N1-UI182, H1N1-PR8, H3N2 and H5N1 viruses from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • Virus culture put the preserved virus solution on ice and slowly thaw it, then inoculate it into a monolayer of MDCK (less than 24 hours), continue to culture for 72-96 hours, and harvest the virus solution according to the cytopathic state. And determine the virus content, with TCID 50 /100 ⁇ L as the calculation unit.
  • Cytotoxicity was determined by MTT assay (Promega). Passaging cells After culturing for 24 hours, the cells were seeded in a 96-well plate (4000 cells per well) and cultured. Cells were tested for sensitivity using the MTT assay according to the manufacturer's instructions. Briefly, 10 ul of MTT solution was added to each well and the OD570 value of each well was determined after 4 hours of incubation and recorded.
  • the drug and virus are mixed and inserted into the cells at the same time: the cells with good growth (A549 and MDCK) are digested with trypsin and the cell content is calculated, inoculated in a 96-well plate, 10 5 cells per well, inoculated within 12 hours and carried out in a monolayer state Research on the effect of drugs.
  • the H1N1-UI182, H1N1-PR8, H3N2 and H5N1 viruses with a virus content of 100 TCID 50 were mixed with taurolidine (20 ⁇ g/mL) and immediately inoculated into plated 6-well plates.
  • a blank cell control and a taurolidine cytotoxicity control were set up, and three replicates were performed; the inoculated cell plate was placed in a 37°C, CO 2 incubator, continued to culture, and cell lesions were observed.
  • MDCK cells were fixed (3.7% PFA) and then cell punched (2% Triton-100). They were blocked with 2% BSA prior to incubation with the primary antibody. It is then incubated with the secondary antibody before staining the nuclei. Finally, the cells were observed under a fluorescence microscope.
  • TRD showed potent anti-influenza virus activity and suppressed the titer of influenza virus H1N1-UI182 strain (Fig. 3A).
  • Fig. 3A After converting the concentration of TRD to logarithm and virus titer to percentage, semi-logarithmic fitting of the curve was performed to calculate the EC50 value. A 50% effective concentration (EC50 value) was observed at low doses, while high doses of TRD were able to reduce influenza virus transmission to 94% ( Figure 3B).
  • H1N1-UI182, H1N1-PR8, H3N2 and H5N1 strains were used as well as A549 and MDCK cells.
  • H1N1-UI182, H1N1-PR8, H3N2 and H5N1 strains were treated with DMSO and TRD (20 ⁇ g/mL) for 24 h, and viral titers were determined.
  • OSTA was used as a positive control drug.
  • the viral titers of the strains were expressed as Log 10 TCID 50 .
  • Taurolidine has broad-spectrum anti-influenza virus effects and can be used for the prevention and treatment of influenza virus.
  • Embodiment 4 Taurolidine inhibits the detection result of pseudorabies virus infectivity
  • Virus is an enveloped DNA virus with a virion diameter of 150nm-180nm.
  • the MDCK cells were stored in liquid nitrogen, taken out and recovered, and passed on for three consecutive generations. After the cells grew well, they were used for experimental research.
  • the preserved virus pseudorabies strain with envelope was placed on ice and slowly thawed, inoculated into a monolayer of MDCK cells (less than 24 hours), and continued to culture for 72-96 hours, and the virus liquid was harvested according to the cytopathic state. And measure its content, take TCID 50 /100 ⁇ l as the calculation unit.
  • the drug was mixed with the pseudorabies virus solution obtained above and then inserted into the cells.
  • the well-growing MDCK cells were digested with trypsin to calculate the cell content, and seeded into a 96-well plate, and the amount of cells in each well was 1 ⁇ 10 5 .
  • Drug effect studies were performed within 12 hours of seeding and when the cells were in a monolayer state.
  • Pseudorabies virus with a virus content of 100 TCID 50 was mixed with 15 ⁇ M taurolidine and 30 ⁇ M taurolidine respectively, and then inoculated into a plated 96-well plate after acting at room temperature for 10 minutes. A blank cell control and a virus control were also established, and three replicates were performed. Cell plates inoculated placed in 37 °C, 5% CO 2 incubator after 72 hours and cultured in Cytopathic, cell proliferation rate is calculated.
  • Embodiment 5 The detection result that taurolidine inhibits the infectivity of parvovirus
  • Parvovirus is a DNA virus without an envelope, with a virion diameter of 18-26 nm.
  • the F81 cells were stored in liquid nitrogen, taken out and recovered, and passed on for three consecutive generations. After the cells grew well, they were used for experimental research.
  • the preserved parvovirus strains were placed on ice and thawed slowly, inoculated into a monolayer of F81 cells (less than 24 hours), and continued to culture for 72-96 hours, and the virus liquid was harvested according to the cytopathic state. And measure its content, take TCID 50 /100 ⁇ l as the calculation unit.
  • the drug is mixed with the above-obtained parvovirus solution and then inserted into the cells.
  • the well-growing F81 cells were digested with trypsin to calculate the cell content, and seeded into a 96-well plate, and the amount of cells in each well was 1 ⁇ 10 5 .
  • Drug effect studies were performed within 12 hours of seeding and when the cells were in a monolayer state.
  • Parvovirus with a virus content of 100 TCID 50 was mixed with 15 ⁇ M taurolidine and 30 ⁇ M taurolidine, respectively, and then inoculated into a plated 96-well plate after exposure to room temperature for 10 minutes. A blank cell control and a virus control were also established, and three replicates were performed. Cell plates inoculated placed in 37 °C, 5% CO 2 incubator after 72 hours and cultured in Cytopathic, cell proliferation rate is calculated.
  • Embodiment 6 The inhibition experiment of taurolidine to canine coronavirus CCV
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • the cat kidney cell line F81 was recovered and passed on for three consecutive generations. After the cells grew well, they were used for experimental research;
  • Virus culture Put the preserved virus solution on ice and slowly thaw it, then inoculate it into the monolayer cat kidney cell line F81 (less than 24 hours), continue to culture for 72-96 hours, and harvest the virus solution according to the cytopathic state. And determine the virus content, with TCID 50 /100 ⁇ L as the calculation unit.
  • the drug and virus were mixed and inserted into the cells at the same time: the well-growing cat kidney cell line F81 was digested with trypsin to calculate the cell content, and inoculated in a 96-well plate, with 10 5 cells per well, and the drug was inoculated within 12 hours and in a monolayer state. effect research.
  • the HCoV-229E virus with a virus content of 200 TCID 50 was mixed with taurolidine (20 ⁇ g/mL taurolidine solution, and the drug doses were 50 ⁇ L, 25 ⁇ L, 12.5 ⁇ L, 7.5 ⁇ L, 3.75 ⁇ L) and inoculated immediately after mixing in plated 6-well plates.
  • a blank cell control and a taurolidine cytotoxicity control were set up, and three replicates were performed;
  • the cell state was observed in a 37 °C, CO 2 incubator for 72 hours, and observed under a microscope, the cells in the 50 ⁇ L and 25 ⁇ L drug inoculation dose groups were in good condition without cytopathic changes. 12.5 ⁇ L, 7.5 ⁇ L and 3.75 ⁇ L showed different degrees of cytopathic changes. There was no difference between the 3.75 ⁇ L and 200TCID 50 inoculation dose group.
  • L929 mouse fibroblasts
  • Virus strain mouse hepatitis virus, from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • the L929 cells were revived, passed on for three consecutive generations, and used for experimental research after the cells grew well;
  • Virus culture Put the preserved virus solution on ice and slowly thaw it, then inoculate it into a monolayer of L929 cells (less than 24 hours), continue to culture for 72-96 hours, and harvest the virus solution according to the cytopathic state. And determine the virus content, with TCID 50 /100 ⁇ L as the calculation unit.
  • the drug and virus are mixed and inserted into the cells at the same time: the well-growing L929 cells are digested with trypsin to calculate the cell content, inoculated in a 96-well plate, 10 5 cells per well, and the drug effect is studied within 12 hours in a monolayer state.
  • the mouse hepatitis virus with a virus content of 200 TCID 50 was mixed with taurolidine (20 ⁇ g/mL taurolidine solution, the drug doses were 50 ⁇ L, 25 ⁇ L, 12.5 ⁇ L, 7.5 ⁇ L, 3.75 ⁇ L) immediately after inoculation. in plated 96-well plates.
  • a blank cell control and a taurolidine cytotoxicity control were set up, and three replicates were performed;
  • the cell state was observed in a 37 °C, CO 2 incubator for 72 hours, and observed under a microscope, the cells in the 50 ⁇ L and 25 ⁇ L drug inoculation dose groups were in good condition without cytopathic changes. 12.5 ⁇ L, 7.5 ⁇ L and 3.75 ⁇ L showed different degrees of cytopathic changes. There was no difference between the 3.75 ⁇ L and 200TCID 50 inoculation dose group.
  • SARS-CoV-2 virus from the Virology Research Office of the Military Veterinary Research Institute
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • Vero-E6 and Huh7 cells were recovered and passed on for three consecutive generations. After the cells grew well, they were used for experimental research;
  • the stored virus solution was slowly thawed on ice and then inoculated into a monolayer of Vero-E6 cells (less than 24 hours), continued to culture for 72-96 hours, and the virus solution was harvested according to the cytopathic state. And determine the virus content, with TCID 50 /100 ⁇ L as the calculation unit.
  • the drug and virus are mixed and inserted into the cells at the same time: the well-growing Vero-E6 cells are digested with trypsin to calculate the cell content, inoculated in a 96-well plate, 10 5 cells per well, and the drug is inoculated within 12 hours in a monolayer state.
  • a blank cell control and a taurolidine cytotoxicity control were set up, and three replicates were performed; the inoculated cell plate was placed in a 37°C, CO 2 incubator, continued to culture, and cell lesions were observed.
  • the drug and virus were mixed and inserted into the cells at the same time: the well-growing Vero-E6 and Huh7 cells were digested with trypsin and the cell content was calculated, inoculated in a six-well plate, 10 6 cells per well, inoculated within 12 hours and in a monolayer state Conduct studies of drug effects.
  • the SARS-CoV-2 virus with a virus content of 100 TCID 50 was mixed with taurolidine, and then inoculated into a paved six-well plate with a 2% taurolidine solution and a drug dose of 50 ⁇ L and 25 ⁇ L.
  • the drug and virus are mixed and inserted into the cells at the same time: the well-growing Vero-E6 and Huh7 cells are digested with trypsin to calculate the cell content, and seeded in a 12-well plate, with 10 6 cells per well, within 12 hours of seeding and in a monolayer state Conduct studies of drug effects.
  • the SARS-CoV-2 virus with a virus content of 100 TCID 50 was mixed with taurolidine (20 ⁇ g/mL taurolidine solution, and the drug dose was 50 ⁇ L and 25 ⁇ L, respectively), and then inoculated on a 12-well plate. middle.
  • DMSO treatment Another established virus control wells (DMSO treatment), the cells were inoculated plates facing 37 °C, CO 2 incubator, and cultured after 24h, using ⁇ -actin as a control, whole cell extracts were analyzed by Western blot. Positive expression of viral NP protein was determined.
  • Vero E6 cells were infected with the original strain of SARS-CoV-2 isolated from Wuhan, China, and 6 h later, they were treated in the absence of TRD or at TRD low-dose (25 ⁇ g/mL) and high-dose (50 ⁇ g/mL) concentrations nourish.
  • SARS-CoV-2 promoted lysis of Vero-E6 cells compared to controls, however this phenomenon was inhibited by administration of TRD and only limited cytopathic effects could be detected at high doses (Fig. 8A, C).
  • NP nucleoprotein
  • Fig. 9A,C fluorescent labeling of nucleoprotein (NP) of SARS-CoV-2 confirmed that TRD caused a significant inhibition of NP accumulation.
  • DMSO-treated control Vero-E6 cells about 57% of cells were NP-positive at low doses, while there was a significant reduction of about 8.5% of NP-positive cells at high doses ( Figure 9B).
  • Hu7 cells about 60% of the cells were NP-positive at low doses, while the NP-positive cells were significantly reduced by about 12% at high doses (Fig. 9D). This indicated that TRD administration significantly inhibited the pathogenic effect of SARS-CoV-2 on cells, and this inhibition was positively correlated with drug dose.
  • Taurolidine has a significant inhibitory effect on SARS-CoV-2 at the cellular level.
  • the test results show that the compound taurolidine can significantly inhibit the cytopathic effects caused by SARS-CoV-2, and can be used for the development of preventive and therapeutic drugs for SARS-CoV-2.
  • H1N1-UI182, H3N2, H5N1 viruses from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • influenza virus H1N1-UI182, H3N2, H5N1
  • the influenza virus was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg).
  • the survival status and body weight changes of the mice were observed and recorded.
  • Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into the mice in the virus control group and the drug treatment group, and in different ways every morning and evening, the mice in the drug treatment group were continuously given drug treatment, 100uL/time (2mg).
  • the lung tissues of mice were collected on the third and fifth days after exposure, respectively, to observe the effect of taurolidine on the lung index of mice caused by influenza virus.
  • Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into the mice in the virus control group and the drug treatment group, and in different ways every morning and evening, the mice in the drug treatment group were continuously given drug treatment, 100uL/time (2mg). The lung tissues of mice were collected on the third and fifth days after drug treatment, respectively, and their viral loads were determined.
  • mice vaccinated with influenza virus significantly lost weight.
  • the weight loss trend of the mice in the drug-treated group was significantly slower, and the survival time of the mice was significantly prolonged.
  • the detection results show that the compound taurolidine can prolong the survival time of influenza virus-infected mice, and can be used for the prevention and treatment of influenza virus.
  • H1N1-UI182, H3N2, H5N1 viruses from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • influenza virus H1N1-UI182, H3N2, H5N1
  • H1N1-UI182, H3N2, H5N1 The influenza virus was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg).
  • Influenza viruses H1N1-UI182, H3N2, H5N1 were inoculated into the mice in the virus control group and the drug treatment group, and in different ways every morning and evening, the mice in the drug treatment group were continuously given drug treatment, 100uL/time (2mg).
  • the lung tissues of mice were collected on the third and fifth day after exposure, respectively, to observe the effect of taurolidine on the lung index of mice caused by influenza virus.
  • Influenza viruses H1N1-UI182, H3N2, H5N1 were inoculated into the mice in the virus control group and the drug treatment group, and in different ways every morning and evening, the mice in the drug treatment group were continuously given drug treatment, 100uL/time (2mg). On the fifth day after drug treatment, the lung tissues of mice were collected and fixed, and histopathological changes were detected.
  • the total lung index (Lung index) can be used as an important indicator to judge the severity of restrictive lung diseases. Therefore, we analyzed the effect of TRD administration on the total lung index in mice before and after administration, including the third and fifth days, respectively.
  • TRD administration significantly decreased H1N1-UI182 (Fig. 12A,B), H3N2 (Fig. 12C,D) and H5N1 (Fig. 12E,F) strains infected for 3 days and Total lung index after 5 days.
  • taurolidine Compared with the control, taurolidine has a significant protective effect on the lungs of mice after administration, and can improve the pathological damage caused by influenza virus to the lung tissue, which can be used for the prevention and treatment of influenza virus.
  • Example 11 The effect of administration of taurolidine on the prognosis of SARS-CoV-2 model mice
  • SARS-CoV-2 (C57MA14 strain), from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • the novel coronavirus (C57MA14) was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg). The survival status and body weight changes of the mice were observed and recorded.
  • the new coronavirus (C57MA14 strain) was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg).
  • the lung tissues of mice were collected on the third and fifth days after exposure, respectively, to observe the effect of taurolidine on the lung index of mice caused by SARS-CoV-2.
  • Influenza viruses H1N1-UI182, H3N2, H5N1 were inoculated into the mice in the virus control group and the drug treatment group, and in different ways every morning and evening, the mice in the drug treatment group were continuously given drug treatment, 100uL/time (2mg). On the fifth day after drug treatment, the lung tissues of mice were collected and fixed, and histopathological changes were detected.
  • mice began to decrease on the 2nd day after virus infection, and the mice died on the 3rd day, and the mortality rate reached 100% on the 7th day (Fig. 14).
  • the above results indicate that the SARS-CoV-2 strain can induce lethal infection in mice.
  • administration of TRD inhibited the reduction of viral mouse body weight in both BALB-C mice (FIG. 14A) and C57BL mice (FIG. 14C).
  • administration of TRD significantly protected mice from lethality during SARS-CoV-2 infection, with a protection rate of 92.31% in BALB-C mice (Fig. 14B) and 84.62% in C57BL mice (Fig. 14D) .
  • SARS-CoV-2 vaccinated mice significantly lost weight compared to controls.
  • the drug treatment protected the mice from weight loss and prolonged the mice's survival time.
  • the test results show that the compound taurolidine can reduce the number of virus RNA copies in the lungs of mice and improve the survival time of SARS-CoV-2 mice, which can be used for the prevention and treatment of new coronavirus.
  • SARS-CoV-2 (C57MA14 strain), from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • the new coronavirus (C57MA14 strain) was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg).
  • the new coronavirus (C57MA14 strain) was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg).
  • the blood of the mice was collected on the third and fifth day after exposure, and the total number of leukocytes was counted.
  • taurolidine Compared with the non-administered group, the administration of taurolidine can significantly increase the number of lymphocytes and monocytes in mice, and can significantly reduce the total number of neutrophils and platelets after virus infection, suggesting that taurolidine Has remission and improved overall prognostic level of novel coronavirus infection.
  • Example 13 Taurolidine improves SARS-CoV-2 damage to mouse lung tissue
  • SARS-CoV-2 (C57MA14 strain), from the Virology Research Office of the Military Veterinary Research Institute;
  • Instrument consumables pipette and matching tips, 1.5mL centrifuge tube, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.
  • the novel coronavirus (C57MA14) was inoculated into the mice in the virus control group and the drug treatment group, and the mice in the drug treatment group were continuously given drug treatment in the morning and evening in different ways, 100uL/time (2mg).
  • Influenza viruses H1N1-UI182, H3N2, H5N1 were inoculated into the mice in the virus control group and the drug treatment group, and in different ways every morning and evening, the mice in the drug treatment group were continuously given drug treatment, 100uL/time (2mg). On the fifth day after drug treatment, the lung tissues of mice were collected and fixed, and histopathological changes were detected.
  • TRD SARS-CoV-2-induced damage in host tissues, including lung, liver, spleen, and kidney
  • BALB-C and C57BL mice intranasally with the virus, 24 hours later.
  • TRD drug treatment was administered, and lungs, livers, spleens and kidneys were harvested on the seventh day after dosing.
  • the results of HE staining showed that the virus infection caused a large area of congestion in the lungs, liver, spleen and kidney, resulting in severe pathological symptoms.
  • this phenomenon was significantly reversed ( Figure 19).
  • pathological analysis showed that viral infection caused internal hemorrhage of the tissue (indicated by black arrows).
  • Taurolidine can significantly improve the pathological damage of lung, liver, spleen and kidney tissue in mice infected with 2019-nCoV, and can reduce the viral load in the above tissues, suggesting that the drug has the effect of preventing and treating 2019-nCoV infection.
  • taurolidine has a very effective inhibitory effect on influenza virus and new coronavirus or similar RNA viruses, and can prolong the survival time of influenza virus and new coronavirus-infected mice and improve the effect of the virus on the lungs of mice.
  • the pathogenic effect of taurolidine indicates that taurolidine can be used for the prevention of this virus and the development of related therapeutic drugs.

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