JP2023533041A - Use of Taurolidine against viruses - Google Patents

Abstract

Uses of Taurolidine against viruses are provided in this application. Specifically, Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof, or Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof in the preparation of an antiviral agent. Uses of compositions comprising acceptable salts are provided in this application. Research results show that Taurolidine can significantly inhibit influenza viruses and coronaviruses at the cellular level. Furthermore, in vivo experiments have confirmed that Taurolidine has a significant protective effect on the lung and can prolong the survival of mice infected with influenza virus or SARS-CoV-2 virus. It has been shown that Taurolidine can be used for the prevention and treatment of lung disease caused by influenza viruses or coronaviruses. The research results of the present application expand the effective range of taurolidine, provide a research base, and open up new directions for research and development of preventive or therapeutic agents for pulmonary infections caused by viruses.

Description

This application consists of Chinese Application No. 202010638926.9 with filing date July 6, 2020, Chinese Application No. 202010639186.0 with filing date July 6, 2020, and filing date September 2020 The priority thereof is claimed based on Chinese Application No. 202010994928.1 dated 21st, and the disclosure content of those Chinese Applications is incorporated into the present application in its entirety.

The present invention relates to the field of drug therapy, in particular to the use of Taurolidine against viruses, especially in the treatment and/or prevention of infections caused by novel coronaviruses and influenza viruses.

Coronaviruses are a large class of viruses that are widespread in nature. In biological classification, coronaviruses belong to the order Nidoviridae, family Coronaviridae, genus Coronavirus, and are RNA viruses with an enveloped and linear, single-stranded positive-stranded genome, with a diameter of 80-120 nm. , the nucleic acid is a non-segmented single-stranded (+) RNA with a length of 27-31 kb. Coronaviruses are the viruses with the longest RNA nucleic acid strands among RNA viruses and contain an important structural feature unique to positive-strand RNA, namely a methylated "cap" at the 5' end of the RNA strand. with a poly A "tail" structure at the 3' end. This structure is very similar to eukaryotic mRNA and is also an important structural basis for the genomic RNA itself to function as a translation template.

Coronaviruses are excreted with respiratory secretions, spread through oral fluids, sneezes, contact, and air droplets, infecting vertebrates such as humans, mice, horses, pigs, cats, dogs, birds, and humans. and cause diseases such as respiratory infections and acute gastroenteritis.

Influenza viruses are the major viruses that cause acute respiratory infections leading to influenza. Influenza viruses belong to the Orthomyxoviridae family of RNA viruses, which mainly include influenza A, B, and C viruses. Influenza A viruses are highly variable, infectious, and pathogenic, and very easily cause seasonal epidemics. Human infection with influenza virus can cause severe pneumonia, acute respiratory distress syndrome, sepsis with shock, etc., and the mortality rate is very high, posing a great threat to social public health safety. Currently, commonly used anti-influenza virus drugs mainly include alkylamine drugs and neuraminidase inhibitors. However, alkylamine drugs are effective only against influenza A virus, and it has been found that influenza viruses can rapidly acquire resistance to these antiviral drugs due to genetic mutation, drug response, and the like. Neuraminidase inhibitors can inhibit viral replication by preventing the release of progeny virus. However, side effects of these drugs are a constant problem in clinical applications, including hallucinations, abnormal behavior, hearing impairment, and visual impairment. Therefore, it is of great importance to search and develop a class of drugs with universal applicability against multiple subtypes of influenza virus.

A novel coronavirus (severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is currently the seventh known coronavirus to infect humans. The virus has a long incubation period, is highly contagious, has a high replication rate, and is difficult to prevent and control. When humans are infected with the new coronavirus, clinical symptoms include fever, malaise, dry cough, and progressive dyspnea, and in severe cases, acute respiratory distress syndrome, septic shock, metabolic acidosis, and coagulation dysfunction, which are difficult to cure and thus can have a serious impact on the national economy and human health. Currently, there are no effective drugs for the treatment of SARS-CoV-2, and the occurrence of complications can only be actively prevented based on symptomatic treatment. Therefore, there is an urgent need to develop effective drugs that can prevent or treat the novel coronavirus, and research and development of such drugs is also attracting interest from researchers around the world.

Taurolidine (chemical _name : 4,4'-methylenebis[tetrahydro-2H-1,2,4 _{- thiadiazine} ]1,1,1',1'-tetraoxide, molecular formula: _{C7H16N4O4S2 )} is a derivative of the amino acid taurine, whose structure is shown below.

Taurolidine has anti-endotoxin, antibacterial, and anti-adhesion properties. With respect to bacteria, Taurolidine can chemically react with cell walls, endotoxins and exotoxins, inhibit microbial adhesion, and play an antibacterial role. Furthermore, with respect to anti-tumor, Taurolidine can induce cytotoxicity in tumor cells by inducing apoptosis, autophagy and necrosis. The extent to which these processes are involved can vary by tumor cell type. By July 2020, there were over 260 foreign publications on Taurolidine research, most of which focused on investigating the role of Taurolidine in tumor-associated signaling pathways, but no reports on its antiviral activity. rice field.

Currently, drug research on Taurolidine in the prior art focuses on its antitumor and antibacterial aspects. The study results of the present invention show that Taurolidine inhibits influenza viruses (eg, H1N1, H3N2, or H5N1) and coronaviruses (eg, novel coronavirus SARS-CoV-2, canine coronavirus CCV, or murine hepatitis virus MHV). levels can be significantly inhibited. In vivo experiments have confirmed that Taurolidine has a significant protective effect on the lung and can prolong the survival of mice infected with influenza virus or coronavirus. The above results demonstrate that Taurolidine can be used to prevent and treat lung disease caused by influenza virus or coronavirus. Accordingly, one object of the present application is to extend the efficacy spectrum of Taurolidine and provide its antiviral use.

In one aspect, the present application provides Taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof, or Taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof in the manufacture of an antiviral medicament. Use of a composition comprising a pharmaceutically acceptable salt is provided.

In another aspect, the present application relates to Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof in the manufacture of a medicament for inhibiting viral replication and/or reproduction in cells, Alternatively, use of a composition comprising taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof is provided.

In another aspect, the present application relates to Taurolidine or its derivatives, prodrugs, solvates, or pharmaceutically acceptable compounds for the manufacture of a medicament for preventing and/or treating diseases or infections caused by viruses. There is provided use of a salt or composition comprising Taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof.

In another aspect, the present application provides a method of inhibiting viral infection, comprising administering to a virally infected cell, a virally susceptible cell, or a subject in need thereof, an effective amount of Taurolidine or a derivative thereof, a A method comprising administering a drug, solvate, or pharmaceutically acceptable salt, or a composition comprising Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof is provided. do.

In some embodiments, the method further comprises administering to the cell or subject an effective amount of an antibiotic, such as erythromycin, acetylspiramycin, or azithromycin. In some embodiments, the antibiotic is not vancomycin. In some embodiments, the antibiotic is administered simultaneously, separately, or sequentially with Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof, or composition thereof.

In another aspect, the present application provides a method of inhibiting viral replication and/or propagation in a cell, comprising treating the cell with an effective amount of Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof. or a composition comprising taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprises contacting the cells with an effective amount of an antibiotic, such as erythromycin, acetylspiramycin, or azithromycin. In some embodiments, the antibiotic is not vancomycin. In some embodiments, the antibiotic is administered to the cells simultaneously, separately, or sequentially with Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof, or composition thereof. be.

In another aspect, the present application provides a method for 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 its derivatives, prodrugs, or a pharmaceutically acceptable salt thereof, or a composition comprising taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof.

In some embodiments, the method further comprises administering to the subject an effective amount of an antibiotic, such as erythromycin, acetylspiramycin, or azithromycin. In some embodiments, the antibiotic is not vancomycin. In some embodiments, the antibiotic is administered to the subject concurrently, separately, or sequentially with Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof, or composition thereof. be.

In some embodiments, the virus of any one of the items above is an influenza virus, a coronavirus, a hepatitis virus, or an RNA virus such as HIV. In some embodiments, the influenza virus is an influenza A virus, influenza B virus, or influenza C virus, eg, H1N1, H2N2, H3N2, H5N1, H7N7, or H9N2. In some embodiments, the coronavirus is novel coronavirus SARS-CoV-2, canine coronavirus CCV, or murine hepatitis virus MHV. In some embodiments, the coronavirus is CCV. In some embodiments, the coronavirus is SARS-CoV-2. In some embodiments, the coronavirus is MHV.

In some embodiments, the cell according to any one of the items above is a mammalian cell. In some embodiments, the mammal is selected from the group consisting of bovine, equine, ovine, porcine, canine, feline, rodent, and primate. In some embodiments, the mammal is human, cat, chicken, pig, or dog. In some embodiments, the cells are human-derived cells or chicken embryo cells.

In some embodiments, the disease or infection according to any one of the above items is pulmonary disease. In some embodiments, the disease or infection according to any one of the above items is influenza, simple infections, pneumonia including severe pneumonia, acute or severe acute respiratory infections, hypoxic selected from respiratory failure, acute respiratory distress syndrome, sepsis, septic shock or severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and novel coronavirus pneumonia (COVID-19). In some embodiments, the simple infection is fever, cough, or sore throat. In some embodiments, the disease or infection is COVID-19.

In some embodiments, the composition according to any one of the items above further comprises an antibiotic such as erythromycin, acetylspiramycin, and azithromycin. In some embodiments, the antibiotic is not vancomycin. In some embodiments, the composition also contains pharmaceutically acceptable agents such as diluents, absorbents, wetting agents, excipients, fillers, binders, disintegrants, surfactants, and any combination thereof. can include a carrier that is In some embodiments, the composition comprises PVP-KF-17. In some embodiments, the composition is an injection, infusion, tablet, capsule, spray, aerosol, or rinse. In some embodiments, the composition is provided in unit dosage form. In some embodiments, the composition comprises 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 Including derivatives, prodrugs, solvates or pharmaceutically acceptable salts.

In some embodiments, a composition may have a formulation of 2 g Taurolidine and 5 g PVP-KF-17 contained per 100 ml of solution. The method of preparation is to first dissolve PVP-KF-17 in water at room temperature, and then add taurolidine at 45°C to 50°C, stir until completely dissolved, and then add appropriate amount of 4% water. Add sodium oxide to adjust the pH of the solution, then add appropriate amount of activated carbon, maintain the above temperature for 30 minutes, and filter while warm. The filtrate is filtered through a 0.25 mm filtration membrane, bottled and capped.

DEFINITIONS OF TERMS In this application, unless otherwise specified, scientific and technical terms used herein have the meanings that are commonly understood by those of ordinary skill in the art. Moreover, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory manipulation steps used herein are all routine steps widely used in the corresponding fields. Meanwhile, definitions and explanations of related terms are provided below for a better understanding of this disclosure.

As used herein, "TRD" refers to Taurolidine.

As used herein, the term "pharmaceutically acceptable salt" includes inorganic or organic acid salts of Taurolidine, as well as, for example, sodium, potassium, calcium, lithium, meglumine, Hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate, phosphate, hydrogen phosphate, acetate, propionate, butyrate, oxalate, trimethylacetate , adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, mesylate, Inorganic or organic base salts such as ethanesulfonate, benzenesulfonate, p-toluenesulfonate, or pamoate are included.

As used herein, "derivative" refers to a sulfonamide compound derived from Taurolidine with a hydrogen atom or other atomic group substituted that has greater than 10% of Taurolidine's biological activity. Analogous compounds are formed, for example, by structural alterations by simply replacing two thioxyl groups in the taurolidine molecule or by adding or removing groups -NH- and -CH ₂ -.

As used herein, a "prodrug" is a modification of the chemical structure of Taurolidine that does not per se exhibit the pharmacological effects of the original drug (i.e., Taurolidine) but is converted to the original drug in vivo to play a role. Refers to derivatives with new physical, chemical and biological properties that are obtained.

Taurolidine may exist in the form of solvates (preferably hydrates) that contain polar solvents, in particular water, methanol or ethanol, as structural constituents of the taurolidine crystals. The amount of polar solvent, especially water, can be present in stoichiometric or non-stoichiometric ratios. Any solvate of Taurolidine used in the treatment of the diseases or infections described herein may provide different properties (including pharmacokinetic properties), but when absorbed by a subject, Taurolidine is obtained. , therefore, the use of Taurolidine should be understood to encompass the use of any solvate of Taurolidine, respectively.

As used herein, the term "therapeutically effective amount" or "prophylactically effective amount" means, within the scope of sound medical judgment, to treat or treat disease in a patient (with a reasonable benefit/risk ratio). It refers to an amount sufficient to prevent but low enough to avoid serious side effects. A therapeutically effective amount of a compound depends upon the particular compound selected (e.g., considering the potency, efficacy, and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, , the age, size, weight, and physical disease of the patient being treated, the medical history of the patient being treated, the duration of treatment, the nature of the concomitant therapy, the desired therapeutic effect, but still routinely by those skilled in the art. can be determined to

In addition, specific dosages and regimens for various patients may vary depending on age, weight, sex, natural health status, patient nutritional status, drug potency, time of administration, metabolic rate, disease severity, and physician subjectivity. It depends on many factors such as personal judgment. A dose of 0.001-1000 mg/kg body weight/day is preferably used herein.

Beneficial Effects of the Invention The present application provides the use of Taurolidine in antiviral agents. Cells such as Vero-E6, MDCK, A549, and Huh7 were used as cell models to evaluate the antiviral effects of taurolidine, virus was inoculated into the cells, and virus fluids were harvested according to the pathological state of the cells. , measured the inhibitory effect of Taurolidine on viruses. The results show that Taurolidine significantly inhibits influenza viruses (e.g., H1N1, H3N2, or H5N1) and coronaviruses (e.g., novel coronavirus SARS-CoV-2, canine coronavirus CCV, or murine hepatitis virus HMV) at the cellular level. showed that it can be done. Furthermore, in vivo experiments have confirmed that Taurolidine has a significant protective effect on the lung and can prolong the survival of mice infected with influenza virus or SARS-CoV-2 virus. This indicated that Taurolidine could be used for prevention and treatment of lung disease caused by influenza virus or coronavirus. The research results of the present application expand the effective range of Taurolidine, provide a research base, and open up new directions for the research and development of prophylactic or therapeutic agents for pulmonary infections caused by viruses.

The accompanying drawings described herein are used to provide a further understanding of the invention and form a part of this application. The exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute undue limitations to the invention. The drawings are as follows.

FIG. 1 shows the test results of sensitivity of MDCK, Vero-E6, F81 cells to taurolidine. FIG. 2 shows the inhibitory effect of 14 μM Taurolidine on influenza virus subtype H3N2. Figure 3 shows the inhibitory effect of TRD on influenza virus subtype H1N1. FIG. 3A: A549 cells were infected with influenza virus strain HIN1-UI182 (MOI=0.1), treated with DMSO and TRD for 12 hours each, and dose-response curves were plotted. FIG. 3B shows EC50 values calculated by semi-logarithmic fitted curves after transforming TRD concentrations to logarithms and virus titers to percentages. FIG. 3C shows the sensitivity of A549 cells measured by MTT after 24 h treatment with solvent DMSO and different TRD concentrations. FIG. 3D: After infection with H1N1-UI182 at the indicated MOIs, cells were treated with vehicle 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 μm). FIG. 3E shows quantification of the number of NP-positive nuclei. FIG. 3F shows total protein expression levels of NP in A549 cells. β-actin was used as a control and whole cell extracts were analyzed by Western blot. FIG. 3G shows quantification of NP expression levels. Data represent mean±SD, n=3 independent experiments. ^* p<0.05, ^** p<0.01, and ^*** p<0.001 compared to control. Figure 4 shows the inhibitory effect of TRD on different influenza virus subtypes. Figures 4A-4D: A549 cells were infected with influenza virus strains H1N1-UI182, H1N1-PR8, H3N2, and H5N1 for 12 hours, after which cells were treated with DMSO and TRD for 24 hours and virus titers were measured. Figures 4E-4H: After infection of MDCK cells with influenza virus strains H1N1-UI182, H1N1-PR8, H3N2, and H5N1, cells were treated with DMSO and TRD for 24 hours and virus titers were measured. OSTA was used as a positive control drug. Viral titers of strains were expressed as Log _{10 TCID50} . Data represent mean±SD and statistical significance was assessed by ANOVA analysis ( ^* p<0.05, ^** p<0.01, and ^*** p<0.001). FIG. 5 shows the inhibitory effect of 15 μM and 30 μM Taurolidine on pseudorabies virus. FIG. 6 shows the inhibitory effect of 15 μM and 30 μM Taurolidine on parvovirus. FIG. 7 shows that TRD inhibited replication of SARS-CoV-2 in cells. FIG. 7A shows detection of viral pathogenicity. FIG. 7B shows that after infection with the novel coronavirus SARS-CoV-2 (MOI=0.1), Vero-E6 cells were treated with DMSO and TRD for 24 hours, respectively, and dose-response curves were plotted. FIG. 7C shows the sensitivity of Vero-E6 cells measured by MTT after treatment with vehicle DMSO and different concentrations of TRD for 24 hours. FIG. 7D shows Vero-E6 cells were treated with DMSO and TRD for 24 hours after infection with novel coronavirus SARS-CoV-2 (100 TCID ₅₀ ) and (200 TCID ₅₀ ), and virus titers were measured. . FIG. 7E shows total protein expression levels of NP in Vero-E6 cells. β-actin was used as a control and whole cell extracts were analyzed by Western blot. FIG. 7F shows quantification of NP expression levels. Data represent mean±SD, n=3 independent experiments. ^* p<0.05, ^** p<0.01, and ^*** p<0.001 compared to control. Figure 8 shows the anti-SARS-CoV-2 activity of TRD. Figures 8A and 8C show that Vero-E6 and Huh7 cells were imaged by light microscopy to detect typical SARS-CoV-2-induced cell lysis. Figures 8B and 8D show quantification of virus yield in cell supernatants by RT-qPCR. Data represent mean±SD, n=3 independent experiments. ^* p<0.05, **p<0.01, ^*** p<0.001 compared to controls. Figure 9 shows the anti-SARS-CoV-2 activity of TRD. Figures 9A and 9C show Vero-E6 (left) and Huh7 (right) cells treated with solvent DMSO and different doses of TRD for 24 h after infection with SARS-CoV-2 at specific MOIs and stained with DAPI. , indicating that infected cells were detected by viral nuclear NP staining (scale bar 10 μm). Figures 9B and 9D show quantification of the number of NP-positive nuclei. Data represent mean±SD, n=3 independent experiments. ^* p<0.05, ^** p<0.01, ^*** p<0.001 compared to control. FIG. 10 shows that progression of influenza virus infection was limited after TRD treatment in vivo. FIG. 10A shows mice in blank control group , virus control group
, and the TRD treatment group
2 and plotted of cumulative body weight change against days post H1N1-UI182 infection. FIG. 10B is a survival curve showing changes in survival of mice infected with H1N1-UI182. Figures 10C and 10D show virus titers in mouse lung tissue measured 3 and 5 days after H1N1-UI182 infection. FIG. 10E shows total protein expression levels of NP in day 5 mouse lung tissue. β-actin was used as a control and whole cell extracts were analyzed by Western blot. FIG. 10F shows 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).
Figure 11 shows that administration of TRD restricted the course of infection in mice with different influenza virus subtypes. FIG. 11A shows mice in blank control group , virus control group
, and the TRD treatment group
and plotted cumulative body weight change against days post H3N2 infection. FIG. 11B is a survival curve showing changes in mouse survival after H3N2 infection. Figures 11C and 11D show virus titers in mouse lung tissue measured 3 and 5 days after H3N2 infection. FIG. 11E shows the cumulative change in body weight plotted against days post H5N1 infection. FIG. 11F is a survival curve showing changes in mouse survival after H5N1 infection. Figures 11G and 11H show virus titers in mouse lung tissue measured 3 and 5 days after H5N1 infection. Data represent mean±SD and statistical significance was assessed by ANOVA analysis ( ^* p<0.05, ^** p<0.01, and ^*** p<0.001).
Figure 12 shows the protective effect of TRD on the lung. Figures 12A and 12B show the protective effect of drugs on lung tissue of BALB-C mice detected on days 3 and 5 after H1N1-UI182 virus infection. Figures 12C and 12D show the protective effect of drugs on lung tissue of BALB-C mice detected on days 3 and 5 after H3N2 virus infection. Figures 12E and 12F show the protective effect of the drug on lung tissue of BALB-C mice detected 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). Figure 13 shows that TRD ameliorated the extent of lung injury. HE staining was performed on lung tissue of mice in control, virus and TRD-treated groups, respectively, and representative pictures of different groups were obtained: left (HIN1-UI182), middle (H3N2), right (H5N1). Figure 14 shows that TRD improved body weight and survival in SARS-CoV-2 infected mice. FIG. 14A shows BALB-C female mice in the control group. , virus control group
and TRD treatment group
, and plotted of cumulative body weight change against days post SARS-CoV-2 infection. FIG. 14B shows survival curves of virus-infected BALB-C mice. FIG. 14C shows C57BL mice in the control group.
, virus control group
and TRD treatment group
, and the cumulative body weight change was plotted against the number of days after virus infection. FIG. 14D is a survival curve showing the survival rate of C57BL mice after viral infection. Data represent mean±SD, n=3 independent experiments. ^* p<0.05, ^** p<0.01, ^*** p<0.001 compared to controls.
Figure 15 shows that TRD improved the prognosis of SARS-CoV-2 infected mice. FIG. 15A shows viral RNA yield in lung tissue of different groups of BALB-C mice measured at 3 and 5 days after SARS-CoV-2 infection. FIG. 15B shows the protective effect of the drug on lung tissue of BALB-C mice detected on days 3 and 5 after virus infection. FIG. 15C shows virus titers in lung tissue of C57BL mice measured on days 3 and 5 after virus infection. FIG. 15D shows the protective effect of drug on lung tissue of C57BL mice detected on days 3 and 5 after virus infection. Data represent mean±SD, n=3 independent experiments. ^* p<0.05, ^** p<0.01, ^*** p<0.001 compared to controls. Figure 16 shows the effect of TRD on leukocytes after SARS-CoV-2 infection. Figures 16A and 16B show the percentage of immune cells in the serum of BALB-C mice measured on days 3 and 5 after virus infection. Figures 16C and 16D show the percentage of immune cells in the sera of C57BL mice determined on days 3 and 5 after 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 17 shows the effect of TRD on host platelets after SARS-CoV-2 infection. Figures 17A and 17B show platelet counts in the serum of BALB-C mice measured on days 3 and 5 after virus infection. Figures 17C and 17D show platelet counts in the serum of C57BL mice on days 3 and 5 after 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. 18 shows that TRD ameliorated SARS-CoV-2 malignancy in lung tissue lesions in mice. FIG. 18A shows the pathological analysis process. FIG. 18B shows that the lung tissue of different groups of mice was sampled and photographed. Black arrows (→) indicate severe tissue lesions. FIG. 19 shows that TRD ameliorated SARS-CoV-2 malignancy in lung, liver, spleen, and kidney tissue lesions in mice. HE staining was performed on lung, liver, spleen, and kidney tissues of control, virus, and TRD-treated mice, respectively, and representative pictures of different groups are shown. Black arrows (→) indicate severe tissue hemorrhage. Figure 20 shows that TRD inhibited the histopathological expression of ACE2 after viral infection. After SARS-CoV-2 virus infection, immunohistochemical detection was performed on the lung tissue of mice (BALB-C and C57BL) in the control, virus and TRD-treated groups, respectively, and representative photographs of the different groups are shown. (left). 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).

Specific Embodiments The technical solutions in the embodiments of the present invention are clearly and completely described in the following embodiments of the present invention with reference to the accompanying drawings. Apparently, the described embodiments are only some but not all of the examples of the present invention. The following description of at least one exemplary embodiment is merely exemplary in nature and is in no way to be construed as limiting the invention, its application or uses. All other examples obtained by persons skilled in the art without creative efforts based on the embodiments of the present invention shall fall within the protection scope of the present invention.

The BALB/c and C57BL/6N mice and Taurolidine used in the Examples are all commercially available and Taurolidine was obtained from Changchun Mailing Bioengineering Co., Ltd. , Ltd. Vero-E6, MDCK, A549, Huh7 cells, and influenza virus were provided by the Institute of Military Veterinary Medicine, Academy of Military Science, and SARS-CoV-2 virus was provided by the Academy of Military Science, Academy of Military Medicine. It is an isolate from Beijing that was preserved at the Veterinary Institute.

Example 1 Effect of Taurolidine on Cell Safety MDCK, Vero-E6, and F81 cells were stored in liquid nitrogen, removed, resuscitated and serially passaged for 3 generations after the cells had grown sufficiently. Used for experimental research. MDCK, Vero-E6, and F81 cells were seeded in 96-well plates at 1×10 ⁵ cells per well and cultured overnight at 37° C. in a 5% CO ₂ thermostatic cell incubator. When the cell density was 60-70%, 2% FBS DMEM with different concentrations of Taurolidine (initial concentration was 4 mg/ml and the test drug was diluted in a 10-fold gradient for a total of 10 concentrations). Maintenance solution was added at 100 μL/well and four replicate wells were measured for each concentration. In addition, a blank control and a PBS control were provided, repeated 4 times, the culture was continued, and the state of the cells was observed under a microscope every day. Two days after administration, 10 μL 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 measured. Data were analyzed using Graphpad Prism 8.0 software to calculate the half-inhibitory concentration ( _IC50 ) of drug against the three cell types.

The test results are shown in FIG. The _IC50 value of Taurolidine on MDCK cells (A) was 22.88 μM, on Vero-E6 cells (B) was 20.38 _μM , and on F81 cells (C) was _36.74 μM. I was able to find out what happened.

Example 2: Detection Results of Influenza Virus Infectivity Inhibited by Taurolidine MDCK cells were stored in liquid nitrogen, removed and resuscitated, serially passaged for three generations, and after the cells had grown sufficiently, experimental studies were performed. used for Stored H3N2 influenza virus strains were slowly thawed on ice, inoculated onto MDCK cell monolayers (up to 24 hours), continued culture for 72-96 hours, and aliquoted with viral fluids depending on the pathological state of the cells. recovered. Virus content was also measured using TCID ₅₀ /100 μl as the unit of calculation.

The drug was mixed with the H3N2 subtype influenza virus solution obtained above and then inoculated into the cells. Well-grown MDCK cells were digested with trypsin to calculate cell volume and seeded at 1×10 ⁵ cells per well in 96-well plates. Drug effect studies were performed within 12 hours of inoculation when the cells were in a monolayer. H3N2 subtype influenza virus with a viral content of 100 TCID ₅₀ was inoculated, mixed with Taurolidine (14 μM), allowed to react for 10 minutes at room temperature, and then plated onto coated 96-well plates. In addition, blank cell controls and virus controls were set up and repeated three times. The seeded cell plate was placed in a 37° C. 5% CO ₂ incubator and continued to be cultured for 72 hours, then the cell lesions were observed and the cell proliferation rate was calculated.

The calculation formula was relative growth rate (P%) of cells: P% = OD experimental group/OD control group x 100%.
The observation results are shown in FIG. A dose of 14 μM Taurolidine was found to have a significant inhibitory effect on influenza virus at the cellular level. The detection results showed that Taurolidine could obviously inhibit the cytopathic effect caused by influenza, and the results showed that Taurolidine could inhibit influenza virus infection.

Example 3 Inhibition of Influenza Virus Reproduction by Taurolidine in Cells 1 . Experimental Materials 1.1 Cells: human lung cancer cells A549 and African green monkey kidney cells MDCK from the Virology Laboratory of the Military Veterinary Institute;
1.2 Virus strains: H1N1-UI182, H1N1-PR8, H3N2, and H5N1 viruses from the Virology Laboratory of the Military Veterinary Institute;
1.3 Reagents: DMEM, 0.25% trypsin, FBS, PBS (pH=7.0);
1.4 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. Experimental methods 2.1 Cell culture: A549 and MDCK were resuscitated and serially passaged for 3 generations, and used for experimental studies after the cells had grown sufficiently.

2.2 Viral Culture: Stored viral fluids are placed on ice to slowly thaw, then inoculated onto MDCK monolayers (up to 24 hours), culture continued for 72-96 hours, and viral fluids subdivided into cell pathology. were collected according to their condition. Virus content was also determined using TCID ₅₀ /100 μL as the unit of calculation.

2.3 Taurolidine toxicity test Cytotoxicity was determined by the MTT assay (Promega). After culturing the subcultured cells for 24 hours, the cells were seeded in 96-well plates (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 incubated for 4 hours before the OD ₅₇₀ value of each well was measured and recorded.

2.4 Inhibitory Effect of Taurolidine Drug and virus were mixed and inoculated into cells simultaneously. Well-grown cells (A549 and MDCK) were digested with trypsin to calculate cell volume, seeded ¹⁰⁵ cells per well in 96-well plates, and within 12 hours of inoculation when the cells were in a monolayer. A drug effect study was performed. 100 TCID ₅₀ of H1N1-UI182, H1N1-PR8, H3N2, and H5N1 were mixed with Taurolidine (20 μg/mL) and immediately seeded onto coated 6-well plates. Additionally, a blank cell control and a taurolidine cytotoxicity control were set up and repeated three times. The seeded cell plates were placed in a CO ₂ incubator at 37° C., culture was continued, and changes in cytopathicity were observed.

2.5 Immunofluorescent detection MDCK cells were fixed (3.7% PFA) and then subjected to cell perforation (2% Triton-100). They were blocked with 2% BSA prior to primary antibody incubation. Nuclei were subsequently stained after incubation with a secondary antibody. Finally, cells were observed under a fluorescence microscope.

3. Experimental Results Correlation between drug dose and infection outcome in MDCK cells after influenza virus infection was observed.

After infection with influenza virus strain HIN1-UI182 (MOI=0.1), MDCK cells were treated with DMSO and TRD for 12 h each and dose-response curves were plotted. TRD exhibited potent anti-influenza virus activity and suppressed the titer of influenza virus H1N1-UI182 strain (Fig. 3A). After transforming TRD concentrations to logarithms and virus titers to percentages, semi-logarithmic fitted curves were used to calculate _EC50 values. A 50% effective concentration (EC50 value) was observed at the low dose, while the high dose of TRD was able to reduce influenza virus infection by 94% (Fig. 3B).

Next, MTT experiments were performed to confirm the effect of TRD on cytotoxicity. A549 cells were treated with DMSO and different concentrations of TRD for 24 hours and then the sensitivity of A549 cells was determined. An inverse correlation was observed between cell proliferation and TRD dose (Fig. 3C). Therefore, a low toxicity concentration (50 μg/mL) was chosen for follow-up studies. To further investigate the potential antiviral effects of TRD, oseltamivir (OSTA), a drug commonly used to treat viral infections, was selected as a positive control. To investigate whether TRD antiviral activity is an early stage of influenza virus infection, a one-cycle infection assay was performed. After infection with H1N1-UI182 at the indicated MOIs, cells were treated with DMSO, TRD, and OSTA for 12 hours each. Nuclei were stained with DAPI and infected cells were detected with nuclear vNP staining (scale bar 10 μm). The nuclear appearance of viral nucleoprotein (NP) was quantified in successfully infected cells (Fig. 3D). As expected, OSTA had a significant inhibitory effect on influenza virus, but in DMSO-treated control cells, 38% of MDCK nuclei were NP-positive, and the percentage of NP-positive cells after TRD treatment was 21%. % significantly decreased (Fig. 3E). Whole cell extracts were subsequently analyzed by Western blot using β-actin as a control, which also showed the same results (FIGS. 3F and 3G).

To further investigate the antiviral efficacy of TRD, different influenza virus subtypes including H1N1-UI182, H1N1-PR8, H3N2 and H5N1 strains were used to infect different cells including A549 and MDCK cells. After A549 cells were infected with influenza virus strains H1N1-UI182, H1N1-PR8, H3N2, and H5N1 for 12 hours, cells were treated with DMSO and TRD (20 μg/mL) for 24 hours and virus titers were measured. OSTA was used as a positive control drug. Viral titers of strains were expressed as Log _{10 TCID50} . Data represent mean±SD and statistical significance was assessed by ANOVA analysis ( _* p<0.05, _** p<0.01, and _*** p<0.001). The results showed that administration of TRD significantly reduced virus titers in A549 cells (FIGS. 4A-4D). Similarly, after infection with influenza virus strains H1N1-UI182, H1N1-PR8, H3N2, and H5N1, MDCK cells were treated with DMSO and TRD for 24 hours and virus titers were determined. OSTA was used as a positive control drug. Viral titers of strains were expressed as Log _{10 TCID50} . Data represent mean±SD and statistical significance was assessed by ANOVA analysis ( ^* p<0.05, ^** p<0.01, and ^*** p<0.001). Results showed that administration of TRD also significantly reduced viral titers in MDCK cells (FIGS. 4E-H).

4. Conclusion Taurolidine has broad-spectrum anti-influenza virus effects and can be used for prevention and treatment of influenza virus.

Example 4 Results of Detection of Pseudorabies Virus Infectivity Inhibited by Taurolidine Virus: Pseudorabies virus is an enveloped DNA virus with a viral particle size of 150-180 nm.

MDCK cells were stored in liquid nitrogen, removed, resuscitated, serially passaged for 3 generations, and used for experimental studies after the cells had grown sufficiently. Stored enveloped pseudorabies virus strains were slowly thawed on ice, inoculated onto monolayers of MDCK cells (up to 24 hours), continued to culture for 72-96 hours, and the viral fluid was analyzed for cell pathology. were collected according to their condition. The content was also determined using TCID ₅₀ /100 μl as the unit of calculation.

The drug was mixed with the pseudorabies virus solution obtained above and then inoculated into the cells. Well-grown MDCK cells were digested with trypsin, cell volume was calculated, and 1×10 ⁵ cells were seeded per well in 96-well plates. Drug effect studies were performed within 12 hours of inoculation when the cells were in a monolayer. 100 TCID ₅₀ pseudorabies virus was mixed with 15 μM Taurolidine and 30 μM Taurolidine, respectively, reacted at room temperature for 10 minutes, and then seeded onto coated 96-well plates. In addition, blank cell controls and virus controls were set up and repeated three times. The seeded cell plate was placed in a 37° C. 5% CO ₂ incubator and continued to be cultured for 72 hours to observe cell lesions and calculate the cell growth rate.

The calculation formula was relative growth rate (P%) of cells: P% = OD experimental group/OD control group x 100%.

The observation results are shown in FIG. It could be found that concentrations of 15 μM and 30 μM Taurolidine had no inhibitory effect on pseudorabies virus at the cellular level. The test results showed that 15 μM and 30 μM Taurolidine could not inhibit the cytopathic effect caused by pseudorabies virus, which indicated that Taurolidine could not inhibit the infection of pseudorabies virus. .

Example 5 Results of Detection of Parvovirus Infectivity Inhibited by Taurolidine Viruses: Parvoviruses are non-enveloped DNA viruses with viral particle diameters of 18-26 nm.

F81 cells were stored in liquid nitrogen, removed, resuscitated, serially passaged for three generations, and used for experimental studies after the cells had grown sufficiently. Slowly thaw stored parvovirus stocks on ice, inoculate monolayers of F81 cells (up to 24 hours), continue culturing for 72-96 hours, and harvest virus fluids depending on cell pathology. bottom. The content was also determined using TCID ₅₀ /100 μl as the unit of calculation.

Drugs were mixed with the parvovirus solution obtained above and then inoculated into cells. Fully grown F81 cells were digested with trypsin, cell volume was calculated, and 1×10 ⁵ cells were seeded per well in 96-well plates. Drug effect studies were performed within 12 hours of inoculation when the cells were in a monolayer. 100 TCID ₅₀ parvovirus was mixed with 15 μM Taurolidine and 30 μM Taurolidine, respectively, reacted at room temperature for 10 minutes, and then seeded onto coated 96-well plates. In addition, blank cell controls and virus controls were set up and repeated three times. The seeded cell plates were placed in a 37° C. 5% CO ₂ incubator and continued to be cultured for 72 hours to observe cell lesions and calculate the cell proliferation rate.

The calculation formula was relative growth rate (P%) of cells: P% = OD experimental group/OD control group x 100%.

The observation results are shown in FIG. It could be found that concentrations of 15 μM and 30 μM Taurolidine had no inhibitory effect on parvovirus at the cellular level. The test results showed that 15 μM and 30 μM Taurolidine could not inhibit the cytopathic effect caused by parvovirus, indicating that Taurolidine could not inhibit parvovirus infection.

Example 6: Taurolidine inhibition experiment against canine coronavirus CCV1. Experiment material:
1.1 Cells: Feline kidney cell line F81 from the Virology Laboratory of the Military Veterinary Institute;
1.2 Strain: Canine coronavirus from the Virology Laboratory of the Military Veterinary Institute;
1.3 Reagents: DMEM, 0.25% trypsin, FBS, PBS (pH=7.0);
1.4 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. experimental method:
2.1 Cell culture: The feline kidney cell line F81 was resuscitated and serially passaged for 3 generations, and well-grown cells were used for experimental studies.

2.2 Virus culture: The stored virus solution is slowly thawed on ice, inoculated into a monolayer feline kidney cell line F81 (24 hours or less), continued culture for 72 to 96 hours, and the pathological state of the cells Virus fluids were collected according to Virus content was also determined using TCID ₅₀ /100 μL as the unit of calculation.

2.3 Inhibitory Effect of Taurolidine Drugs and virus were mixed and inoculated into cells simultaneously. The well-grown feline kidney cell line F81 was digested with trypsin to calculate the cell amount, seeded in 96-well plates at 10 ⁵ cells per well, and drug effect studies were performed on the inoculum in which the cells were in a monolayer. Went within 12 hours. 200 TCID ₅₀ HCoV-229E virus was mixed with Taurolidine (20 μg/mL Taurolidine solution, drug doses were 50 μL, 25 μL, 12.5 μL, 7.5 μL, 3.75 μL) and immediately seeded onto coated 6-well plates. bottom. A blank cell control and a taurolidine cytotoxicity control were set up and repeated three times. The seeded cell plates were placed in a CO ₂ incubator at 37° C., culture was continued, and changes in cytopathicity were observed.

3. Experimental Results According to the experimental method described above, the state of cells was observed under a microscope in a CO ₂ incubator at 37° C. for 72 hours. The cells in the 50 μL and 25 μL drug-inoculated groups were in good condition and no cytopathic changes occurred, while the cells in the 12.5 μL, 7.5 μL, and 3.75 μL groups all showed different degree of cell Degeneration was shown, and there was no difference in cytopathicity between the group treated with 3.75 μL Taurolidine and the group inoculated with 200 _TCID50 .

4. Conclusion From the above experimental results, the inoculation doses of 50 μL and 25 μL can completely inhibit 200 TCID ₅₀ , and the inoculation doses of 12.5 μL and 7.5 μL can completely inhibit the infection of 200 TCID ₅₀ virus into cells. It was determined that an inoculum dose of 3.75 μL was unable to inhibit infection of cells with 200 TCID ₅₀ virus.

Example 7: Taurolidine inhibition experiment against mouse hepatitis virus HMV1. Experiment material:
1.1 Cells: L929 (mouse fibroblasts) from the Virology Laboratory of the Military Veterinary Institute;
1.2 Strain: Mouse Hepatitis Virus from the Virology Laboratory of the Military Veterinary Institute;
1.3 Reagents: DMEM, 0.25% trypsin, FBS, PBS (pH=7.0);
1.4 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. experimental method:
2.1 Cell culture: L929 cells were resuscitated and serially passaged for 3 generations, and well-grown cells were used for experimental studies.

2.2 Virus culture: Slowly thaw the stored virus solution on ice, inoculate the monolayer of L929 cells (24 hours or less), continue culturing for 72 to 96 hours, and depending on the pathological state of the cells Virus fluid was collected. Virus content was also determined using TCID ₅₀ /100 μL as the unit of calculation.

2.3 Inhibitory Effect of Taurolidine Drug and virus were mixed and inoculated into cells simultaneously. Well-grown L929 cells were digested with trypsin to calculate cell volume, seeded ¹⁰ cells per well in 96-well plates, and drug effect studies were performed within 12 hours of seeding when the cells were in a monolayer. gone. 200 TCID ₅₀ mouse hepatitis virus was mixed with Taurolidine (20 μg/mL Taurolidine solution, drug doses were 50 μL, 25 μL, 12.5 μL, 7.5 μL, 3.75 μL) and then immediately seeded onto coated 96-well plates. bottom. A blank cell control and a taurolidine cytotoxicity control were set up and repeated three times. The seeded cell plates were placed in a CO ₂ incubator at 37° C., culture was continued, and changes in cytopathicity were observed.

3. Experimental Results According to the experimental method described above, the state of cells was observed under a microscope in a CO ₂ incubator at 37° C. for 72 hours. The cells in the groups with 50 μL and 25 μL inoculum volumes were in good condition and no cytopathic changes occurred. 12.5 μL, 7.5 μL, and 3.75 μL cells had different degrees of cytopathicity, the difference in cytopathicity between the group treated with 3.75 μL Taurolidine and the group inoculated with 200 TCID ₅₀ was I didn't.

4. Conclusion From the above experimental results, the inoculation doses of 50 μL and 25 μL can completely inhibit 200 TCID ₅₀ , and the inoculation doses of 12.5 μL and 7.5 μL can completely inhibit the infection of 200 TCID ₅₀ virus into cells. It was determined that an inoculum volume of 3.75 μL was unable to inhibit infection of cells with 200 TCID ₅₀ virus.

Example 8: Inhibition of Coronavirus SARS-CoV-2 by Taurolidine1. Experiment material:
1.1 Cells: Vero-E6 and Huh7 cells from the Virology Laboratory of the Military Veterinary Institute;
1.2 Strain: SARS-CoV-2 virus from the Virology Laboratory of the Military Veterinary Institute.
1.3 Reagents: DMEM medium, 0.25% trypsin, FBS, PBS (pH=7.0);
1.4 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. experimental method:
2.1 Cell culture: Vero-E6 and Huh7 cells were resuscitated and serially passaged for 3 generations and well grown cells were used for experimental studies.

2.2 Virus culture: Slowly thaw the stored virus solution on ice, inoculate it into a monolayer of Vero-E6 cells (24 hours or less), continue culturing for 72 to 96 hours, and allow the cells to reach a pathological state. Virus fluids were collected accordingly. Virus content was also determined using TCID ₅₀ /100 μL as the unit of calculation.

2.3 Inhibitory Effect of Taurolidine Drug and virus were mixed and inoculated into cells simultaneously. Well-grown Vero-E6 cells were digested with trypsin to calculate cell volume, seeded 10 ⁵ cells per well in 96-well plates, and drug effect studies were performed 12 hours after seeding when the cells were in a monolayer state. went within 200 TCID ₅₀ SARS-CoV-2 virus was mixed with Taurolidine (20 μg/mL Taurolidine solution, drug doses were 50 μL, 25 μL, 12.5 μL, 7.5 μL, 3.75 μL) and then coated 96 wells. was inoculated into Additionally, a blank cell control and a taurolidine cytotoxicity control were set up and repeated three times. The seeded cell plates were placed in a CO ₂ incubator at 37° C., culture was continued, and changes in cytopathicity were observed.

2.4 Inhibition of SARS-CoV-2 Viral Load by Taurolidine in Vero-E6 and Huh7 Cells Drugs and virus were mixed and inoculated into cells simultaneously. Well-grown Vero-E6 and Huh7 cells were digested with trypsin to calculate cell volume, seeded ¹⁰ cells per well in 6-well plates, and drug effect studies were performed by inoculating cells in a monolayer state. Went within hours. 100 TCID ₅₀ of SARS-CoV-2 virus was mixed with Taurolidine and then seeded in coated 6-well plates, 2% Taurolidine solution and drug dose had doses of 50 μL and 25 μL. In addition, blank cell controls and virus controls were also set up and repeated 6 times. The seeded cell plate was placed in a CO2 incubator at 37° C., and culture was continued for 24 hours, after which viral RNA in all cells (including supernatant) was collected and the amount of virus was measured.

2.5 Replication Inhibition of SARS-CoV-2 by Taurolidine in Vero-E6 and Huh7 Cells Drugs and viruses were mixed and inoculated into cells simultaneously. Well-grown Vero-E6 and Huh7 cells were digested with trypsin to calculate the cell amount, seeded 10 ⁶ cells per well in 12-well plates, and drug effect studies were performed on the inoculum in which the cells were in a monolayer. Went within 12 hours. 100 TCID ₅₀ of SARS-CoV-2 virus was mixed with Taurolidine (20 μg/mL Taurolidine solution, drug doses were 50 μL and 25 μL, respectively) and then seeded onto coated 12-well plates. In addition, virus control wells (DMSO-treated) were also set up and the seeded cell plates were placed in a _CO2 incubator at 37°C and culture continued for 24 hours, then β-actin was used as a control and whole cell extracts were analyzed by Western blotting. analyzed. Positive expression of viral NP protein was determined.

3. Experimental Results To further investigate the antiviral effect of TRD against SARS-CoV-2, virus inoculation experiments with Vero-E6 cells were performed. Viral amplification results showed that SARS-CoV-2 was superior at both low (MOI=0.05) and high (MOI=0.1) multiplicity of infection after infecting cells for 48 hours. virulence, and the two MOI values were shown to result in very similar virus production (Fig. 7A). Therefore, an MOI of 0.05 was used in the following experiments.

Note that there was a negative correlation between SARS-CoV-2 virulence and TRD dose, i.e., viral virulence to cells gradually diminished as dose of drug increased. Deserved. After infection with the novel coronavirus SARS-CoV-2 (MOI=0.1), Vero-E6 cells were treated with DMSO and TRD for 24 h, respectively, and dose-response curves were plotted (FIG. 7B). Considering the effects of drugs on cell proliferation, MTT experiments were performed. Sensitivity of Vero-E6 cells was measured by MTT after treatment with DMSO and different TRD concentrations for 24 hours. The results showed that even with a drug dose of 100 μg/mL, there was no significant change in cell proliferation (Fig. 7C). Vero-E6 cells were infected with novel coronavirus SARS-CoV-2 (100 TCID ₅₀ ) and (200 TCID ₅₀ ), respectively, then the cells were treated with DMSO and TRD for 24 hours and virus titers were measured. Indeed, TRD showed excellent antiviral effects regardless of 100 _TCID50 or ₂₀₀ TCID50 viruses (Fig. 7D).

A less toxic dose (50 μg/mL) was then selected for a follow-up study. Importantly, TRD administration was able to significantly inhibit viral nucleoprotein (NP) expression in cells, and this inhibition was positively correlated with drug dose (Figs. 7E and 7F). Because there were many copies of NP for each RNA molecule in the virion, and consistent immunoreactive bands were detectable at different doses, the presence of viral proteins at the start of dosing may explain the dose-dependent observed during treatment. may reflect masking of significant inhibitory effects.

Next, the potential anti-SARS-CoV-2 effect of TRD was evaluated. VeroE6 cells were infected with the original strain of SARS-CoV-2 isolated from Wuhan, China, and 6 hours later they were treated either in the absence of TRD or at low (25 μg/mL) and high (50 μg) doses. /mL) of TRD. SARS-CoV-2 promoted the lysis of Vero-E6 cells compared to the control group, but this phenomenon was suppressed by administration of TRD, and only limited cytopathic effects could be detected at high doses (Fig. 8A and 8C). A significant inhibitory effect of TRD on viral particle production was demonstrated by quantification of viral RNA copy number in Vero-E6 cell culture supernatants, i.e., nearly doubled at low dose compared to untreated infected cells. decreased, approximately 15,000-fold at high doses (Figs. 8B and 8D).

Moreover, fluorescent labeling of the nuclear protein (NP) of SARS-CoV-2 confirmed that TRD caused significant inhibition of NP accumulation (FIGS. 9A and 9C). In the DMSO control Vero-E6 cells, approximately 57% of the cells were NP-positive at the low dose, whereas the high dose significantly reduced NP-positive cells to approximately 8.5% (Fig. 9B). On the other hand, in Hu7 cells, approximately 60% of the cells were NP-positive at the low dose, but the number of NP-positive cells was significantly reduced to approximately 12% at the high dose (Fig. 9D). This indicated that TRD administration significantly inhibited the pathogenic effects of SARS-CoV-2 on cells, and this inhibitory effect was positively correlated with drug dose.

4. Conclusion Taurolidine had a significant inhibitory effect against SARS-CoV-2 at the cellular level. The test results showed that Taurolidine significantly inhibited the cytopathic changes caused by SARS-CoV-2, and could be used in the research and development of drugs for prevention and treatment of SARS-CoV-2.

Given that TRD showed significant antiviral effects in cell culture models, we shifted our focus to animal models to further evaluate the therapeutic properties of this drug.

Example 9: Resistance to Lethal Influenza Virus Infection by Taurolidine Administration 1 . Experimental materials 1.1 Experimental animals: Beijing Vital River Laboratory Animal Technology Co., Ltd. , Ltd. BALB/c mice from
1.2 Strains: H1N1-UI182, H3N2, H5N1 viruses from the Virology Laboratory of the Military Veterinary Institute;
1.3 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. Experimental method 2.1 Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control group and drug-treated group mice, and drugs (100 uL/time ( 2 mg)) were administered continuously. The living conditions and weight changes of the mice were observed and recorded.

2.2 Inhibitory Effect of Taurolidine Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control group and drug-treated mice, and drugs (100 uL/ (2 mg)) were administered continuously. Mouse lung tissue was collected on days 3 and 5 after inoculation, respectively, to observe the effect of Taurolidine on mouse lung index caused by influenza virus.

2.3 Inhibition of pulmonary viral load by taurolidine Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control group and drug-treated mice, and drug-treated mice were infected every morning and evening by different routes, respectively. Drugs (100 uL/dose (2 mg)) were administered continuously. In addition, mouse lung tissue was collected on days 3 and 5 after drug treatment, and the viral load was measured.

3. Experimental Results For additional polarizing treatment doses between different species, a factor-by-factor dose method based on the ectopic measurement method and the species conversion factor was used. Improved survival was found to be related to the amount of body weight lost during treatment of UI182 infection (FIGS. 10A and 10B). Furthermore, similar results were found with H3N2 (FIGS. 11A and 11B) and H5N1 (FIGS. 11E and 11F).

To further study the protective effect of TRD treatment on the host after influenza virus infection, virus titers were examined in lung tissue 3 and 5 days after infection with strain UI82. Results showed that virus titers were significantly reduced after TRD treatment (Figures 10C and 10D). This effect persisted after infection with the H3N2 strain (Figures 11C and 11D) or the H5N1 strain (Figures 11G and 11H). Western blot results also confirmed that TRD administration significantly inhibited viral replication in mouse lung tissue (FIGS. 10E and 10F). These results showed that TRD treatment significantly increased survival and significantly decreased mortality during influenza virus infection.

4. CONCLUSIONS Mice inoculated with influenza virus lost significantly more weight than controls. Compared with the virus control group, the mice in the drug-treated group showed a significantly smaller tendency to lose weight, and the survival period of the mice was significantly prolonged. The test results showed that Taurolidine prolongs survival of influenza virus-infected mice and can be used for prevention and treatment of influenza virus.

Example 10: Effect of Taurolidine on Influenza Virus Pathogenesis in Mice. Experimental materials 1.1 Experimental animals: Beijing Vital River Laboratory Animal Technology Co., Ltd. , Ltd. BALB/c mice from
1.2 Strains: H1N1-UI182, H3N2, H5N1 viruses from the Virology Laboratory of the Military Veterinary Institute;
1.3 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. Experimental method 2.1 Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control group and drug-treated group mice, and drugs (100 uL/time ( 2 mg)) were administered continuously.

2.2 Inhibitory Effect of Taurolidine Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control group and drug-treated mice, and drugs (100 uL/ (2 mg)) were administered continuously. Mouse lung tissue was collected on days 3 and 5 after inoculation, respectively, to observe the effect of Taurolidine on mouse lung index caused by influenza virus. The calculation formula was lung index=(mouse lung tissue weight/mouse weight)×100%.

2.3 Taurolidine Ameliorates Influenza Virus-Induced Lung Injury in Mice Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control and drug-treated mice, and drug-treated mice were vaccinated every morning and 10 days after treatment, respectively. Drugs (100 uL/dose (2 mg)) were administered continuously each evening by a different route. Five days after drug treatment, mouse lung tissue was fixed to detect histopathological changes.

3. Experimental Results The lung index level could be used as an important index to determine the severity of restrictive lung disease. Therefore, we analyzed the effects on the pulmonary index of mice before and after TRD administration, including days 3 and 5, respectively. Influenza virus infection results in increased lung index, while days 3 and 5 after infection with H1N1-UI182 (FIGS. 12A and 12B), H3N2 (FIGS. 12C and 12D), and H5N1 (FIGS. 12E and 12F) strains. , found that drug treatment significantly decreased lung index. These results indicate that administration of TRD has a positive protective effect on mouse lung tissue.

Moreover, drug treatment was able to significantly ameliorate influenza virus damage to the lungs of mice (Fig. 13). These observations clearly suggest that the antiviral effects observed in vitro did indeed occur in vivo, and that at least the antimicrobial TRD has potential antiviral effects in the treatment of indoor viral airborne infections. are doing.

4. CONCLUSION Compared with the control, the administration of Taurolidine has a significant protective effect on the lungs of mice and can ameliorate the pathological damage of lung tissue caused by influenza virus, preventing and preventing influenza virus. could be used for treatment.

Example 11: Effect of Taurolidine administration on the prognosis of SARS-CoV-2 model mice. Experiment material:
1.1 Experimental animals: Beijing Vital River Laboratory Animal Technology Co., Ltd. , Ltd. BALB/c and C57BL/6N mice from
1.2 Strain: SARS-CoV-2 (C57MA14 strain) from the Virology Laboratory of the Military Veterinary Institute;
1.3 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. experimental method:
2.1 The novel coronavirus (C57MA14) was inoculated into mice in the virus control group and the drug treatment group, and the drugs (100 uL/time (2 mg)) were continuously administered to the mice in the drug treatment group by different routes every morning and evening, respectively. dosed. The living conditions and weight changes of the mice were observed and recorded.

2.2 Inhibitory effect of Taurolidine Novel coronavirus (C57MA14 strain) was inoculated into virus control group and drug-treated group mice, and drugs (100 uL/time (2 mg) ) were administered continuously. In addition, the lung tissue of mice was collected on days 3 and 5 after inoculation, respectively, to observe the effect of Taurolidine on the lung index of mice caused by SARS-CoV-2. The calculation formula was lung index=(mouse lung tissue weight/mouse weight)×100%.

2.3 Taurolidine Ameliorates Influenza Virus-Induced Lung Injury in Mice Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control and drug-treated mice, and drug-treated mice were vaccinated every morning and 10 days after treatment, respectively. Drugs (100 uL/dose (2 mg)) were administered continuously each evening by a different route. Five days after drug treatment, mouse lung tissue was fixed to detect histopathological changes.

3. Experimental Results Our research team succeeded in establishing SARS-CoV-2 animal infection models including rhesus monkeys, cynomolgus monkeys, mice, golden hamsters, dogs, cats, pangolins, etc. in early stages. Previous results indicated that TRD has a good inhibitory effect against SARS-CoV-2 in vitro. To further evaluate the antiviral efficacy of TRD in vivo, a mouse infection model (including BALB-C female mice and C57BL male mice) was performed. Model animals were dosed by injection using a factor-by-factor dosing method based on the ectopic measurement method and species conversion factors. Experimental results showed that mice began to lose weight on day 2 after virus infection, mice died on day 3, and mortality reached 100% on day 7 (FIG. 14). The above results indicated that the SARS-CoV-2 strain was able to induce lethal infection in mice. Importantly, administration of TRD inhibited weight loss of virus-infected mice in both BALB-C mice (Fig. 14A) and C57BL mice (Fig. 14C). Moreover, 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).

To investigate the relationship between antiviral efficacy of TRD and time, infected mice were sampled and analyzed on days 3 and 5 after administration. A significant inhibitory effect of drug treatment on viral particle production was confirmed by quantification of viral RNA copy number in mouse lung tissue (FIGS. 15A and 15C). In particular, administration of TRD showed a superior protective effect on lung tissue after SARS-CoV-2 infection (Figures 15B and 15D). Viral infection usually results in the host's own immune defense, which may well explain the lower production of viral RNA in lung tissue 6 days after viral infection than 4 days after viral infection. Nevertheless, administration of TRD had a significant suppressive effect on virus production in the lung.

4. Conclusion Compared with the control group, mice inoculated with SARS-CoV-2 had significantly decreased body weight. Drug treatment could protect mice from weight loss and prolong their survival. Test results show that Taurolidine can reduce the number of viral RNA copies in the lungs of mice and improve the survival time of SARS-CoV-2 mice, thus it can be used for the prevention and treatment of novel coronavirus. It has been shown.

Example 12: Taurolidine improves the prognosis of SARS-CoV-2 infection in mice. Experiment material:
1.1 Experimental animals: Beijing Vital River Laboratory Animal Technology Co., Ltd. , Ltd. BALB/c and C57BL/6N mice from
1.2 Strain: SARS-CoV-2 (C57MA14 strain) from the Virology Laboratory of the Military Veterinary Institute;
1.3 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. experimental method:
2.1 The novel coronavirus (C57MA14 strain) was inoculated into mice in the virus control group and the drug treatment group, and the drug (100 uL/time (2 mg)) was continuously administered to the mice in the drug treatment group every morning and evening by different routes. was administered to

2.2 Inhibitory effect of Taurolidine Novel coronavirus (C57MA14 strain) was inoculated into virus control group and drug-treated group mice, and drugs (100 uL/time (2 mg) ) were administered continuously. Mice were bled 3 and 5 days after inoculation and total white blood cells were counted.

3. Experimental Results Next, the effect of TRD on clinical outcome after SARS-CoV-2 infection was further evaluated. Consistent with the expected results, 3 days (FIG. 16A) and 5 days (FIG. 16B) after drug treatment of BALB-C mice, the percentage of lymphocytes in the serum increased and the percentage of neutrophils increased. was found to decrease. This phenomenon also occurred in C57BL mice (Figures 16C and 16D).

Furthermore, the results also showed that SARS-CoV-2 infection resulted in a rapid increase in platelet counts in the host serum. However, after 3 and 5 days of drug treatment in BALB-C mice, the number of platelets in serum was significantly reduced (Figures 17A and 17B). Importantly, the same results were obtained in C57BL mice (Figures 17C and 17D). These findings indicated that important biological phenotypes arise from the pathogenic novel coronavirus after TRD treatment.

4. CONCLUSION: Compared with the untreated group, taurolidine treatment can significantly increase the numbers of lymphocytes and monocytes in mice, and significantly decrease the total number of neutrophils and platelets after viral infection. This suggested that Taurolidine could reduce and improve the overall prognosis of COVID-19 infection.

Example 13: Taurolidine ameliorates lung tissue damage in mice caused by SARS-CoV-2. Experiment material:
1.1 Experimental animals: Beijing Vital River Laboratory Animal Technology Co., Ltd. , Ltd. BALB/c and C57BL/6N mice from
1.2 Strain: SARS-CoV-2 (C57MA14 strain) from the Virology Laboratory of the Military Veterinary Institute;
1.3 Instruments and consumables: pipettes and support tips, 1.5 mL centrifuge tubes, ice box, ice maker, biological safety cabinet, carbon dioxide incubator.

2. experimental method:
2.1 The novel coronavirus (C57MA14) was inoculated into mice in the virus control group and the drug treatment group, and the drugs (100 uL/time (2 mg)) were continuously administered to the mice in the drug treatment group by different routes every morning and evening, respectively. dosed.

2.2 Improvement of Taurolidine against Lung Injury in Mice Caused by Novel Coronavirus Influenza viruses (H1N1-UI182, H3N2, H5N1) were inoculated into virus control group and drug-treated mice, and drug-treated mice were inoculated each morning. and drugs (100 uL/dose (2 mg)) were continuously administered by a different route every evening. Five days after drug treatment, mouse lung tissue was fixed to detect histopathological changes.

3. Experimental Results Previous results indicated that administration of TRD can protect lung tissue from SARS-CoV-2 and improve clinical outcome of viral infection. To further determine the role of TRD in lung injury induced by SARS-CoV-2, BALB-C and C57BL mice were inoculated with virus by intranasal injection and administered TRD 24 h and 3 days post-dose. Lung tissue was collected at day 1 and day 5 (Fig. 18A). The results showed that the viral infection caused large areas of congestion within the lung tissue, resulting in severe pathological symptoms. Importantly, this phenomenon was reversed by administration of TRD (Fig. 18B).

To further determine the role of TRD in ameliorating or treating host tissue (including lung, liver, spleen, and kidney) damage induced by SARS-CoV-2, BALB-C was administered by intranasal injection of viral TRD. and C57BL mice. TRD was administered 24 hours later, and lungs, livers, spleens, and kidneys of mice were harvested 7 days after administration. HE staining results showed that lungs, liver, spleen, and kidneys were massively congested after viral infection, resulting in severe pathological symptoms. Importantly, this phenomenon was significantly reversed after administration of TRD (Figure 19). In addition, pathological analysis showed that viral infection caused tissue internal bleeding (indicated by black arrows).

To further investigate changes in histopathological expression before and after drug treatment, we detected changes in expression of SARS-CoV-2 nuclear protein NP in lung, liver, spleen, and kidney of BALB-C and C57BL mice. After SARS-CoV-2 infection, the percentage of positive cells expressing NP was significantly increased in lung, liver, spleen, and kidney of mice. Notably, the above phenomena were suppressed by TRD treatment (Fig. 20). This indicated that administration of TRD effectively inhibited further replication of the virus in tissues, thereby ameliorating the pathological damage of host tissues caused by SARS-CoV-2.

4. CONCLUSION: Taurolidine can significantly ameliorate histopathological damage in the lung, liver, spleen, and kidney of novel coronavirus-infected mice, and reduce the viral load in these tissues, thereby making this drug It was suggested that has the effect of preventing and treating new coronavirus infection.

Based on the above results, Taurolidine has a highly effective inhibitory effect against influenza virus and novel coronavirus or similar RNA viruses, and prolongs survival of mice infected with influenza virus and novel coronavirus. , could ameliorate the pathogenic effects of the virus on the lungs of mice, suggesting that Taurolidine could be used in the research and development of preventive and related therapeutics against this class of viruses.

Various modifications of the invention, in addition to those described herein, will become apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in this application, including all patents, patent applications, journal articles, books, and any other publications, is hereby incorporated by reference in its entirety.

Claims (14)

Taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof, or Taurolidine or a derivative, prodrug, solvate or pharmaceutically acceptable salt thereof in the manufacture of an antiviral drug Use of compositions comprising. Taurolidine or its derivatives, prodrugs, solvates or pharmaceutically acceptable salts or Taurolidine or its derivatives, prodrugs, solvents in the manufacture of a medicament for inhibiting viral replication and/or propagation in cells Use of a composition comprising a solute or a pharmaceutically acceptable salt,
Preferably said cells are mammalian cells, preferably said mammals are selected from the group consisting of bovine, equine, ovine, porcine, canine, feline, rodent and primate, preferably said mammal is a human, cat, chicken, pig, or dog, preferably said cells are human cells or chicken embryo cells;
use. Taurolidine or its derivatives, prodrugs, solvates or pharmaceutically acceptable salts or Taurolidine or its derivatives, pros in the manufacture of medicaments for the prevention and/or treatment of diseases or infections caused by viruses. Use of compositions containing drugs, solvates, or pharmaceutically acceptable salts. the virus is an influenza virus, a coronavirus, a pneumonia virus, or an RNA virus such as HIV;
Preferably, said influenza virus is an influenza A virus, an influenza B virus, or an influenza C virus, such as H1N1, H2N2, H3N2, H5N1, H7N7, or H9N2,
Preferably, said coronavirus is SARS-CoV-2, CCV or MHV, preferably said coronavirus is SARS-CoV-2, preferably said coronavirus is CCV, preferably is the coronavirus is MHV,
Use according to any one of claims 1-3. The disease or infection is influenza, simple infection, pneumonia including severe pneumonia, acute or severe acute respiratory tract infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis syndrome, septic shock, and novel coronavirus pneumonia (COVID-19), preferably the simple infection is fever, cough, or sore throat;
Preferably, said disease or infection is COVID-19.
Use according to claim 3. the composition comprises a pharmaceutically acceptable carrier such as diluents, absorbents, wetting agents, excipients, fillers, binders, disintegrants, surfactants, and any combination thereof;
Preferably, said composition comprises PVP-KF-17,
Use according to any one of claims 1-5. The composition is an injection, an infusion, a tablet, a capsule, a spray, an aerosol, or a rinse,
Preferably said composition is present in unit dosage form, preferably said composition is in the range of 1-1000 mg (eg 1-800 mg, 1-500 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-50 mg, 1-1000 mg, 1-500 mg, 1-50 mg, 1-50 mg, ~20 mg, or 1-10 mg) of taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof;
Use according to any one of claims 1-6. A method of inhibiting viral infection comprising administering to a cell infected with said virus, a cell susceptible to infection with said virus, or a subject in need thereof, an effective amount of Taurolidine or a derivative, prodrug, solvate, or thereof. administering a pharmaceutically acceptable salt, or a composition comprising Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof;
Method. A method of inhibiting viral replication and/or propagation in a cell comprising treating said cell with an effective amount of Taurolidine or a derivative thereof, prodrug, solvate or pharmaceutically acceptable salt, or Taurolidine or a derivative thereof; contacting with a composition comprising a prodrug, solvate, or pharmaceutically acceptable salt;
Preferably said cells are mammalian cells, preferably said mammals are selected from the group consisting of bovine, equine, ovine, porcine, canine, feline, rodent and primate, preferably said mammal is a human, cat, chicken, pig, or dog, preferably said cells are human cells or chicken embryo cells;
Method. A method for 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, solvate, or pharmaceutically acceptable thereof. or a composition comprising Taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof;
Method. the virus is an influenza virus, a coronavirus, a pneumonia virus, or an RNA virus such as HIV;
Preferably, said influenza virus is an influenza A virus, an influenza B virus, or an influenza C virus, such as H1N1, H2N2, H3N2, H5N1, H7N7, or H9N2,
Preferably, said coronavirus is SARS-CoV-2, CCV or MHV, preferably said coronavirus is SARS-CoV-2, preferably said coronavirus is CCV, preferably is the coronavirus is MHV,
The method according to any one of claims 8-10. The disease or infection is influenza, simple infection, pneumonia including severe pneumonia, acute or severe acute respiratory infection, hypoxic respiratory failure, acute respiratory distress syndrome, sepsis syndrome, septic shock, or novel coronavirus is selected from pneumonia (COVID-19), preferably said simple infection is fever, cough or sore throat;
Preferably, said disease or infection is COVID-19.
11. The method of claim 10. the composition comprises a pharmaceutically acceptable carrier such as diluents, absorbents, wetting agents, excipients, fillers, binders, disintegrants, surfactants, and any combination thereof;
Preferably, said composition comprises PVP-KF-17,
The method according to any one of claims 8-12. The composition is an injection, infusion, tablet, capsule, spray, aerosol, or rinse,
Preferably said composition is present in unit dosage form, preferably said composition is in the range of 1-1000 mg (eg 1-800 mg, 1-500 mg, 1-200 mg, 1-100 mg, 1-50 mg, 1-50 mg, 1-1000 mg, 1-500 mg, 1-50 mg, 1-50 mg, ~20 mg, or 1-10 mg) of taurolidine or a derivative, prodrug, solvate, or pharmaceutically acceptable salt thereof;
The method according to any one of claims 8-13.