阅读说明：本技术 一种化合物在治疗和预防新型冠状病毒和流感病毒药物方面的应用 (Application of compound in medicine for treating and preventing novel coronavirus and influenza virus ) 是由 高玉伟 苏忠 王铁成 周彤 李元果 吕朝相 孙伟洋 王雪峰 周博 向海洋 冯娜 于 2020-09-21 设计创作，主要内容包括：本发明公开了一种化合物在抗病毒药物方面的应用,具体涉及牛黄罗定在治疗和预防新型冠状病毒或流感病毒药物方面的应用。所述药物为预防和治疗由病毒引起肺部疾病的药物。所述病毒包括流感病毒、冠状病毒等RNA病毒。本发明拓展了牛磺罗定的功效范围,证实在细胞水平上能够显著抑制流感病毒和SARS-CoV-2的复制,提示牛磺罗定对流感病毒和SARS-CoV-2具有非常积极的抑制作用,为制备预防和治疗由流感病毒和新型冠状病毒引起肺部感染药物提供了研究基础和开辟了新方向。并通过体外实验证实牛磺罗定对肺脏具有显著的保护作用,能延长流感病毒对小鼠的生存时间,可用于流感病毒引起的肺部疾病的预防和治疗。(The invention discloses application of a compound in antiviral drugs, and particularly relates to application of taurolidine in drugs for treating and preventing novel coronavirus or influenza virus. The medicine is used for preventing and treating lung diseases caused by viruses. The virus includes influenza virus, coronavirus and other RNA virus. The invention expands the efficacy range of taurolidine, proves that the taurolidine can obviously inhibit the replication of influenza virus and SARS-CoV-2 at the cellular level, prompts that the taurolidine has very positive inhibition effect on the influenza virus and SARS-CoV-2, provides research basis for preparing medicaments for preventing and treating lung infection caused by the influenza virus and novel coronavirus and opens up a new direction. In vitro experiments prove that the taurolidine has obvious protective effect on the lung, can prolong the survival time of influenza virus to mice, and can be used for preventing and treating lung diseases caused by the influenza virus.)

1. The application of the compound in the aspect of preparing antiviral drugs is characterized in that the compound is taurolidine or a derivative of the taurolidine or a compound based on the molecular structure of the taurolidine, and the virus is RNA virus.

2. The use according to claim 1, wherein said taurolidine compound based on the molecular structure thereof is obtained by simply replacing or adding or subtracting the groups-NH-and-CH-from the two sulfoxy groups of the taurolidine molecule₂The resulting structural changes form analogous compounds.

3. The use according to claim 1, wherein the medicament is a medicament for the prophylaxis or treatment of pulmonary diseases caused by viruses.

4. The use of claim 1, wherein the medicament is an agent that inhibits replication or propagation of a virus in a cell or in vivo.

5. The use according to claim 1, wherein the virus is an influenza virus or a coronavirus.

6. The use according to claim 5, wherein the coronavirus is SARS-CoV-2.

7. A pharmaceutical composition for preventing or treating SARS-CoV-2, which comprises taurolidine or a derivative thereof, or a compound based on the molecular structure of taurolidine.

8. The pharmaceutical composition for preventing or treating anti-SARS-CoV-2 according to claim 7, wherein the pharmaceutical composition is prepared into an injection, a spray, an aerosol or a lotion by adding a pharmaceutically acceptable carrier compound, a salt-soluble or water-soluble compound.

9. The pharmaceutical composition for preventing or treating anti-SARS-CoV-2 according to claim 8, wherein the carrier compound comprises one or more of a diluent, an absorbent, a wetting agent, an excipient, a filler, a binder, a disintegrant, a surfactant.

10. The pharmaceutical composition for preventing or treating anti-SARS-CoV-2 according to claim 9, wherein the carrier compound is PVP-KF-17 as a solubilizer and stabilizer.

Technical Field

The invention relates to the technical field of compound medicine application, in particular to application of a compound in the aspect of medicines for treating and preventing novel coronavirus and influenza virus.

Background

Coronaviruses (Coronaviruses) are a large group of viruses that are widely found in nature. In biological classification, coronaviruses belong to the order of the nested viruses (Nidovirales), the family of Coronaviridae (Coronaviridae) and the genus Coronaviridae (Coronavir), and are RNA viruses with envelope and linear single-stranded positive strands in genome, the diameter of the viruses is 80-120 nm, and nucleic acids are non-segmented single-stranded (+) RNA and the length of the viruses is 27-31 kb. Coronaviruses are the longest strands of RNA nucleic acids in RNA viruses, containing important structural features unique to positive strand RNA: namely, the 5 'end of the RNA chain is provided with a methylated cap, and the 3' end is provided with a polyA tail structure. This structure is very similar to eukaryotic mRNA and is an important structural basis for the genomic RNA itself to function as a translation template.

Coronaviruses can be excreted from the body through respiratory secretions, and are transmitted through oral fluid, sneeze, contact, and air droplets, thereby infecting vertebrates and humans and causing respiratory infections and acute gastroenteritis, such as human, mouse, horse, pig, cat, dog, poultry, etc.

Influenza viruses (inflenza viruses) are the major viruses causing acute respiratory infections, leading to Influenza. Influenza virus belongs to the family of Orthomyxoviridae (Orthomyxoviridae), is an RNA virus, and mainly includes influenza a virus, influenza B virus, and influenza C virus. The influenza A virus has high variability, is most highly transmissible and pathogenic, and is very easy to cause seasonal epidemics. Severe pneumonia, acute respiratory distress syndrome, septicemia with shock and the like can be caused after human beings are infected with influenza virus, the death rate is very high, and the method has great threat to social public health safety. The drugs commonly used against influenza viruses at present mainly include alkylamine drugs and neuraminidase inhibitor drugs. However, alkylamine drugs are only effective against influenza a viruses, and influenza viruses are found to rapidly develop resistance to such antiviral drugs by means of genetic variation or drug reaction. Neuraminidase inhibitor drugs can inhibit viral replication by preventing the release of progeny virus. However, the side effects of such drugs have been problematic in clinical applications, including hallucinations, behavioral abnormalities, hearing and vision disorders. Therefore, the search for various subtypes of influenza viruses and the development of a class of drugs with universal applicability are of great significance.

The novel Coronavirus (SARS-CoV-2) is a new strain of Coronavirus found in 2019 in human body, is the 7 th Coronavirus which is known to infect human at present, and has the characteristics of long latency, strong infectivity, high replication rate, difficulty in prevention and control and the like. After the human being is infected with the novel coronavirus, the clinical manifestations of the coronavirus are fever, hypodynamia, dry cough and gradual dyspnea, the severe patients manifest acute respiratory distress syndrome, sepsis shock, metabolic acidosis and blood coagulation dysfunction which are difficult to correct, and the national economy and the human health are seriously affected. At present, no effective medicine for treating SARS-CoV-2 exists, and the medicine can only be used for actively preventing and treating complications on the basis of symptomatic treatment. Therefore, the development of effective drugs for preventing or treating novel coronaviruses is not slow enough, and the development of such drugs is also drawing attention of researchers in various countries in the world.

Taurolidine (the English name is Taurolidine, the chemical name is 4,4' -methylenebis [ tetrahydro-2H-1, 2, 4-thiadiazine)]1,1,1',1' -tetraoxide with the molecular formula of C₇H₁₆N₄O₄S₂) Is derivative of amino acid taurine, and has antiendotoxin, antibacterial and antiadhesion properties. In the aspect of bacteria, taurolidine can generate chemical reaction with cell walls, endotoxin and exotoxin to inhibit the adhesion of microorganisms and play a role in resisting bacteria. In addition, in anti-tumor terms, 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 7 months 2020, about 260 foreign literature searches reported in taurolidine research have been reported, most research focuses on the exploration of the action of taurolidine on tumor-related signaling pathways, and no research report is found on the application of taurolidine in antiviral activity.

Because the existing disease treatment research on taurolidine focuses on the aspects of tumor resistance and sterilization, the efficacy range of the taurolidine is limited. The invention develops a new therapeutic drug application of taurolidine, namely the application of taurolidine in antiviral drugs.

Disclosure of Invention

The invention aims to provide application of a compound in the aspect of preparing antiviral drugs, wherein the compound is taurolidine or a derivative of the taurolidine or a compound based on the molecular structure of the taurolidine.

The compound based on the molecular structure of taurolidine is obtained by simply replacing two sulfo-oxo groups of the taurolidine molecule or increasing or decreasing the groups-NH-and-CH₂The resulting structural changes form analogous compounds.

The medicine is used for preventing and treating lung diseases caused by viruses.

Alternatively, the medicament is an agent that inhibits replication or propagation of the virus in cells or in vivo.

The virus is RNA virus, including influenza virus, coronavirus, and hepatitis virus.

Specifically, the coronavirus is SARS-CoV-2.

The invention also provides a pharmaceutical composition for preventing or treating SARS-CoV-2, which comprises taurolidine or its derivatives, or compounds based on the molecular structure of taurolidine.

The pharmaceutical composition is added with a pharmaceutically acceptable carrier compound, a salt-soluble or water-soluble compound, and prepared into an injection, a spray, an aerosol or a flushing agent by adopting the prior art.

The carrier compound comprises one or more of diluents, absorbents, humectants, excipients, fillers, binders, disintegrants, surfactants.

The carrier compound is PVP-KF-17 and is used as a solubilizer and a stabilizer.

Specifically, the formulation of the above formulation may be: every 100ml of solution contains 2g of taurolidine and 175 g of PVP-KF; the preparation process comprises the following steps: dissolving PVP-KF-17 in water at room temperature, adding taurolidine at 45-50 ℃ after complete dissolution, stirring until complete dissolution, adding a proper amount of 4% sodium hydroxide solution to adjust the pH, adding a proper amount of activated carbon, preserving heat for 30 minutes, and filtering while hot. Filtering the filtrate with 0.25mm filter membrane, bottling, and capping.

In addition, taurolidine may also be used in combination with antibiotics (except for vancomycin).

Compared with the prior art, the invention has the beneficial effects that:

the MDCK, Vero-E6 and F81 cells are used as a cell model of the antiviral effect of taurolidine, viruses are inoculated in the cells, virus liquid is harvested according to the cytopathic state, and the inhibitory effect of the taurolidine on the viruses is measured. The invention expands the efficacy range of taurolidine, proves that the taurolidine can obviously inhibit the replication of influenza virus and SARS-CoV-2 at the cellular level, prompts that the taurolidine has very positive inhibition effect on the influenza virus, SARS-CoV-2 and other RNA viruses, provides research basis for preparing medicaments for preventing and treating lung infection caused by the influenza virus and novel coronavirus and opens up a new direction. Meanwhile, two DNA viruses are selected as representative strains, and a non-enveloped parvovirus and an enveloped pseudorabies virus are taken as examples to prove that the taurolidine can not inhibit the replication of the canine parvovirus and the pseudorabies virus on a cellular level, so that the taurolidine is prompted to have no inhibition effect on the DNA viruses. In addition, in-vitro experiments prove that the taurolidine has obvious protective effect on the lung, can prolong the survival time of the influenza virus on mice, and can be used for preventing and treating lung diseases caused by the influenza virus.

Drawings

In order to more clearly illustrate the embodiments of the present application or technical solutions in the prior art, the drawings needed to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings can be obtained by those skilled in the art according to the drawings.

FIG. 1 is a graph showing the results of detecting the sensitivity of taurolidine to MDCK, Vero-E6 and F81 cells, which is provided in example 1 of the present invention;

FIG. 2 is a graph showing the results of detection of the influenza virus infectivity inhibited by taurolidine in example 2 of the present invention;

FIG. 3 is a graph showing the results of detecting the pseudorabies virus infectivity inhibited by taurolidine in example 3 of the present invention;

FIG. 4 is a graph showing the results of testing the ability of taurolidine to inhibit canine parvovirus infection as provided in example 4 of the present invention;

FIG. 5 is a graph showing the effect of taurolidine on the body weight of influenza virus model mice as provided in example 5 of the present invention;

FIG. 6 is a graph showing the effect of taurolidine on the survival rate of influenza virus model mice as provided in example 5 of the present invention;

FIG. 7 is a graph showing the effect of taurolidine on the pathogenic effect of influenza virus in mice, as provided in example 6 of the present invention;

FIG. 8 is a graph showing the results of detecting the SARS-CoV-2 infectivity suppressed by taurolidine in example 6 of the present invention.

Detailed Description

In order to make the technical solutions of the present invention better understood by those skilled in the art, the present invention will be further described in detail with reference to the accompanying drawings and examples. The BALB-C mouse, MDCK, Vero-E6, F81 and taurolidine are all commercial products, wherein the taurolidine is provided by Changchun Meiling bioengineering limited company, MDCK, Vero-E6, F81, influenza virus, rabies virus and canine parvovirus are from military veterinary institute of military medical institute of military academy of sciences, and SARS-CoV-2 virus is from Beijing isolate and is stored in military veterinary institute of military medical institute of military academy of sciences.

Example 1 Effect of Taurolidine on cell safety

MDCK, Vero-E6 and F81 cells are respectively stored in liquid nitrogen, taken out and revived, continuously transmitted for three generations, and used for experimental study after the cells grow well. MDCK, Vero-E6 and F81 cells were inoculated into 96-well plates with a cell amount of 1 × 10 per well, respectively⁵At 37 ℃ 5% CO₂The cells were incubated overnight in a thermostatted cell incubator. When the cell density is 60-70%, adding 2% FBS DMEM maintenance solution containing taurolidine (initial concentration 4mg/ml, 10 times gradient to dilute the drug to be tested, total 10 concentrations) with different concentrations 100 μ L/wellAnd 4 duplicate wells were measured for each concentration. Blank cell control and PBS control were also set up and cultured for 4 replicates and the cell status was observed under the microscope every day. On day 2, 10. mu.L of MTT solution (5mg/ml) was added thereto at 37 ℃ with 5% CO₂Incubated for 1 hour under the conditions, and OD was measured₅₇₀The value is obtained. Data were analyzed using Graphpad prism5.0 software and the half maximal Inhibitory Concentration (IC) of drug on cells was calculated₅₀)。

The detection result is shown in figure 1, and IC of the taurolidine on MDCK cells (figure 1A) can be found₅₀IC of 22.88. mu.M, Vero-E6 cells (FIG. 1B)₅₀IC of F81 cells (FIG. 1C) with a value of 20.38. mu.M₅₀The value was 36.74. mu.M.

Example 2 results of examining the ability of taurolidine to inhibit influenza Virus infection

MDCK cells are stored in liquid nitrogen, taken out and revived, continuously transmitted for three generations, and used for experimental study after the cells grow well. The preserved H3N2 influenza virus strain was placed on ice and thawed slowly, seeded on a monolayer of MDCK cells (no more than 24 hours), cultured for an additional 72-96 hours, and virus fluid was harvested according to the cytopathic state. And determining the content thereof as TCID₅₀The unit of calculation is/100. mu.l.

The drug is mixed with H3N2 subtype influenza virus and then inoculated into cells. Digesting well-grown MDCK cells with pancreatin, calculating cell content, inoculating to 96-well plate with 1 × 10 cells per well⁵. The study of the effect of the drug was performed within 12 hours of inoculation and with the cells in the monolayer state. Inoculation virus content of 100TCID₅₀The influenza virus subtype H3N2 was mixed with taurolidine (14. mu.M), allowed to act at room temperature for 10 minutes, and then inoculated into a well-plated 96-well plate. A blank cell control and a virus control were also set for 3 replicates. The inoculated cell plates were placed at 37 ℃ in 5% CO₂After the culture in the incubator was continued for 72 hours, the cell lesion was observed, and the cell proliferation rate was calculated.

The calculation formula is as follows:

relative proliferation rate of cells OD experimental group/OD control group × 100%

The observation results are shown in figure 2. It can be found that taurolidine with the content of 14 mu M has obvious inhibition effect on influenza virus on the cellular level. The detection result shows that the compound taurolidine can obviously inhibit cytopathic effect caused by influenza, and the result indicates that the taurolidine can inhibit the infection of the influenza virus.

Example 3 results of measurement of potency of taurolidine to inhibit Pseudorabies Virus infection

Virus: the pseudorabies virus is a DNA virus with an envelope, and the diameter of a virus particle is 150nm-180 nm.

MDCK cells are stored in liquid nitrogen, taken out and revived, continuously transmitted for three generations, and used for experimental study after the cells grow well. The virus pseudorabies strain with the preserved envelope is placed on ice to be slowly melted, is inoculated to a single-layer MDCK cell (no more than 24 hours), is continuously cultured for 72 to 96 hours, and is harvested according to the cytopathic state. And determining the content thereof as TCID₅₀The unit of calculation is/100. mu.l.

The drug is mixed with pseudorabies virus and then inoculated into cells. Digesting well-grown MDCK cells with pancreatin, calculating cell content, inoculating to 96-well plate with 1 × 10 cells per well⁵. The study of the effect of the drug was performed within 12 hours of inoculation and with the cells in the monolayer state. Inoculation virus content of 100TCID₅₀The pseudorabies virus was mixed with 15. mu.M taurolidine and 30. mu.M taurolidine, reacted at room temperature for 10 minutes, and inoculated into a well-plated 96-well plate. A blank cell control and a virus control were also set up and 3 replicates were performed. The inoculated cell plates were placed at 37 ℃ in 5% CO₂After the culture in the incubator was continued for 72 hours, the cell lesion was observed, and the cell proliferation rate was calculated.

The calculation formula is as follows:

relative proliferation rate of cells OD experimental group/OD control group × 100%

The observation results are shown in FIG. 3. It was found that taurolidine at levels of 15. mu.M and 30. mu.M had no inhibitory effect on pseudorabies virus at the cellular level. The detection results show that the taurolidine compounds with the content of 15 mu M and 30 mu M cannot inhibit the cytopathic effect caused by the pseudorabies virus, and the results indicate that the taurolidine cannot inhibit the infection of the pseudorabies virus.

Example 4 results of measurement of the ability of taurolidine to inhibit canine parvovirus infectivity

Virus: parvovirus is DNA virus without capsule membrane, and the diameter of virus particle is 18-26 nm.

F81 cells are stored in liquid nitrogen, taken out and revived, continuously transmitted for three generations, and used for experimental study after the cells grow well. The preserved canine parvovirus strain is placed on ice to be slowly thawed, inoculated to a monolayer of F81 cells (no more than 24 hours), continuously cultured for 72 to 96 hours, and virus liquid is harvested according to the cytopathic state. And determining the content thereof as TCID₅₀The unit of calculation is/100. mu.l.

The drug is mixed with canine parvovirus and then inoculated into cells. Digesting well grown F81 cells with pancreatin, calculating cell content, inoculating to 96-well plate with 1 × 10 cell per well⁵. The study of the effect of the drug was performed within 12 hours of inoculation and with the cells in the monolayer state. Inoculation virus content of 100TCID₅₀The canine parvovirus is respectively mixed with 15 mu M taurolidine and 30 mu M taurolidine, acted for 10 minutes at room temperature and then inoculated into a paved 96-well plate. A blank cell control and a virus control were also set up and 3 replicates were performed. The inoculated cell plates were placed at 37 ℃ in 5% CO₂After the culture in the incubator was continued for 72 hours, the cell lesion was observed, and the cell proliferation rate was calculated.

The calculation formula is as follows:

relative proliferation rate of cells OD experimental group/OD control group × 100%

The observation results are shown in FIG. 4. It was found that taurolidine at levels of 15. mu.M and 30. mu.M had no inhibitory effect on canine parvovirus at the cellular level. The detection result shows that the taurolidine compounds with the content of 15 mu M and 30 mu M can not inhibit the cytopathic effect caused by the canine parvovirus, and the result indicates that the taurolidine can not inhibit the infection of the canine parvovirus.

Example 5 Effect of taurolidine on weight and survival Rate of influenza Virus infected mice

BALB-C mice were divided into a blank control group, a virus control group, a nebulized treatment group, and an intramuscular injection group, with 7 mice per group. The influenza virus is inoculated to mice of a virus control group, an atomization treatment group and an intramuscular injection group, and the mice of the atomization treatment group and the intramuscular injection group are respectively and continuously treated with the taurolidine drug every day by adopting different modes. The body weights of the mice of each group were measured every day, and the survival status of the mice was observed.

The results of the above-described observation are shown in FIGS. 5 and 6. Influenza virus vaccinated mice lost significantly in weight compared to the blank control group (figure 5). Although drug treatment did not save the body weight of the mice, the survival time of the mice could be extended, with the intramuscular injection group > the nebulization treatment group (fig. 6). The detection result shows that the compound taurolidine can prolong the survival time of influenza virus to mice and can be used for preventing and treating the influenza virus.

Example 6 Effect of taurolidine on the pathogenicity of influenza Virus in mice

Experimental method similar to example 5, influenza virus was inoculated to mice of virus control group, nebulized treatment group, and intramuscular injection group, and drug treatment was continuously administered to the mice of nebulized treatment group and intramuscular injection group, respectively, every day in different ways. And respectively taking mouse lung tissues on the third day and the fifth day after inoculation, and observing the influence of the taurolidine on the mouse lung index caused by the influenza virus.

The calculation formula is as follows: lung index ═ (weight of mouse lung tissue ÷ mouse body weight) × 100%

The results of the above-described method are shown in FIG. 7. Compared with virus control, the taurolidine treated (atomized treated group and intramuscular injected group) has obvious protection effect on the lung of the mouse.

EXAMPLE 5 titration assay of taurolidine for inhibition of SARS-CoV-2 infectivity

The Vero-E6 cells are stored in liquid nitrogen, taken out and recovered, continuously transmitted for three generations, and used for experimental study after the cells grow well. The preserved SARS-CoV-2 liquid is placed on ice to be melted slowly, inoculated to a single layer of Vero-E6 cells (no more than 24 hours), cultured for 72 to 96 hours continuously, and then the SARS-CoV-2 liquid is harvested according to the cytopathic state. And determining the content thereof as TCID₅₀The unit of calculation is/100. mu.l.

The drug is mixed with SARS-CoV-2 and then inoculated into cells. The Vero-E6 cells were trypsinized and cell content was calculated, and seeded into 96-well plates at 1X 10 cells/well⁵. The study of the effect of the drug was performed within 12 hours of inoculation and with the cells in the monolayer state. Inoculation virus content of 100TCID₅₀After the SARS-CoV-2 virus and the taurolidine are mixed, the mixture is inoculated into a paved 96-well plate after acting for 10 minutes at room temperature, and the medicine dosage is 5, 15, 25 and 50 mul/mL in sequence. A blank cell control and taurolidine cytotoxicity control were also set up and 3 replicates were performed. The inoculated cell plates were placed at 37 ℃ in 5% CO₂After further 72 hours in the incubator, cytopathic effects were observed. And calculating the half maximal Effective Concentration (EC) of the drug against influenza virus₅₀)。

The calculation formula is as follows:

infection rate ═ infection rate (drug treatment OD-virus control OD) ÷ (cell control OD-virus control OD) × 100%

The observation results are shown in figure 8. It can be found that taurolidine has significant inhibitory effect on SARS-CoV-2 at the cellular level. The detection result shows that the compound taurolidine can obviously inhibit cytopathic effect caused by SARS-CoV-2, and can be used for the research and development of medicaments for preventing and treating SARS-CoV-2.

The results are combined, and the taurolidine can obviously inhibit influenza viruses and novel coronaviruses, prolong the survival time of influenza virus mice and improve the pathogenic effect of the influenza viruses on the lungs of the mice. However, taurolidine at doses of 15. mu.M and 30. mu.M, respectively, was not able to inhibit the replication of canine parvovirus and pseudorabies virus.

These results suggest that taurolidine has no inhibitory effect on pseudorabies virus and canine parvovirus or on this class of DNA viruses. However, taurolidine has very effective inhibitory action on RNA viruses such as influenza virus and novel coronavirus, and can be used for the prevention of the viruses and the research and development of related therapeutic drugs.

The above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: it is to be understood that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof, but such modifications or substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.