CN112043707B

CN112043707B - Application of clofazimine and/or pharmaceutically acceptable derivatives thereof in preparation of anti-saxavirus medicine

Info

Publication number: CN112043707B
Application number: CN202010939267.2A
Authority: CN
Inventors: 刘愈杰; 徐国东; 袁冰
Original assignee: Canvest Wuhan Biotechnology Co ltd
Current assignee: Canvest Wuhan Biotechnology Co ltd
Priority date: 2020-09-09
Filing date: 2020-09-09
Publication date: 2021-07-30
Anticipated expiration: 2040-09-09
Also published as: CN112043707A

Abstract

The invention provides an application of clofazimine and/or a medicinal derivative thereof in preparing a medicament for resisting a salsa virus. The invention detects the cytotoxicity of clofazimine on Vero cells and BHK-21 cells and the antiviral activity of clofazimine on arenavirus, shows that the clofazimine has stronger effect of resisting the arenavirus at the cellular level, and provides a new direction for preventing and treating arenavirus infectious diseases.

Description

Application of clofazimine and/or pharmaceutically acceptable derivatives thereof in preparation of anti-saxavirus medicine

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to application of clofazimine and/or pharmaceutically acceptable derivatives thereof in preparation of a medicine for resisting arenavirus.

Background

Mammalian arenaviruses (Mammarenavirus) belong to the Arenaviridae (Bunyavirales) family of bunyaviridae (Arenaviridae), and consist mainly of old and new world arenaviruses, including many virulent viruses that pose a significant threat to humans, such as Lassa virus (LASV), vernonia virus (Jun virus, JUNV), machupulo virus (Machupo virus, MACV), and the like. Wherein LASV is prevalent in West Africa countries such as Nigeria, Libiria, Serioleon, and Guinea, where 10-30 million people infect each year and about 5000 people die. JUNV is prevalent mainly in the argentina Pampa area, causing argentina hemorrhagic fever with a mortality rate of 15% -30%. In addition, Lymphocytic choriomeningitis virus (LCMV) exists in our country, mainly in mice, and humans are infected by contact with mouse excreta and outbreaks in immunocompromised individuals, causing central nervous system infections, one of the important pathogens that has been overlooked. Among the pathogenic microorganisms of the interpersonal infection established by the ministry of health of the people's republic of China, the arenavirus family accounts for 8.

Arenaviruses are widespread and seriously threaten human health, and there is currently no FDA-approved vaccine or drug specific for arenaviruses. The current main treatment method is mainly limited to ribavirin, but the treatment effect is limited and the ribavirin has strong side effects. Therefore, it is of great importance to research and develop effective anti-arenavirus infection drugs.

Clofazimine (clofazimine), also known as clofazimine, has an inhibitory effect on leprosy bacillus, and has an action mechanism of interfering nucleic acid metabolism and inhibiting mycoprotein synthesis, which is slower than dapsone. However, at present, there is no report on the finding that clofazimine has an anti-arenavirus effect.

Disclosure of Invention

The invention aims to solve the problems of limited treatment effect and strong side effect of the existing anti-arenavirus medicine.

Therefore, the invention provides the application of clofazimine and/or pharmaceutically acceptable derivatives thereof in preparing the anti-arenavirus medicament.

Further, the arenavirus is lymphocytic choriomeningitis virus.

Furthermore, the concentration of the anti-arenavirus activity of the clofazimine is 0.25-64 mu M.

Furthermore, the anti-arenavirus medicine comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient comprises clofazimine and/or pharmaceutically acceptable derivatives thereof.

In the invention, clofazimine (also called chlorophenoxazine) is a drug mainly used for treating the dapsone-resistant leprosy bacillus infection at present, and the chemical structural formula is as follows:

。

the invention has the beneficial effects that:

the invention detects the cytotoxicity of clofazimine on Vero cells and BHK-21 cells and the antiviral activity of clofazimine on arenavirus, shows that the clofazimine has stronger effect of resisting the arenavirus at the cellular level, and provides a new direction for preventing and treating arenavirus infectious diseases.

The present invention will be described in further detail below with reference to the accompanying drawings.

Drawings

FIG. 1 is a graph of cell viability of clofazimine on Vero cells at various concentrations in example 1 of the present invention;

FIG. 2 is a graph showing the cell viability of clofazimine at various concentrations in example 1 of the present invention on BHK-21 cells;

FIG. 3 is a graph of cell viability on Vero cells for different concentrations of ribavirin according to example 1 of the present invention;

FIG. 4 is a graph of cell viability for different concentrations of ribavirin on BHK-21 cells in example 1 of the present invention;

FIG. 5 is a graph of the inhibition rate of clofazimine at different concentrations on Vero cells in example 2 of the present invention for inhibiting LCMV viral replication;

FIG. 6 is a graph showing the inhibition rate of various concentrations of clofazimine in example 2 of the present invention on BHK-21 cells for inhibiting LCMV viral replication;

FIG. 7 is a graph of inhibition of LCMV viral replication on Vero cells by different concentrations of ribavirin in example 2 of the invention;

FIG. 8 is a graph of the inhibition rate of different concentrations of ribavirin on BHK-21 cells for LCMV viral replication in example 2 of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

the cytotoxicity of clofazimine as a target compound is researched by the embodiment, which comprises the following specific steps:

BHK-21 and Vero cells were as 1.5X 10, respectively⁴Cells/well were seeded in 96-well cell culture plates at 37 ℃ in 5% CO₂After culturing for 12-16 h in the incubator, the culture medium is replaced by DMEM containing 2% FBS, and then clofazimine with the concentration of 0.25 MuM, 0.5 MuM, 1 MuM, 2 MuM, 4 MuM, 8 MuM, 16 MuM, 32 MuM and 64 MuM is usedTreatment was performed by adding the same volume of DMSO to the negative control, setting three duplicate wells for each concentration, incubating for 36h at 37 ℃, and setting zero-setting wells with no cells in the medium. Discarding the supernatant, washing with PBS once, adding 50 μ l of 0.5% MTT PBS solution into each well, standing at 37 deg.C for 4h in a dark condition, observing the occurrence of bluish-purple crystals at the bottom of the plate, terminating the culture, carefully sucking the supernatant, adding 100 μ l DMSO into each well, and placing on a shaking table to slowly shake for 10min to fully dissolve the crystals. The absorbance of each well was measured using a microplate reader at OD570nm, the cytotoxicity of each well was calculated from the values of DMSO control wells after zeroing the wells and calibration, as shown in FIGS. 1 and 2, respectively, and the CC of clofazimine on Vero cells and BHK-21 cells was calculated by plotting the cell viability curves at different concentrations₅₀55.05 μ M and 35.03 μ M, respectively.

In addition, compared experiments of treating BHK-21 cells and Vero cells with ribavirin show that the treatment process is basically the same as that of clofazimine except that the treatment concentrations of ribavirin are 1 μ M, 3.3 μ M, 10 μ M, 33 μ M, 100 μ M, 330 μ M and 1000 μ M; the absorbance of each well was measured using a microplate reader at OD570nm, the cytotoxicity of each well was calculated from the values of the DMSO control wells after zeroing the wells, as shown in FIGS. 3 and 4, respectively, and the CC of ribavirin on Vero cells and BHK-21 cells was calculated by plotting the cell viability curves at different concentrations₅₀Are respectively as>1000. mu.M and 100.4. mu.M.

Example 2:

the antiviral activity of clofazimine, a target compound, on arenavirus (LCMV) is studied in the example, which comprises the following steps:

BHK-21 and Vero cells were as 1.5X 10, respectively⁴Cells/well were seeded in 96-well cell culture plates at 37 ℃ in 5% CO₂After culturing for 12-16 h in an incubator, the culture medium is replaced by a DMEM culture medium containing 2% FBS, then the DMEM culture medium is treated by using chlorine method zimine with the concentration of 0.25 mu M, 0.5 mu M, 1 mu M, 2 mu M, 4 mu M, 8 mu M, 16 mu M, 32 mu M and 64 mu M, the DMSO with the same volume is added into a negative control, and three multiple wells are arranged at each concentration. After 1h incubation, cells were infected with LCMV. After 36h, the mixture is passedThe effect of clofazimine on inhibiting virus replication is detected by measuring the titer of the supernatant, the results on two cells are respectively shown in fig. 3 and fig. 4, and the compound clofazimine has obvious inhibition effect on LCMV under different concentration conditions. By drawing the inhibition rate curve of the clofazimine inhibiting viruses under different concentrations, the IC of the clofazimine inhibiting LCMV in Vero and BHK-21 cells is calculated₅₀1.704. mu.M and 0.9338. mu.M, respectively.

The comparative experiment was conducted with respect to the antiviral activity of ribavirin against arenavirus (LCMV), and the treatment process was substantially identical to that of clofazimine described above, except that the treatment concentrations of ribavirin were 1. mu.M, 3.3. mu.M, 10. mu.M, 33. mu.M, 100. mu.M, 330. mu.M, and 1000. mu.M. The results of the inhibition of virus replication by ribavirin are shown in FIGS. 7 and 8, respectively, and the IC of ribavirin on Vero cells and BHK-21 cells was calculated by plotting the inhibition rate curves of ribavirin against virus at different concentrations₅₀40.13 μ M and 4.26 μ M, respectively.

The above examples are merely illustrative of the present invention and should not be construed as limiting the scope of the invention, which is intended to be covered by the claims and any design similar or equivalent to the scope of the invention.

Claims

1. The application of clofazimine as the only active component in preparing the medicine for resisting the arenavirus, wherein the arenavirus is lymphocyte choriomeningitis virus.

2. The use of claim 1, wherein: the active concentration of the clofazimine against the arenavirus is 0.25-64 mu M.

3. The use of claim 1, wherein: the anti-arenavirus medicine comprises an active ingredient and pharmaceutically acceptable auxiliary materials, wherein the active ingredient is clofazimine.