Application of Ttebulin in preparation of chikungunya virus infection resisting medicine

Abstract

The invention relates to the technical field of medicines, in particular to application of Tilbanibulin in preparation of a medicine for treating chikungunya virus (CHIKV) infection. The medicine for resisting CHIKV infection is a medicine composition which takes the tetra-brulin as a unique active ingredient or contains the tetra-brulin, and the medicine for resisting CHIKV infection refers to a medicine for preventing or treating CHIKV infection. Candidate small molecule drugs capable of inhibiting CHIKV infection are screened from a clinically approved drug small molecule library by using an experimental operation system of CHIKV susceptible cells, and the screened tebusbulin can effectively reduce the death rate of mice infected with lethal CHIKV, can be used as a candidate anti-CHIKV drug and has application prospects.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the application of tibanibulin in the preparation of drugs against chikungunya virus infection. Background Technology

[0002] Tibanibulin is a microtubule polymerization inhibitor and Src kinase inhibitor that targets the peptide substrate binding site of Src kinase. Its molecular formula is C26H29N3O3, and its molecular weight is 431.53. Tibanibulin was approved for marketing in the United States in December 2020 and in the European Union in July 2021, primarily for the topical treatment of actinic keratosis (AK) of the face or scalp. Furthermore, its potential applications in solid cancers such as gastric cancer, breast cancer, anterior lacrimal gland cancer, and psoriasis are in clinical trials. In preclinical animal models of cancer, oral administration of tibanibulin inhibited the growth of primary tumors and suppressed metastasis. Simultaneously, tibanibulin has also been found to inhibit certain commercially available drug-resistant leukemia cells, such as leukemia cell-derived cells carrying the T315I mutation.

[0004] Chikungunya fever virus (CHIKV) belongs to the genus Alphavirus of the family Occultviridae. It is a single-stranded positive-sense RNA virus, primarily transmitted by Aedes mosquitoes, and causes chikungunya fever. Although the mortality rate is low, infected individuals may experience a prolonged decline in quality of life and work capacity due to arthritis. In rare cases, it can affect the central nervous system (CNS), leading to severe illness and even death. CHIKV was first isolated in Tanzania, Africa, in 1952-1953, and has since experienced multiple outbreaks in Africa, Asia, and South America. In particular, the A226V mutation in the envelope glycoprotein E1 of the East Central and Southern African type of CHIKV after 2005 has adapted it to replicate in Aedes albopictus mosquitoes, posing a threat to populations in subtropical and temperate regions where Aedes albopictus is widely present. In July 2025, a local outbreak of chikungunya fever caused by imported cases occurred in Guangdong Province, my country. However, there is currently no vaccine or specific treatment for CHIKV infection. Summary of the Invention

[0005] The present invention aims to overcome the above-mentioned defects and provide a new use for tebanbulin.

[0006] The chemical structural formula of tembambarin is as follows:

[0007] This invention utilizes an experimental system for CHIKV infection of susceptible cells to screen candidate small molecule drugs that can inhibit CHIKV infection from a clinically approved drug library. Terbanibrine was found to effectively inhibit CHIKV infection of human liver cancer cells Huh7 and mouse hippocampal neurons HT22, exhibiting low cytotoxicity and potential as an anti-CHIKV drug with promising application prospects.

[0008] This invention suggests the use of tebanbulin in the preparation of drugs against chikungunya virus infection.

[0009] This invention suggests the application of terbanumin in the preparation of drugs for the prevention of chikungunya virus infection.

[0010] This invention suggests the use of tebanbulin in the preparation of medicaments for treating chikungunya virus infection.

[0011] This invention suggests the application of terbanumin in the preparation of drugs that inhibit viral infection of human hepatocellular carcinoma cells Huh7.

[0012] This invention suggests the application of terbanumin in the preparation of drugs that inhibit viral infection of hippocampal neurons at HT22.

[0013] In the above applications, the drug is either terbanumlin as the sole active ingredient or a pharmaceutical composition containing terbanumlin.

[0014] In the above applications, a pharmaceutical composition containing terbanum refers to a pharmaceutical composition consisting of terbanum and one or more pharmaceutically permissible excipients. Attached Figure Description

[0015] Figure 1 The effect of tebanbulin on protecting BHK cells against chikungunya virus infection; BHK cells were infected with chikungunya virus and terbanum (final concentration 10 mM) or DMSO (final concentration 0.12%) was added, or they were not infected with chikungunya virus (no virus was added). After 72 hours, CCK8 reagent was added and the absorbance at 450 nm was measured.

[0016] Figure 2 The toxicity of tebanbulin to Huh7 and HT22 cells; Huh7 and HT22 cells were treated with terbanolol (final concentration 20 mM) and DMSO (final concentration 0.5%), respectively. After 72 hours, CCK8 reagent was added and the absorbance at 450 nm was measured.

[0017] Figure 3 Inhibitory effect of tebanbulin (final concentration 5 mM) on chikungunya virus in Huh7 and HT22 cell infection models.

[0018] Figure 4 Effect of administration of terbanumin on changes in body weight in mice induced by chikungunya virus infection.

[0019] Figure 5 Effect of administration of terbanumin on mortality in mice infected with chikungunya virus. Detailed Implementation

[0020] To more clearly illustrate the present invention, the following description, in conjunction with preferred embodiments, further clarifies the invention. Those skilled in the art should understand that the specific descriptions below are illustrative rather than restrictive, and should not be construed as limiting the scope of protection of the present invention.

[0021] All the tebanbulin used in the embodiments of this invention can be purchased commercially.

[0022] I. Viruses, drugs, reagents and other materials 1. Virus: Chikungunya virus, specifically the Middle East-South Africa type Indian Ocean lineage LR2006 strain, synthesized by the Department of Biomedical Protection, Naval Medical University of the Chinese People's Liberation Army using reverse genetics technology, and amplified and cultured from young hamster kidney BHK cells. All experimental procedures involving viral infection were performed in the P3 laboratory of the Naval Medical University.

[0023] 2. Compounds: The FDA-approved libraries of 2,580 and 698 chemical drug molecules were purchased from Selleck, Inc. in the United States.

[0024] 3. Human hepatocellular carcinoma cell line Huh7, mouse hippocampal neuronal cell line HT22, and young hamster kidney BHK cells were purchased from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences, and preserved by the Department of Biomedical Protection, Naval Medical University of the Chinese People's Liberation Army.

[0025] 4. The DMEM cell culture medium was a product of Hyclone, USA. When using it, 10% fetal bovine serum, non-essential amino acids, ampicillin and streptomycin (100 U / ml each) were added. All culture medium additives were products of Thermo Fisher, USA.

[0026] 5. Cell digestion solution containing 0.25% trypsin, prepared with phosphate buffer.

[0027] 6. The CCK8 cell viability and proliferation assay kit was a product of MedChemExpress, Inc., USA.

[0028] 7. The polyclonal antibody against Chikungunya virus was prepared by the Department of Biomedical Protection, Naval Medical University of the Chinese People's Liberation Army, by immunizing mice with formaldehyde-inactivated virus.

[0029] 8. The Alexa Fluor 488-labeled anti-rabbit IgG is a product of Thermo Fisher Scientific, Inc.

[0030] II. Experimental Methods: (i) Screening anti-Chikungunya virus drugs from two FDA small molecule drug libraries (a total of 3278 compounds). Young hamster kidney BHK cells were passaged in T75 cell culture flasks using complete DMEM medium and seeded into 96-well plates at 10,000 cells per well with 100 mL of DMEM medium. The cells were cultured for 12 hours. Then, 50 mL of complete DMEM medium containing 1000 PFU (plaque-forming units) of Chikungunya virus was added to each well; simultaneously, 50 mL of complete DMEM medium containing an FDA small molecule chemical drug was added to each well at a final concentration of 10 mM. Each concentration was repeated in triplicate. An equal volume of DMSO was added as a control without the drug. The plates were incubated at 37°C in a 5% CO2 incubator. After 72 hours, microscopic examination revealed that all cells in the wells containing DMSO had become rounded or detached. 10 mL of CCK8 cell viability and proliferation assay reagent was added to each well and incubated at 37°C with 5% CO2. After 30 minutes, the absorbance at 450 nm was measured using a multi-functional microplate reader. The protective rate of the drug against cells was calculated as: (absorbance at 450 nm of drug-treated cells - absorbance at 450 nm of cells treated with DMSO) / absorbance at 450 nm of cells treated with DMSO but without virus * 100%. This yielded the protective rate of each drug against chikungunya virus-infected cells at a concentration of 10 mM. The results showed that terbanumin had a significant protective effect on cells. The absorbance at 450 nm of cells treated with terbanumin compared to the DMSO control was as follows: Figure 1 As shown, the calculated cell protection rate of terbanum was 82.3%.

[0031] (II) Cell toxicity of terbanum Human hepatocellular carcinoma cell line Huh7 and mouse hippocampal neuronal line HT22 were seeded into 96-well plates at 10,000 cells per well with 100 mL of culture medium. After 12 hours, the original culture medium was aspirated, and 100 mL of serially diluted terbambulin in complete DMEM medium was added to each well. The concentrations of terbambulin were 2.5, 5, 10, 20, 40, and 80 mM, with each concentration replicated in triplicate. The 80 mM drug solution in DMSO served as a control without drug. The plates were incubated at 37°C in a 5% CO2 incubator. After 48 hours, 10 mL of CCK8 cell viability and proliferation assay reagent was added to each well, and the plates were incubated at 37°C in a 5% CO2 incubator. After 30 minutes, the absorbance at 450 nm was measured using a multi-mode microplate reader. The cytotoxicity of the drug was evaluated based on the difference in absorbance at 450 nm between the drug-treated wells and the solvent-treated wells.

[0032] The results showed that when the concentration was equal to or below 20 mM, there was no significant difference between the two cell types treated with terbanum and those treated with DMSO solvent. Figure 2 The absorbance at 450 nm of Huh7 and HT22 cells treated with terbanum at 20 mM is compared with that of cells treated with DMSO solvent.

[0033] (III) Inhibitory effect of tebanbulin on chikungunya virus in a cell infection model Human hepatocellular carcinoma cell line Huh7 and mouse hippocampal neuronal line HT22 were seeded into 96-well plates, 10,000 cells per well, with 100 mL of culture medium, and cultured for 12 hours. Then, 50 mL of culture medium containing 1000 PFU of chikungunya virus was added to each well; simultaneously, 50 mL of complete DMEM culture medium containing tebanbulin was added to each well, with a final drug concentration of 5 mM. Each concentration was repeated in triplicate. An equal volume of DMSO was added as a control without drug. The plates were incubated at 37°C in a 5% CO2 incubator.

[0034] Twenty hours later, the viral infection status of the cells was detected using immunofluorescence. The specific procedure was as follows: The culture medium in the culture plate was aspirated, and 100 mL of methanol was added to each well. The culture plate was placed at -20°C. After 20 minutes, the culture plate was removed, the methanol was aspirated, and each well was washed once with phosphate-buffered saline (PBS). Then, 100 mL of PBS containing 3% bovine serum albumin (BSA) (hereinafter referred to as 3% BSA-PBS) was added, and the plate was placed on a horizontal shaker and slowly shaken at room temperature for 1 hour. The 3% BSA-PBS in the culture plate was aspirated, and 100 mL of 1% BSA-PBS containing anti-Chikungunya virus polyclonal antibody (antibody diluted 500 times) was added to each well. The plate was slowly shaken at room temperature for 1 hour. The anti-Chikungunya virus polyclonal antibody working solution in the culture plate was aspirated, and each well was washed 3 times with PBS. Then, 100 mL of Alexa Fluor was added. 488-labeled anti-rabbit IgG was incubated in 1% BSA-PBS (fluorescein antibody diluted 1500-fold) at room temperature in the dark with gentle shaking for 1 hour. The fluorescein antibody working solution was then removed from the culture plate. 100 mL of DAPI nuclear staining solution was added to each well, and the plate was incubated at room temperature in the dark with gentle shaking for 10 minutes. The DAPI nuclear staining solution was then removed from the culture plate. Each well was washed three times with PBS. The fluorescence distribution of cells in each well was photographed using a BioTek Cytation 5 Imaging Reader. Four fields of view were photographed for each well. The percentage of green fluorescent positive cells, i.e., the virus infection rate, was analyzed and calculated.

[0035] The results are as follows Figure 3 As shown, compared with the control group treated with DMSO solvent, the viral infection rate of terbumulin treatment was significantly reduced in both Huh7 and HT22 cells. The scale bar indicates a length of 200 μm. These results demonstrate that at a concentration of 5 mM, terbumulin significantly inhibits the infection of Huh7 and HT22 cells by chikungunya virus.

[0036] (iv) Tebanbulin can effectively protect mice against chikungunya virus infection and reduce mouse mortality. We first conducted a preliminary study on the viral challenge dose in mice, using 1*102 4 Mice were infected with PFU via nasal drops, and the mortality criterion was a decrease in body weight of more than 25%. Mice died as early as 7 days after infection, with a mortality rate of about 70%.

[0037] Tebanbulin powder was initially dissolved in DMSO, then fully dissolved in PBS containing 40% PEG300-5% Tween 80. It was administered via gavage at a dose of 10 mg / kg / day, once daily. Twenty 6-week-old female C57BL / 6 mice were randomly divided into three groups: 1. Drug treatment group: Administration began one day before viral challenge, administered via gavage on the day of challenge, followed by intranasal viral infection (8 mice); 2. DMSO group: Administered DMSO via gavage 24 hours after viral challenge, followed by daily DMSO administration (8 mice); 3. Blank control group: No viral challenge or drug administration (3 mice). Viral challenge was administered via intranasal infection at a dose of 1*10 mg / kg / day. 4 PFU (Chikungunya virus)

[0038] Mice were weighed starting on the day of viral attack (before nasal administration of the virus). They were then weighed daily before gavage administration and their survival was observed twice daily (once during gavage administration and once 12 hours after administration). Changes in mouse weight and survival were recorded.

[0039] Mouse weight changes as follows Figure 4 As shown: The body weight of mice in the DMSO group began to decrease from day 5 after viral attack, while the body weight of mice in the drug-treated group did not show a significant decrease.

[0040] Mouse survival status as follows Figure 5 As shown, mice in the DMSO group began to die on day 6 after viral challenge, with a mortality rate reaching 67% by day 11; all mice in the drug-treated group survived. The results indicate that terbanumin is effective against chikungunya virus infection in mice, providing both prevention and treatment.

[0041] The above in vitro and in vivo experimental results both indicate that terbanumin has significant activity against chikungunya virus infection and can be used to prepare drugs against chikungunya virus infection for the prevention and treatment of chikungunya virus infection.

[0042] The foregoing has shown and described the main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of terbanumin in the preparation of drugs against Chikungunya virus infection.

2. Application of terbanumin in the preparation of drugs for the prevention of chikungunya virus infection.

3. Application of terbanumin in the preparation of drugs for treating chikungunya virus infection.

4. Application of terbanumin in the preparation of drugs that inhibit viral infection of human hepatocellular carcinoma cells Huh7.

5. Application of terbanumin in the preparation of drugs that inhibit viral infection of hippocampal neurons at HT22.

6. The application as described in any one of claims 1-5, characterized in that: The drug is a drug in which terbanum is the sole active ingredient, or a drug composition containing terbanum.

7. The application as described in claim 6, characterized in that: The pharmaceutical composition containing terbanum refers to a pharmaceutical composition consisting of terbanum and one or more pharmaceutically permissible excipients.

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