Application of Eltrombopag in the preparation of drugs for preventing and treating rotavirus

Abstract

The present invention belongs to the field of biotechnology, in particular to the application of Eltrombopag in the preparation of drugs for preventing and treating rotavirus. The CC in cells infected with rotavirus and healthy cells is measured. 50 Value and IC 50 The study found that eltrombopag can act as a rotavirus inhibitor, demonstrating excellent antiviral effects in rotavirus-infected cells. Further immunofluorescence analysis revealed that eltrombopag at a concentration of 2.5-10 μmol / L effectively inhibited rotavirus replication in cells, providing a new medicinal raw material for the prevention and treatment of rotavirus.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to application of Eltrombopag in preparing medicines for preventing and treating rotavirus. Background Art

[0002] Rotavirus (RV), a non-enveloped, double-stranded RNA virus of the Reoviridae family, is the most important cause of severe, potentially life-threatening viral gastroenteritis. Worldwide, rotavirus infection affects nearly all children under five years of age, the elderly, and those with chronic illnesses. Rotavirus typically replicates in mature intestinal cells and enters the bloodstream, causing antigenemia and viremia. It infects 130 million children and kills over 200,000 people annually, causing significant losses to the livestock industry. Currently, two vaccines are available in China: live oral rotavirus vaccine (Lanzhou Institute of Biological Products), launched in China in 2001, and RotaTeq™ (Merck Sharp & Dohme, USA), launched in China in 2018. Although the introduction of rotavirus immunization programs has reduced mortality by 60%, vaccine efficacy is low in developing countries. Currently, oral and intravenous rehydration remain the mainstays of rotavirus treatment. However, these treatments are symptomatic and fail to mitigate the duration and severity of diarrhea. Therefore, the development of specific anti-rotavirus drugs is urgently needed.

[0003] Eltrombopag, a US Food and Drug Administration-approved drug, is used to treat immunocompromised patients with low platelet counts infected with hepatitis C virus (HCV). Its safety and side effects have been thoroughly evaluated and demonstrated in clinical practice, with no reports of serious neurological side effects. Furthermore, eltrombopag is a potent autophagy inhibitor targeting TFEB and can also induce apoptosis by directly activating BAK. Eltrombopag has also demonstrated significant efficacy against pathogenic infections. For example, eltrombopag exhibits potent inhibitory effects against multidrug-resistant Staphylococcus aureus and Salmonella typhi. Furthermore, studies have demonstrated eltrombopag's antiviral activity against a variety of viruses, including fever with thrombocytopenia syndrome virus (SFTSV), severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and hepatitis C virus (HCV).

[0004] Rotavirus is a zoonotic disease that infects 130 million children and kills more than 200,000 people each year, and causes significant losses to the livestock industry. However, there is no specific drug to treat rotavirus clinically at home and abroad. Therefore, research on therapeutic drugs for rotavirus has important scientific and practical significance. Summary of the Invention

[0005] In order to solve the above technical problems, the present invention provides the use of Eltrombopag in the preparation of a medicament for preventing and treating rotavirus.

[0006] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0007] One of the technical solutions of the present invention is the use of eltrombopag as the sole active ingredient in the preparation of a drug for preventing and treating rotavirus.

[0008] The second technical solution of the present invention is a drug for preventing and treating rotavirus, comprising eltrombopag and, if necessary, a pharmaceutically acceptable carrier and / or excipient.

[0009] Preferably, the dosage form of the drug is one of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

[0010] Compared with the prior art, the present invention measures the CC of cells infected with rotavirus. 50 Value and IC 50 values, and found that eltrombopag could be an inhibitor of rotavirus; further immunofluorescence showed that eltrombopag had a good inhibitory effect on rotavirus at a concentration of 2.5-10 µmol / L, and when the concentration reached 10 µmol / L, it almost completely inhibited the replication of rotavirus, providing a new candidate drug for the prevention and treatment of rotavirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 Eltrombopag inhibits CC of rotavirus infection 50 and IC 50 Value determination diagram: A is CC 50 Determination diagram; B is IC 50 Determination diagram.

[0012] Figure 2 Figure 3 shows the inhibitory effect of eltrombopag on the rotavirus genome copy number: A is the rotavirus genome copy number in Caco2 cells; B is the rotavirus genome copy number in MA104 cells.

[0013] Figure 3 Figure 3 shows the inhibitory effect of eltrombopag on the expression of rotavirus VP6 protein: A shows the expression of rotavirus VP6 protein in Caco2 cells; B shows the expression of rotavirus VP6 protein in MA104 cells.

[0014] Figure 4 Figure 3 shows the results of eltrombopag inhibiting rotavirus immunofluorescence observation: A is Caco2 cell rotavirus immunofluorescence; B is MA104 cell rotavirus immunofluorescence. DETAILED DESCRIPTION

[0015] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0016] The Caco2 cells used in the following examples are maintained by the Guangdong Provincial Key Laboratory of Animal Molecular Design and Precision Breeding. Caco2 cells are a cell line derived from human colon adenocarcinoma that can spontaneously differentiate into a polar monolayer similar to small intestinal epithelial cells and are an optimal in vitro model for studying anti-rotavirus drugs. All other relevant reagents used, unless otherwise specified, are commercially available, and the methods involved, unless otherwise specified, are all well-known methods.

[0017] The compound Eltrombopag was purchased from Selleck, and its chemical molecular structure is shown below:

[0018] .

[0019] The rotavirus strains used were: rotavirus vaccine strain LLR (G10P

[15] ), which was maintained by the Viral Diarrhea Laboratory of the Chinese Center for Disease Control and Prevention and subcultured on MA104 cells; MA104 cells (African green monkey embryonic kidney cells), which were maintained by the Viral Diarrhea Laboratory of the Chinese Center for Disease Control and Prevention.

[0020] In order to verify whether Eltrombopag can be used as the sole active ingredient to prepare drugs for the prevention and treatment of rotavirus, the following experiments were conducted:

[0021] 1. Toxicity of Eltrombopag in Caco2 cells

[0022] Caco2 cells were plated in 96-well plates (100 μL of cell suspension per well) using DMEM medium supplemented with 10% FBS, 1% amphotericin, and 1% gentamicin, with 0.1 mL of medium added per well. The cells were cultured to a cell density of approximately 70%. Eltrombopag was diluted with culture medium to obtain concentrations of 1 μM, 2.5 μM, 5 μM, 10 μM, and 20 μM eltrombopag solutions, designated as the experimental drug groups. A blank control group was also established. 10 μL of each experimental drug concentration was added to each well, and the cells were incubated at 37°C, 5% CO2, for 24 hours. 10 μL of CCK-8 solution was added to each well, and the cells were incubated in a 37°C incubator for 4 hours in the dark. The absorbance at 450 nm was measured using a microplate reader. Cell viability was calculated using the formula to determine the optimal drug concentration. Cell survival rate = [(As-Ab] / (Ac-Ab)] × 100%, inhibition rate = [(Ac-As] / (Ac-Ab)] × 100%.

[0023] The results are as follows Figure 1 As shown in Figures A and B, eltrombopag solution showed no significant toxicity to Caco2 cells at concentrations below 10 μM. The IC50 value of eltrombopag in Caco2 cells was 4.35 μM, indicating that eltrombopag exerts potent anti-rotavirus activity even at low concentrations. These results suggest that eltrombopag is a potential rotavirus inhibitor.

[0024] 2. Effect of the compound Eltrombopag on rotavirus replication

[0025] Effects on the copy number of rotavirus in cells

[0026] Caco2 and MA104 cells (2×10 6 Cells were inoculated with rotavirus (MOI = 0.01) at 37°C, 5% CO₂ for 2 h. The experimental group was treated with eltrombopag at different concentrations (1, 2.5, 5, and 10 μM). The infection control group was treated with rotavirus (MOI = 0.01) mixed with DMSO. A blank control group remained untreated. The cell plates were incubated at 37°C, 5% CO₂ for 24 h. Total cellular RNA was then extracted using the Omega Viral RNA Extraction Kit (R6874-02) and reverse-transcribed into cDNA.

[0027] Finally, viral genome copy number was determined using fluorescent quantitative PCR targeting the rotavirus NSP3 gene. The total volume of the fluorescent quantitative PCR reaction system was 25 μL, consisting of 12.5 μL of Premix Ex Taq (2×), 0.5 μL of upstream and downstream primers, 0.5 μL of probe, 2 μL of template, and sterile deionized water to 25 μL. The upstream primer sequence was 5'-ACCATCTWCACRTRACCCTCTATGAG-3'; the downstream primer sequence was 5'-GGTCACATAACGCCCCTATAGC-3'; and the probe sequence was 5'-FAM-AGTTAAAAGCTAACACTGTCAAA-MGB3'.

[0028] The viral copy number calculation results of Caco2 and MA104 cells after rotavirus infection and treatment with different concentrations of Eltrombopag are as follows: Figure 2 As shown in A and B. The results showed that in Caco2 and MA104 cells, compared with the control group, the rotavirus genome copy number gradually decreased with the increase of eltrombopag concentration, indicating that eltrombopag can inhibit rotavirus replication in a dose-dependent manner.

[0029] Effects on the expression of rotavirus VP6 protein in cells

[0030] Caco2 and MA104 cells (2×10 6 The experimental group was inoculated with rotavirus (MOI = 0.01) and adsorbed for 2 h under 5% CO2. The cells were then treated with different concentrations of eltrombopag (1, 2.5, 5, and 10 μM). The infection control group was treated with rotavirus (MOI = 0.01) mixed with DMSO. The blank control group received no treatment. The cell plates were incubated at 37°C, 5% CO2 for 24 h, the cell culture supernatant was discarded, the cells were rinsed three times with PBS, and 1× SDS-PAGE loading buffer was added for protein denaturation (100°C, 10 min). The cells were centrifuged for 10 min (12,000 rpm), homogenized, and loaded into the sample tank of the gel. Electrophoresis (120 V, 30 min) was performed. After the bromophenol blue dye fell to the edge of the gel, the membrane was transferred to a 0.2 μm nitrocellulose membrane (NC membrane) (100 V, 1 h). The membrane was blocked with 5% skim milk powder (1 h), the skim milk powder was discarded, and the NC membrane was washed with 1× TBST. The primary antibody dilution (murine rotavirus VP6 monoclonal antibody, 1:1000 dilution) was added and the membrane was refrigerated at 4°C overnight. The NC membrane was washed with 1× TBST (5 times / 5 min), the corresponding secondary antibody dilution was added (incubated for 1 h), and the membrane was washed with 1× TBST (5 times / 5 min). Finally, the membrane was analyzed by ECL. The luminescent liquid was exposed to detect the expression of rotavirus VP6 protein.

[0031] The results are as follows Figure 3 As shown in Figures A and B, in Caco2 and MA104 cells, compared with the control group, as the concentration of eltrombopag solution increased, the expression of rotavirus VP6 viral protein decreased, indicating that eltrombopag can inhibit rotavirus replication in a dose-dependent manner.

[0032] Effects on rotavirus immunofluorescence in cells

[0033] Caco2 cells (2 × 10 6The experimental group was inoculated with rotavirus (MOI = 0.01) and, after 2 hours of adsorption under 5% CO₂, treated with different concentrations of eltrombopag (1, 2.5, 5, and 10 μM). The infection control group was treated with rotavirus (MOI = 0.01) mixed with DMSO (referred to as the DMSO group). The blank control group remained untreated (referred to as the mock group). The cell plates were incubated at 37°C and 5% CO₂ for 24 hours. The cell culture supernatant was discarded, and the cells were rinsed three times with PBS and fixed with 4% paraformaldehyde for 30 minutes. The plates were then permeabilized with 0.3% Triton X-ray for 10 minutes. Blocking was performed with 5% BSA for 1 hour at room temperature. The blocking buffer was then discarded, and the primary antibody diluted in 5% BSA (mouse rotavirus VP6 monoclonal antibody, 1:200 dilution) was added directly and incubated overnight at 4°C (protected from light). Fluorescein isothiocyanate (FITC)-conjugated secondary antibody was then incubated at 37°C for 1 hour. After incubation, the antibody was discarded and the cells were washed three times with PBST before observation under an inverted fluorescence microscope.

[0034] The results are as follows Figure 4 As shown in Figures A and B, in Caco2 and MA104 cells, compared with the control group, as the concentration of eltrombopag solution increased, the rotavirus fluorescence decreased significantly, indicating that eltrombopag can inhibit rotavirus replication in a dose-dependent manner.

[0035] In summary, eltrombopag can be used as the sole active ingredient in the preparation of drugs for the prevention and treatment of rotavirus. Of course, the prepared drugs contain eltrombopag and also contain pharmaceutically acceptable carriers and / or excipients; the dosage form of the drugs is one of tablets, sprays, granules, capsules, oral solutions, injections, and suspensions.

[0036] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of eltrombopag as the sole active ingredient in the preparation of drugs for the prevention and treatment of rotavirus.

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