Application of sulindacyl trifluoromethyl selenide in the preparation of anti-enteroviral drugs

Abstract

The present invention discloses the use of sulindac acid trifluoromethyl selenide in the preparation of anti-enteroviral drugs, belonging to the field of biomedicine technology. The present invention first discovered that sulindac acid trifluoromethyl selenide can significantly inhibit the viral titer of enterovirus 71 and coxsackievirus A16, and can also significantly inhibit the viral replication of enterovirus 71 and coxsackievirus A16, as well as significantly inhibit the expression of 3D protein of enterovirus 71 and the expression of VP1 protein of coxsackievirus A16, and the above inhibition is dose-dependent. Therefore, the sulindac acid trifluoromethyl selenide has good anti-enteroviral activity, providing new ideas and solutions for the development of anti-enteroviral drugs and clinical treatment.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to the application of sulindacyl trifluoromethyl selenide in the preparation of anti-enteroviral drugs. Background Art

[0002] Enteroviruses are small, icosahedral viruses with a diameter of 24-30 nm. They lack lipids, have single-stranded RNA cores, are resistant to ether and other lipid solvents, are acid-resistant, and are resistant to various antibiotics, antivirals, and detergents. Most enteroviruses produce cytopathic effects in cell culture. Enteroviruses belong to the Picornaviridae family and are naked viruses. Different enteroviruses can cause the same symptoms, and the same virus can cause different clinical manifestations. Enterovirus infections often present as insidious infections, causing symptoms such as a mild upper respiratory tract infection, abdominal discomfort, and diarrhea. They occasionally invade the central nervous system, causing flaccid paralysis.

[0003] Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the primary pathogens causing hand, foot, and mouth disease (HFMD) in infants and young children in the Asia-Pacific region. Although an EV71 vaccine is available, it offers no cross-protection against other enteroviruses. HFMD remains a serious public health problem, yet there are no effective preventive measures or treatments. Therefore, the search for antiviral drugs is urgent.

[0004] At present, there is no report that sulindacyl trifluoromethyl selenide has anti-enteroviral efficacy. Summary of the Invention

[0005] The present invention aims to provide the use of sulindac ester trifluoromethyl selenide in the preparation of anti-enteroviral drugs. The present invention first discovered that sulindac ester trifluoromethyl selenide can significantly inhibit the viral titers of enterovirus 71 and coxsackievirus A16, and has good anti-enteroviral activity, providing new ideas and solutions for the development of anti-enteroviral drugs and clinical treatment.

[0006] In a first aspect, the present invention provides the use of sulindacyl trifluoromethyl selenide in the preparation of an anti-enteroviral drug, wherein sulindacyl trifluoromethyl selenide has a structure shown in the following formula (I):

[0007] .

[0008] In the application of the sulindac acid ester trifluoromethyl selenide provided by the present invention in the preparation of anti-enteroviral drugs, the sulindac acid ester trifluoromethyl selenide is prepared according to the method in the literature (Nie Yousong. Synthesis and anti-tumor activity study of novel non-steroidal anti-inflammatory drug organic selenium derivatives [D]. Wuhan University of Technology, 2023.).

[0009] In the present invention, the inventors discovered for the first time that sulindac ester-based trifluoromethyl selenide can inhibit the viral titer of enterovirus and has good anti-enteroviral activity.

[0010] In some embodiments, the enterovirus comprises at least one of enterovirus 71 and coxsackievirus A16.

[0011] It is understood that enterovirus may include a variety of viruses in the prior art. For example, in the present invention, enterovirus preferably includes at least one of enterovirus 71 and coxsackievirus A16.

[0012] In some embodiments, sulindac ester trifluoromethyl selenide inhibits the proliferation of enterovirus by inhibiting at least one of the viral titer of enterovirus, the replication of enterovirus, and the protein expression of enterovirus.

[0013] In some embodiments, the enterovirus protein includes at least one of the 3D protein of enterovirus 71 and the VP1 protein of coxsackievirus A16.

[0014] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned sulindac ester trifluoromethyl selenide.

[0015] In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0016] In the present invention, the term "pharmaceutically acceptable carrier" refers to an excipient widely used in the field of drug production. Excipients are mainly used to provide a safe, stable and functional pharmaceutical composition. They can also provide methods to dissolve the active ingredient at a desired rate after administration to a subject, or promote effective absorption of the active ingredient after administration of the composition to a subject. Pharmaceutical excipients can be inert fillers, or provide certain functions, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. Pharmaceutical excipients can include one or more of the following excipients: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0017] The pharmaceutical compositions provided herein can be prepared according to the disclosed content using any method known to those skilled in the art, including, but not limited to, conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding, or lyophilizing processes.

[0018] In some embodiments, the dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semisolid preparation, and a liquid preparation.

[0019] In some embodiments, the solid preparation includes at least one of tablets, capsules, powders, pills, granules, and suppositories; the semisolid preparation includes at least one of ointments, creams, and patches; and the liquid preparation includes at least one of injections, oral solutions, syrups, and inhalants.

[0020] The pharmaceutical compositions provided herein can be administered in any form, including intravenous, mucosal, oral (solid and liquid formulations), inhalation, ophthalmic, rectal, topical, or parenteral (infusion, injection, implant, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical compositions of the present invention can also be in controlled-release or sustained-release dosage forms (e.g., liposomes or microspheres). Examples of solid oral formulations include, but are not limited to, powders, capsules, caplets, softgels, and tablets. Examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs, and solutions. Examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops, or serums. Examples of formulations for parenteral administration include, but are not limited to, solutions for injection, dry powder formulations that can be dissolved or suspended in a pharmaceutically acceptable carrier, suspensions for injection, and emulsions for injection. Examples of other suitable formulations of pharmaceutical compositions include, but are not limited to, eye drops and other ophthalmic preparations; aerosols, such as nasal sprays or inhalers; liquid dosage forms suitable for parenteral administration; suppositories; and lozenges.

[0021] In a third aspect, the present invention provides use of any of the above pharmaceutical compositions in the preparation of a medicament for preventing and / or treating diseases caused by enterovirus infection.

[0022] In some embodiments, the enterovirus comprises at least one of enterovirus 71 and coxsackievirus A16.

[0023] The present invention has the following beneficial effects: Unlike the prior art, the present invention first discovered that sulindac ester trifluoromethyl selenide can significantly inhibit the viral titer of enterovirus 71 and coxsackievirus A16, and can also significantly inhibit the viral replication of enterovirus 71 and coxsackievirus A16, as well as the expression of the 3D protein of enterovirus 71 and the expression of the VP1 protein of coxsackievirus A16, and the above inhibition is dose-dependent. Therefore, the sulindac ester trifluoromethyl selenide has good anti-enteroviral activity, providing new ideas and solutions for the development of anti-enteroviral drugs and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram showing the results of a cytotoxicity test of sulindacyl trifluoromethyl selenide in Example 1 of the present invention;

[0025] Figure 2This is a fluorescence image of sulindacyl trifluoromethyl selenide inhibiting enterovirus 71 in Example 2 of the present invention;

[0026] Figure 3 This is a flow cytometric test result of sulindacyl trifluoromethyl selenide inhibiting enterovirus 71 in Example 2 of the present invention;

[0027] Figure 4A This is a graph showing the results of sulindacyl trifluoromethyl selenide inhibiting enterovirus 71 RNA expression in Example 2 of the present invention;

[0028] Figure 4B This is a western blot result showing that sulindac ester trifluoromethyl selenide inhibits the expression of 3D protein of enterovirus 71 in Example 2 of the present invention;

[0029] Figure 5 This is a western blot result showing that sulindac ester trifluoromethyl selenide inhibits the expression of VP1 protein of Coxsackievirus A16 in Example 3 of the present invention. DETAILED DESCRIPTION

[0030] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0031] For experimental methods in the examples where specific conditions are not specified, generally conventional conditions and conditions described in the manual or conditions recommended by the manufacturer were followed. The general equipment, materials, reagents, etc. used were all commercially available unless otherwise specified.

[0032] Example 1 Cytotoxicity Test of Sulindate-Based Trifluoromethyl Selenium

[0033] This example tests the cytotoxicity of sulindacyl trifluoromethyl selenide.

[0034] Specifically, RD cells were plated on a 96-well plate and cultured in a 37°C, 5% CO2 incubator until a monolayer was grown. The cell culture medium was discarded, and different concentrations (0, 6.25, 12.5, 25, 50, 100, 150, 200 μM) of sulindacyl trifluoromethyl selenide were added. The cells were then cultured in the incubator. Samples were taken after 24 hours, and the cell viability was determined using the CCK8 assay. The results are shown in Figure 2. Figure 1 shown.

[0035] from Figure 1It can be seen that sulindac acid ester trifluoromethyl selenide has low toxicity to cells and has good biosafety.

[0036] Example 2: Sulindate-based trifluoromethyl selenium anti-enterovirus type 71 test

[0037] 2.1 Testing of Sulindate-Based Trifluoromethylselenide Anti-Enterovirus 71 Activity

[0038] RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP virus (kindly provided by the Wuhan Institute of Virology, Chinese Academy of Sciences) was added at an MOI of 1 to infect the RD cells. After 2 hours, the culture medium was discarded and different concentrations of sulindole trifluoromethyl selenide (12.5 μM, 25 μM, and 50 μM) were added to treat the RD cells. After 24 hours, the expression of viral green light was observed by fluorescence microscopy and detected by flow cytometry. The results are as follows: Figure 2 and 3 shown

[0039] from Figure 2 and 3 It can be seen that after adding sulindac acid ester trifluoromethyl selenide, the detected green fluorescence signal becomes weaker, and as the concentration of sulindac acid ester trifluoromethyl selenide increases, the detected green fluorescence signal becomes weaker. The results show that in the present invention, sulindac acid ester trifluoromethyl selenide can significantly inhibit the viral titer of enterovirus type 71, and is dose-dependent.

[0040] 2.2 Sulindate-based trifluoromethylselenide inhibits the replication of enterovirus 71

[0041] RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP virus (kindly provided by the Wuhan Institute of Virology, Chinese Academy of Sciences) was added at an MOI of 1 to infect the RD cells. After 2 hours, the culture medium was discarded and different concentrations of sulindole trifluoromethyl selenide (12.5 μM, 25 μM, and 50 μM) were added to treat the RD cells. After 24 hours, the cells were collected and total RNA in each treatment group was extracted using Trizol reagent. The corresponding cDNA was synthesized using M-MLV reverse transcriptase. qPCR primers specifically for detecting EV71 virus and primers for the internal reference gene GAPDH were designed. The expression level of EV71 viral RNA in cells was detected by qPCR. The results are shown in the following table. Figure 4A shown.

[0042] The nucleotide sequences of the qPCR primers for specific detection of EV71 virus and the primers for the internal reference gene GAPDH are as follows:

[0043] EV71-F: 5′-GCAGCCCAAAAGAACTTCAC-3′ (SEQ ID NO: 1);

[0044] EV71-R: 5′-ATTTCAGCAGCTTTGGAGTGC-3′ (SEQ ID NO: 2);

[0045] GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO: 3);

[0046] GAPDH-R: 5′-GAAGATGGGTGATGGGATTTCC-3′ (SEQ ID NO: 4).

[0047] from Figure 4A It can be seen that as the concentration of sulindac acid trifluoromethyl selenide increases, the RNA expression level of enterovirus 71 is significantly reduced. The results show that sulindac acid trifluoromethyl selenide can significantly inhibit the replication of enterovirus 71 in a dose-dependent manner.

[0048] 2.3 Inhibition of enterovirus 71 viral protein expression by sulindacyl trifluoromethyl selenide

[0049] RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP virus (kindly provided by the Wuhan Institute of Virology, Chinese Academy of Sciences) was added at an MOI of 1 to infect the RD cells. After 2 hours, the culture medium was discarded, and different concentrations of sulindole trifluoromethyl selenide (12.5 μM, 25 μM, and 50 μM) and 20 μM of the broad-spectrum antiviral drug ribavirin were added to treat the RD cells. After 24 hours, the culture supernatant was discarded, and the cells were collected after trypsin digestion and resuspended in cell lysis buffer. The cells were placed on ice for 20 minutes, centrifuged at 4°C and 13,000×g for 20 minutes, and the supernatant was collected. The target bands were analyzed by western blot. The results are as follows. Figure 4B shown.

[0050] from Figure 4B It can be seen that compared with the high expression of enterovirus 71 3D protein in the EV71-GFP virus infection group and the low expression of enterovirus 71 3D protein in the positive control group (ribavirin treatment group), in the sulindac-based trifluoromethylselenium treatment group, as the concentration of sulindac-based trifluoromethylselenium increased, the expression level of enterovirus 71 3D protein significantly decreased. The results showed that sulindac-based trifluoromethylselenium can significantly inhibit the expression of enterovirus 71 3D protein in a dose-dependent manner.

[0051] Example 3 Inhibition of Coxsackievirus A16 Viral Protein Expression by Sulindate-Based Trifluoromethyl Selenide

[0052] RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, CA16 virus (isolated from a clinical setting) was added at an MOI of 1 to infect the RD cells. After 2 hours, the culture medium was discarded and different concentrations of sulindole trifluoromethyl selenide (12.5 μM, 25 μM, and 50 μM) and 20 μM ribavirin were added to treat the RD cells. After 24 hours, the target bands were analyzed by western blot analysis. The results are as follows: Figure 5 shown.

[0053] from Figure 5 It can be seen that compared with the high expression of VP1 protein of Coxsackievirus A16 in the CA16 virus infection group and the low expression of VP1 protein of Coxsackievirus A16 in the positive control group (ribavirin treatment group), in the sulindac-based trifluoromethylselenium treatment group, as the concentration of sulindac-based trifluoromethylselenium increased, the expression of VP1 protein of Coxsackievirus A16 was significantly reduced. The results showed that sulindac-based trifluoromethylselenium can significantly inhibit the expression of VP1 protein of Coxsackievirus A16, and is dose-dependent.

[0054] In summary, the present invention discovered for the first time that sulindacyl trifluoromethyl selenide can significantly inhibit the viral titers of enterovirus 71 and coxsackievirus A16 in a dose-dependent manner, and has good anti-enteroviral activity, providing new ideas and solutions for the development of anti-enteroviral drugs and clinical treatment.

[0055] It should be noted that the above embodiments all belong to the same inventive concept, and the description of each embodiment has its own focus. For any details not described in individual embodiments, reference may be made to the description in other embodiments.

[0056] The above-described embodiments merely illustrate the implementation methods of the present invention. While the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make various modifications and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. The use of sulindacyl trifluoromethyl selenide in the preparation of anti-enteroviral drugs, characterized in that: The sulindacyl trifluoromethyl selenide has a structure shown in the following formula (I): ; The enterovirus is at least one of enterovirus 71 and coxsackievirus A16.

2. The use according to claim 1, characterized in that The sulindac ester trifluoromethyl selenide inhibits the proliferation of the enterovirus by inhibiting at least one of the viral titer of the enterovirus, the replication of the enterovirus, and the protein expression of the enterovirus.

3. The use according to claim 2, characterized in that The enterovirus protein is at least one of the 3D protein of enterovirus 71 and the VP1 protein of coxsackievirus A16.

4. The use according to claim 1, characterized in that The anti-enteroviral drug further comprises a pharmaceutically acceptable carrier.

5. The use according to claim 1, characterized in that The dosage form of the anti-enteroviral drug includes at least one of a solid preparation, a semi-solid preparation, and a liquid preparation.

6. The use according to claim 5, characterized in that The solid preparation includes at least one of tablets, capsules, powders, pills, granules, and suppositories; the semi-solid preparation includes at least one of ointments, creams, and patches; and the liquid preparation includes at least one of injections, oral solutions, syrups, and inhalants.

Images