Application of sulinate trifluoromethyl selenium in preparation of anti-enterovirus drugs

Abstract

The invention discloses application of sulindac ester trifluoromethyl selenium in preparation of anti-enterovirus drugs, and belongs to the technical field of biological medicines. It is found for the first time that sulindaate trifluoromethyl selenium can significantly inhibit virus titers of enterovirus 71 and coxsackie virus A16, can significantly inhibit virus replication of the enterovirus 71 and coxsackie virus A16, and can significantly inhibit 3D protein expression of the enterovirus 71 and VP1 protein expression of the coxsackie virus A16. And the inhibition is dose-dependent. Therefore, the sulindac ester trifluoromethyl selenium has good anti-enterovirus activity, and a new thought and scheme are provided for development and clinical treatment of anti-enterovirus drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to the application of sulindac acid ester trifluoromethyl selenium in the preparation of anti-enterovirus drugs. Background Art

[0002] Enterovirus is a type of virus with small particles, icosahedral, 24~30nm in diameter, no lipids, single-stranded RNA in the core, resistance to ether and other lipid solvents, acid resistance, and resistance to various antibiotics, antiviral drugs, and detergents. Most enteroviruses produce cytopathic effects in cell culture. Enterovirus belongs to the Picornaviridae family and is a naked virus. Different enteroviruses can cause the same symptoms, and the same virus can cause different clinical manifestations. Enteroviruses are often latent infections, which can cause symptoms such as mild upper respiratory tract infection, abdominal discomfort, and diarrhea. Occasionally, they invade the central nervous system and cause flaccid paralysis.

[0003] Enterovirus 71 (EV71) and Coxsackievirus A16 (CA16) are the main pathogens causing hand, foot and mouth disease in infants and young children in the Asia-Pacific region. Although EV71 vaccines are available, they have no cross-protection against other enteroviruses. Hand, foot and mouth disease is still a very serious public health problem, but there are no effective preventive measures or treatments so far, so the search for antiviral drugs is urgent.

[0004] At present, there is no report that sulindac ester trifluoromethyl selenium has anti-enteroviral efficacy. Summary of the invention

[0005] The purpose of the present invention is to provide an application of sulindac acid ester trifluoromethyl selenium in the preparation of anti-enteroviral drugs. The present invention firstly finds that sulindac acid ester trifluoromethyl selenium can significantly inhibit the viral titer of enterovirus 71 and coxsackievirus A16, has good anti-enteroviral activity, and provides 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 sulindac acid ester trifluoromethyl selenium in the preparation of anti-enteroviral drugs, wherein sulindac acid ester trifluoromethyl selenium has a structure shown in the following formula (I): .

[0007] In the application of the sulindac acid ester trifluoromethyl selenium provided by the present invention in the preparation of anti-enteroviral drugs, the sulindac acid ester trifluoromethyl selenium is prepared according to the method in the reference (Nie Yousong. Synthesis and anti-tumor activity study of novel non-steroidal anti-inflammatory drug organic selenium derivatives [D]. Wuhan University of Technology, 2023.).

[0008] In the present invention, the inventors discovered for the first time that sulindac acid ester trifluoromethyl selenium can inhibit the viral titer of enterovirus and has good anti-enterovirus activity.

[0009] In some embodiments, the enterovirus includes at least one of enterovirus 71 and coxsackievirus A16.

[0010] It is understood that the enterovirus may include a variety of viruses in the prior art. For example, in the present invention, the enterovirus preferably includes at least one of enterovirus 71 and coxsackievirus A16. 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.

[0011] In some embodiments, the enterovirus protein includes at least one of the 3D protein of enterovirus type 71 and the VP1 protein of coxsackievirus type A16.

[0012] In a second aspect, the present invention provides a pharmaceutical composition comprising the above-mentioned sulindac ester trifluoromethyl selenium.

[0013] In some embodiments, the pharmaceutical composition further includes a pharmaceutically acceptable carrier.

[0014] In the present invention, the term "pharmaceutically acceptable carrier" refers to an adjuvant widely used in the field of drug production. The adjuvant is mainly used to provide a safe, stable and functional pharmaceutical composition, and can also provide a method to dissolve the active ingredient at a desired rate after the subject receives the administration, or promote the effective absorption of the active ingredient after the subject receives the composition. Pharmaceutical adjuvants can be inert fillers, or provide a certain function, such as stabilizing the overall pH value of the composition or preventing the degradation of the active ingredient of the composition. Pharmaceutical adjuvants can include one or more of the following adjuvants: binders, suspending agents, emulsifiers, diluents, fillers, granulating agents, adhesives, disintegrants, lubricants, anti-adhesive agents, glidants, wetting agents, gelling agents, absorption delay agents, dissolution inhibitors, enhancers, adsorbents, buffers, chelating agents, preservatives, colorants, flavoring agents and sweeteners.

[0015] The pharmaceutical composition provided by the present invention can be prepared by any method known to those skilled in the art according to the disclosed content, including but not limited to conventional mixing, dissolving, granulating, emulsifying, grinding, encapsulating, embedding or lyophilizing processes.

[0016] 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.

[0017] In some embodiments, the solid preparation includes at least one of a tablet, a capsule, a powder, a pill, a granule, and a suppository; the semisolid preparation includes at least one of an ointment, a cream, and a patch; and the liquid preparation includes at least one of an injection, an oral solution, a syrup, and an inhalant.

[0018] The pharmaceutical composition provided by the present invention can be administered in any form, including injection (intravenous), mucosal, oral (solid and liquid preparations), inhalation, ocular, rectal, topical or parenteral (infusion, injection, implantation, subcutaneous, intravenous, intraarterial, intramuscular) administration. The pharmaceutical composition of the present invention can also be a controlled release or sustained release dosage form (e.g., liposomes or microspheres). Examples of solid oral preparations include, but are not limited to, powders, capsules, caplets, soft capsules and tablets. Examples of liquid preparations for oral or mucosal administration include, but are not limited to, suspensions, emulsions, elixirs and solutions. Examples of topical preparations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum preparations. Examples of preparations for parenteral administration include, but are not limited to, solutions for injection, dry powder preparations 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.

[0019] 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 a disease caused by enterovirus infection.

[0020] In some embodiments, the enterovirus includes at least one of enterovirus 71 and coxsackievirus A16.

[0021] The beneficial effects of the present invention are as follows: Different from the prior art, the present invention firstly finds that sulindac acid ester trifluoromethyl selenium can significantly inhibit the viral titer of enterovirus 71 and coxsackievirus A16, and can significantly inhibit the viral replication of enterovirus 71 and coxsackievirus A16, and can 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 ester trifluoromethyl selenium has good anti-enterovirus activity, and provides new ideas and solutions for the development of anti-enterovirus drugs and clinical treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the cytotoxicity test results of sulindac acid ester trifluoromethyl selenium in Example 1 of the present invention; Figure 2 This is a fluorescence image of sulindac ester trifluoromethyl selenium inhibiting enterovirus type 71 in Example 2 of the present invention; Figure 3 This is a flow cytometric test result of sulindac acid ester trifluoromethyl selenium inhibiting enterovirus type 71 in Example 2 of the present invention; Figure 4A This is a graph showing the results of sulindac ester trifluoromethyl selenium inhibiting the RNA expression of enterovirus 71 in Example 2 of the present invention; Figure 4B This is a western blot result diagram of the inhibition of 3D protein expression of enterovirus 71 by sulindacyl trifluoromethyl selenide in Example 2 of the present invention; Figure 5 This is a graph showing the western blot results of sulindac ester trifluoromethyl selenium inhibiting the expression of VP1 protein of Coxsackievirus A16 in Example 3 of the present invention. DETAILED DESCRIPTION

[0023] 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0024] The experimental methods without specific conditions in the examples are usually carried out according to conventional conditions and conditions described in the manual, or according to conditions recommended by the manufacturer. The general equipment, materials, reagents, etc. used are all available from commercial channels unless otherwise specified.

[0025] Example 1 Cytotoxicity test of sulindacyl trifluoromethyl selenium This example tests the cytotoxicity of sulindacyl trifluoromethyl selenium.

[0026] Specifically, RD cells were plated in 96-well plates and incubated at 37°C and 5% CO 2 After the cells were fully grown in the incubator, the cell culture medium was discarded and different concentrations (0, 6.25, 12.5, 25, 50, 100, 150, 200 μM) of sulindacyl trifluoromethyl selenium were added. The cells were then cultured in the incubator. Samples were taken after 24 hours and the cell survival rate was determined using the CCK8 method. The results are shown in Table 1. Figure 1 shown.

[0027] from Figure 1 It can be seen that sulindac acid ester trifluoromethyl selenium has low toxicity to cells and has good biosafety.

[0028] Example 2 Test of Sulindac Acid-Based Trifluoromethyl Selenium against Enterovirus Type 71 2.1 Test of Sulindac Acid-Based Trifluoromethyl Selenium Anti-Enterovirus 71 Activity RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP virus (kindly donated by Wuhan Institute of Virology, Chinese Academy of Sciences) with MOI=1 was added to infect RD cells. After 2 hours, the culture medium was discarded and different concentrations of sulindole trifluoromethyl selenium (12.5μM, 25μM and 50μM) were added to treat 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 from Figure 2 and 3 It can be seen that after adding sulindac acid ester trifluoromethyl selenium, the detected green fluorescence signal becomes weaker, and as the concentration of sulindac acid ester trifluoromethyl selenium increases, the detected green fluorescence signal becomes weaker. The results show that in the present invention, sulindac acid ester trifluoromethyl selenium can significantly inhibit the viral titer of enterovirus type 71, and is dose-dependent.

[0029] 2.2 Sulindac ester-based trifluoromethyl selenium inhibits the replication of enterovirus 71 RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP virus (kindly donated by Wuhan Institute of Virology, Chinese Academy of Sciences) with MOI=1 was added to infect RD cells. After 2 hours, the culture medium was discarded, and different concentrations of sulindole trifluoromethyl selenium (12.5μM, 25μM and 50μM) were added to treat RD cells. After 24 hours, the cells were collected, and then the total RNA in each treatment group was extracted using Trizol reagent, and the corresponding cDNA was synthesized using M-MLV reverse transcriptase. qPCR primers for specific detection of EV71 virus and primers for the internal reference gene GAPDH were designed, and qPCR was used to detect the expression of EV71 virus RNA in cells. The results are shown in Figure 2. Figure 4A shown.

[0030] Among them, 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: EV71-F: 5′-GCAGCCCAAAAGAACTTCAC-3′ (SEQ ID NO: 1); EV71-R: 5′-ATTTCAGCAGCTTTGGAGTGC-3′ (SEQ ID NO: 2); GAPDH-F: 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO: 3); GAPDH-R: 5′-GAAGATGGGTGATGGGATTTCC-3′ (SEQ ID NO: 4).

[0031] from Figure 4A It can be seen that as the concentration of sulindac acid ester trifluoromethyl selenium increases, the RNA expression of enterovirus 71 is significantly reduced. The results show that sulindac acid ester trifluoromethyl selenium can significantly inhibit the replication of enterovirus 71, and is dose-dependent.

[0032] 2.3 Inhibition of enterovirus 71 viral protein expression by sulindacyl trifluoromethyl selenide RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, EV71-GFP virus (kindly donated by Wuhan Institute of Virology, Chinese Academy of Sciences) with MOI=1 was added to infect RD cells. After 2 hours, the culture medium was discarded, and different concentrations of sulindole trifluoromethyl selenium (12.5μM, 25μM and 50μM) and 20μM of the broad-spectrum antiviral drug ribavirin were added to treat RD cells respectively. After 24 hours, the culture supernatant was discarded, the cells were collected after trypsin digestion, and the cells were resuspended with cell lysis buffer and placed on ice for 20 minutes. The supernatant was collected after centrifugation at 4℃ and 13000×g for 20 minutes, and the target band was analyzed by western blot. The results are shown as follows: Figure 4B shown.

[0033] from Figure 4B It can be seen that compared with the high expression of EV71 3D protein in the EV71-GFP virus infection group and the low expression of EV71 3D protein in the positive control group (ribavirin treatment group), in the sulindac acid ester trifluoromethyl selenium treatment group, as the concentration of sulindac acid ester trifluoromethyl selenium increased, the expression level of EV71 3D protein was significantly reduced. The results showed that sulindac acid ester trifluoromethyl selenium could significantly inhibit the expression of EV71 3D protein in a dose-dependent manner.

[0034] Example 3 Inhibition of Coxsackievirus A16 Viral Protein Expression by Sulindate-Based Trifluoromethyl Selenium RD cells were seeded in 12-well plates. The next day, when the cells grew to 90%, the CA16 virus (isolated from clinical sites) with an MOI of 1 was added 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. The results are shown in the figure. Figure 5 shown.

[0035] from Figure 5It 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 acid ester trifluoromethyl selenium treatment group, as the concentration of sulindac acid ester trifluoromethyl selenium increased, the expression level of VP1 protein of Coxsackievirus A16 was significantly reduced. The results showed that sulindac acid ester trifluoromethyl selenium can significantly inhibit the expression of VP1 protein of Coxsackievirus A16, and it is dose-dependent.

[0036] In summary, the present invention discovered for the first time that sulindac acid ester trifluoromethyl selenide can significantly inhibit the viral titers of enterovirus type 71 and coxsackievirus type 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.

[0037] 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 matters that are not described in detail in some embodiments, reference may be made to the description in other embodiments.

[0038] The above-mentioned embodiments only express the implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. The use of sulindac acid ester trifluoromethyl selenium in the preparation of anti-enteroviral drugs, characterized in that: The sulindac acid ester trifluoromethyl selenium has a structure shown in the following formula (I): 。 2. The use according to claim 1, characterized in that: The enterovirus comprises at least one of enterovirus 71 and coxsackievirus A16.

3. The use according to claim 1, characterized in that: The sulindac ester trifluoromethyl selenium 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.

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

5. A pharmaceutical composition, characterized in that It includes the sulindac acid ester trifluoromethyl selenium as claimed in claim 1.

6. The pharmaceutical composition according to claim 5, characterized in that Also included are pharmaceutically acceptable carriers.

7. The pharmaceutical composition according to claim 5, characterized in that The dosage form of the pharmaceutical composition includes at least one of a solid preparation, a semisolid preparation, and a liquid preparation.

8. The pharmaceutical composition according to claim 7, characterized in that 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 liquids, syrups, and inhalants.

9. Use of the pharmaceutical composition according to any one of claims 5 to 8 in the preparation of a medicament for preventing and / or treating diseases infected by enterovirus.

10. The use according to claim 9, characterized in that: The enterovirus comprises at least one of enterovirus 71 and coxsackievirus A16.

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