Application of compound NMI 8739 in preparation of medicine for preventing or treating porcine epidemic diarrhea

Abstract

The invention belongs to the technical field of animal epidemic disease prevention and control, and particularly relates to application of a compound NMI 8739 in preparation of a medicine for preventing or treating porcine epidemic diarrhea. It is accidentally found that the compound NMI 8739 can inhibit PEDV proliferation, has a certain inhibition effect on PEDV, can inhibit infection and amplification of PEDV in cells, can be used for preparing drugs for preventing or treating porcine epidemic diarrhea, lays a certain technical foundation for prevention and control of PED, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the technical field of prevention and control of animal diseases, and particularly relates to the application of a compound NMI 8739 in the preparation of a drug for preventing or treating porcine epidemic diarrhea. Background Art

[0002] Porcine epidemic diarrhea (PED) is a viral infectious disease caused by Porcine epidemic diarrhea virus (PEDV). Diseased pigs mainly show clinical symptoms such as diarrhea, vomiting, anorexia, dehydration, and weight loss. Pigs of all ages can be infected by PEDV and show different degrees of disease characteristics. Generally, adult pigs show mild infection symptoms or no symptoms, while piglets are mostly severely infected, and the fatality rate can be as high as 100%, causing huge economic losses to the live pig breeding industry. Due to the relatively fast mutation of PEDV, the prevention and control effect of vaccines is relatively limited. Therefore, the development of relevant preventive and therapeutic drugs is of great technical significance for the prevention and control of PED.

[0003] Compound NMI 8739 is an agonist of dopamine D2 autoreceptor and an amine conjugate of docosahexaenoic acid and the neurotransmitter dopamine. Currently, laboratory research mainly focuses on neurological diseases, and there are no reports related to viruses.

[0004] The present invention unexpectedly discovers that compound NMI 8739 can significantly inhibit the replication of PEDV, reduce the PEDV titer, can be used to prepare a drug for anti-PEDV infection, and is used for preventing or treating PED, with broad application prospects. Summary of the Invention

[0005] In view of the above technical problems, the present invention unexpectedly discovers that compound NMI 8739 can significantly inhibit the replication of PEDV and can be used to prepare a drug for anti-PEDV infection. Specifically, it includes the following contents:

[0006] In a first aspect, the present invention provides the application of a compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, and the structural formula of the compound NMI 8739 is shown as the following formula (Ⅰ):

[0007]

[0008] Preferably, the virus is Porcine epidemic diarrhea virus.

[0009] Preferably, the compound NMI 8739 or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0010] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0011] In a second aspect, the present invention provides the use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing porcine epidemic diarrhea, and the structural formula of the compound NMI 8739 is shown as the following formula (I):

[0012]

[0013] Preferably, the compound NMI 8739 or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0014] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0015] In a third aspect, the present invention provides the use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating porcine epidemic diarrhea, and the structural formula of the compound NMI 8739 is shown as the following formula (I):

[0016]

[0017] Preferably, the compound NMI 8739 or a pharmaceutically acceptable salt thereof is added with a pharmaceutically acceptable carrier and / or excipient to form any pharmaceutically acceptable dosage form.

[0018] Preferably, the dosage form includes tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

[0019] The beneficial effect of the present invention is that: the present invention unexpectedly discovers that the compound NMI 8739 can significantly inhibit the replication and proliferation of PEDV, can be used to prepare drugs against PEDV infection, for preventing or treating PED, and has broad application prospects. Description of the Drawings

[0020] Figure 1 Effect of pretreating PEDV virions with gradient concentrations of compound NMI 8739 on PEDV N RNA in Vero cells;

[0021] Figure 2 Effect of simultaneously adding gradient concentrations of compound NMI 8739 and PEDV virions on PEDV N RNA in Vero cells;

[0022] Figure 3Effect of post - treatment of PEDV virus particles with gradient - concentration compound NMI 8739 on PEDV N RNA in Vero cells.

[0023] Figure 4 Effect of treatment with compound NMI 8739 on the viability of Vero cells. Detailed implementation manners

[0024] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

[0025] The PEDV strain is the G I - type strain CH / SX / 2015, GenBank No. MT783684, which is independently isolated and preserved by the Animal Immunization and Metabolism Innovation Team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0026] Example 1 Detection of PEDV N RNA after pretreatment of PEDV virus particles with gradient - concentration NMI 8739

[0027] 1. Method

[0028] 1) Digest and passage Vero cells, and inoculate them at 1×10 5 cells / mL into a 12 - well cell culture plate at 1 mL / well. Culture in an incubator at 37°C and 5% CO2.

[0029] 2) When the cell density reaches about 70%, use compound NMI8739 at 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1 mM, 2 mM respectively and 10 4 TCID 50 of PEDV virus particles, and co - incubate at 37°C for 30 min. At this time, the volume is 10 μL.

[0030] 3) Use the treated PEDV to infect the cells for 1 h. At this time, the final concentrations of compound NMI8739 are 20 nM, 40 nM, 80 nM, 160 nM, 320 nM, 625 nM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM respectively. After 1 h, replace the medium with fresh DMEM medium containing 5% serum, and collect the cells 12 h after virus infection.

[0031] 4) Use trizol lysis solution to lyse and extract the RNA in the cells. The detailed operation is carried out according to the trizol instruction manual.

[0032] 5) Reverse transcribe the extracted RNA in an amount of 500 ng / 10 μL, and the detailed operation is carried out according to the reverse transcription kit instructions.

[0033] 6) Perform real-time fluorescence quantitative PCR detection on the reverse-transcribed samples. The detailed operation is carried out according to the fluorescence quantitative PCR polymerase instructions, and the information of the fluorescence quantitative primers is as follows:

[0034] PEDV N-F: TGGTGGCTGCTGTCAAGG,

[0035] PEDV N-R: TTTTCGACAAATTCCGCAT;

[0036] Actin-F: ATCGTGCGTGACATTAAG,

[0037] Actin-R: ATTGCCAATGGTGATGAC.

[0038] 2. Results

[0039] The results are as Figure 1 shown. Real-time fluorescence quantitative PCR detection shows that at 12 h after PEDV infection, the PEDV N RNA in the compound NMI 8739 pretreatment group was significantly lower than that in the control group, and showed a concentration gradient-dependent inhibition of PEDV infection.

[0040] Example 2 Detection of PEDV N RNA by simultaneous addition of gradient concentrations of NMI 8739 and PEDV virus particles 1. Method

[0041] 1) Digest and passage Vero cells, and inoculate them into a 12-well cell culture plate at 1×10 5 cells / mL at 1 mL / well. Culture in a 37 °C, 5% CO2 incubator.

[0042] 2) When the cell density reaches about 70%, premix with compound NMI8739 at 62.5 μM, 125 μM, 250 μM, 500 μM, 1 mM and 10 4 TCID 50 of PEDV virus particles, and the volume is 10 μL at this time.

[0043] 3) Directly add the premixed virus solution to the cell supernatant to infect the cells for 1 h. At this time, the final concentrations of compound NMI8739 are 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM respectively.

[0044] 4) After 1 h, replace the culture medium with fresh DMEM medium containing 5% serum, and collect the cells 12 h after virus infection.

[0045] 5) Use trizol lysis solution to lyse and extract RNA from the cells, and perform the detailed operation according to the trizol instruction manual.

[0046] 6) Reverse transcribe the extracted RNA in an amount of 500 ng / 10 μL, and perform the detailed operation according to the reverse transcription kit instruction manual.

[0047] 7) Perform real-time fluorescence quantitative PCR detection on the reverse-transcribed samples. The detailed operation is carried out according to the fluorescence quantitative PCR polymerase instruction manual. The information of the fluorescence quantitative primers is as follows:

[0048] PEDV N-F: TGGTGGCTGCTGTCAAGG,

[0049] PEDV N-R: TTTTCGACAAATTCCGCAT;

[0050] Actin-F: ATCGTGCGTGACATTAAG,

[0051] Actin-R: ATTGCCAATGGTGATGAC.

[0052] 2. Results

[0053] The results are as Figure 2 shown. Real-time fluorescence quantitative PCR detection shows that 12 h after PEDV infection, the PEDV N RNA in the compound NMI 8739 treatment group is significantly lower than that in the control group, and shows concentration gradient-dependent inhibition of PEDV infection.

[0054] Example 3 Detection of PEDV N RNA by adding NMI 8739 at gradient concentrations at 6 h after PEDV infection

[0055] 1. Method

[0056] 1) Digest and passage Vero cells, and inoculate them at 1×10 5 cells / mL into a 12-well cell culture plate at 1 mL / well. Culture in a 37 °C, 5% CO2 incubator.

[0057] 2) When the cell density reaches about 70%, infect the cells with 10 4 TCID 50 of PEDV virus particles for 6 h.

[0058] 3) Add compound NMI 8739 at 6 h post-infection to final concentrations of 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, respectively, and harvest the cells at 12 h post-viral infection.

[0059] 4) Lyse the cells with trizol lysis buffer to extract RNA, and perform the detailed operation according to the trizol instruction manual.

[0060] 5) Reverse transcribe the extracted RNA in an amount of 500 ng / 10 μL, and perform the detailed operation according to the reverse transcription kit instruction manual.

[0061] 6) Perform real-time fluorescence quantitative PCR detection on the reverse-transcribed samples. The detailed operation is carried out according to the fluorescence quantitative PCR polymerase instruction manual. The information of the fluorescence quantitative primers is as follows:

[0062] PEDV N-F: TGGTGGCTGCTGTCAAGG,

[0063] PEDV N-R: TTTTCGACAAATTCCGCAT;

[0064] Actin-F: ATCGTGCGTGACATTAAG,

[0065] Actin-R: ATTGCCAATGGTGATGAC.

[0066] 2. Results

[0067] The results are as Figure 3 shown. Real-time fluorescence quantitative PCR detection showed that the PEDV N RNA in the treatment groups with compound NMI 8739 at 6 h post-PEDV infection was significantly lower than that in the control group, and showed concentration-gradient-dependent inhibition of PEDV infection.

[0068] Example 4 Detection of the cytotoxicity of compound NMI 8739 on Vero cells.

[0069] 1. Method

[0070] 1) Digest and passage Vero cells, and inoculate them into a 96-well cell culture plate at 1 × 10 5 cells / mL, 0.1 mL / well. Culture in a 37 °C, 5% CO2 incubator.

[0071] 2) When the cell density reaches about 70%, treat the cells with NMI 8739 at a final concentration of 10 μM for 24 h.

[0072] 3) At 24 h, use the CCK8 kit to detect the changes in cell proliferation, and perform the detailed operation according to the CCK8 kit instruction manual.

[0073] 2. Results

[0074] The results are as Figure 4 shown. The results detected by the microplate reader indicate that treating cells with 10 μM of NMI 8739 for 24 h does not affect cell viability.

[0075] The above results indicate that the compound NMI 8739 of the present invention can significantly inhibit the replication and proliferation of PEDV, can be used to prepare drugs against PEDV infection, for preventing or treating PED, and has broad application prospects.

[0076] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the broadest scope consistent with the principles and novel features disclosed herein.

Claims

1. Use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug, wherein the structural formula of the compound NMI 8739 is shown in the following formula (I):

2. The use according to claim 1, characterized in that The virus is porcine epidemic diarrhea virus.

3. The use according to claim 1 or 2, characterized in that: The compound NMI 8739 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.

4. The use according to claim 3, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

5. Use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing porcine epidemic diarrhea, wherein the structural formula of the compound NMI 8739 is shown in the following formula (I):

6. The use according to claim 5, characterized in that The compound NMI 8739 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.

7. The use according to claim 6, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

8. Use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating porcine epidemic diarrhea, wherein the structural formula of the compound NMI 8739 is shown in the following formula (I):

9. The use according to claim 8, characterized in that The compound NMI 8739 or a pharmaceutically acceptable salt thereof is added to a pharmaceutically acceptable carrier and / or excipient to prepare any pharmaceutically acceptable dosage form.

10. The use according to claim 9, characterized in that The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

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