Use of the compound NMl 8739 for the preparation of a medicament for the prevention or treatment of porcine epidemic diarrhea

Abstract

The application 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. The application surprisingly finds that the compound NMI 8739 can inhibit proliferation of PEDV, has certain inhibitory effect on PEDV, can inhibit infection and amplification of PEDV in cells, can be used for preparing the medicine for preventing or treating porcine epidemic diarrhea, lays certain technical foundation for prevention and control of PED, and has wide application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of animal epidemic 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. BACKGROUND

[0002] Porcine epidemic diarrhea (PED) is a viral infectious disease caused by porcine epidemic diarrhea virus (PEDV), and pigs suffering from the disease mainly show clinical symptoms such as diarrhea, vomiting, anorexia, dehydration and weight loss. PEDV can infect pigs of all ages and show different degrees of disease characteristics. Generally speaking, adult pigs show mild infection symptoms or no symptoms, and piglets are mostly severely infected, with a mortality rate of up to 100%, which brings huge economic losses to the pig breeding industry. Due to the rapid variation of PEDV, the prevention and control effect of the vaccine is limited. Therefore, the development of related prevention and treatment drugs has very important technical significance for preventing and controlling PED.

[0003] The compound NMI 8739 is an agonist of dopamine D2 autoreceptor and is an amine combination of docosahexaenoic acid and neurotransmitter dopamine. At present, it is mainly focused on neurological diseases in laboratory research, and no virus-related reports have been found.

[0004] The application surprisingly finds that the compound NMI 8739 can significantly inhibit the replication of PEDV and reduce the titer of PEDV, and can be used for preparing a medicine against PEDV infection, for preventing or treating PED, and has a broad application prospect. SUMMARY

[0005] In view of the above technical problems, the application surprisingly finds that the compound NMI 8739 can significantly inhibit the replication of PEDV and can be used for preparing a medicine against PEDV infection. Specifically, the following contents are included:

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

[0007]

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

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

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

[0011] In a second aspect, the present application provides the use of compound NMI 8739 or its pharmaceutically acceptable salt in the preparation of a medicine for preventing porcine epidemic diarrhea, the structural formula of the compound NMI 8739 is as follows:

[0012]

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

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

[0015] In a third aspect, the present application provides the use of compound NMI 8739 or its pharmaceutically acceptable salt in the preparation of a medicine for treating porcine epidemic diarrhea, the structural formula of the compound NMI 8739 is as follows:

[0016]

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

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

[0019] The beneficial effects of the present application are: the present application unexpectedly finds that compound NMI 8739 can significantly inhibit the replication and proliferation of PEDV, and can be used for preparing a medicine for resisting PEDV infection, for preventing or treating PED, and has a broad application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Effect of gradient concentration of compound NMI 8739 pretreatment of PEDV virions on PEDV N RNA of Vero cells;

[0021] Figure 2 Effect of simultaneous addition of gradient concentration of compound NMI 8739 and PEDV virions on PEDV N RNA of Vero cells;

[0022] Figure 3Effect of gradient concentration of compound NMI 8739 on Vero cell PEDV N RNA after treating PEDV virion.

[0023] Figure 4 Effect of compound NMI 8739 treatment on Vero cell activity. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be clearly and completely described below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0025] The PEDV strain is a G I type strain CH / SX / 2015, GenBank No. MT783684, which is independently isolated and preserved by the animal immunity and metabolism innovation team of Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences.

[0026] Example 1: Detection of PEDV N RNA of gradient concentration NMI 8739 pretreated PEDV virion

[0027] 1. Method

[0028] 1) Vero cells were digested and passaged, and inoculated into 12-well cell culture plates at 1×10 5 / mL per well in 1 mL per well. Cultured in a 37℃, 5% CO2 incubator.

[0029] 2) When the cell density was about 70%, 2 μM, 4 μM, 8 μM, 16 μM, 32 μM, 62.5 μM, 125 μM, 250 μM, 500 μM, 1 mM, 2 mM of compound NMI8739 and 10 4 TCID 50 of PEDV virion were used, and 37℃ was incubated for 30 min, and the volume was 10 μL.

[0030] 3) The treated PEDV was used to infect the cells for 1 h, and the final concentration of compound NMI8739 was 20 nM, 40 nM, 80 nM, 160 nM, 320 nM, 625 nM, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM. After 1 h, the culture medium was replaced with fresh DMEM medium containing 5% serum, and the cells were collected 12 h after virus infection.

[0031] 4) The RNA in the cells was lysed and extracted using trizol lysis solution, and the detailed operation was performed according to the instructions of trizol.

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

[0033] 6) Real-time fluorescent quantitative PCR detection was performed on the reverse-transcribed sample. The detailed operation was performed according to the fluorescent quantitative PCR polymerase instructions, and the information of the fluorescent quantitative primer was 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 shown in Figure 1 that the fluorescent quantitative PCR detection showed that the PEDV N RNA of the compound NMI 8739 pretreatment group was significantly lower than that of the control group 12 h after PEDV infection, and showed concentration gradient-dependent inhibition of PEDV infection.

[0040] Example 2 Gradient concentration NMI 8739 and PEDV virion were added simultaneously to detect PEDV N RNA 1. Method

[0041] 1) Vero cells were digested and passaged, and inoculated into 12-well cell culture plates at 1×10 5 / mL 1 mL / well. Incubated in a 37℃, 5% CO2 incubator.

[0042] 2) When the cell density was about 70%, 62.5 μM, 125 μM, 250 μM, 500 μM, and 1 mM of compound NMI8739 and 10 4 TCID 50 of PEDV virion were used for pre-mixing, the volume was 10 μL.

[0043] 3) The pre-mixed virus solution was directly added to the cell supernatant to infect the cells for 1 h, and the final concentration of compound NMI8739 was 625 nM, 1.25 μM, 2.5 μM, 5 μM, and 10 μM, respectively.

[0044] 4) 1h later, the culture medium was replaced with fresh DMEM medium containing 5% serum, and the cells were collected 12h after virus infection.

[0045] 5) The RNA in the cells was lysed and extracted using trizol lysis solution, and the detailed operation was performed according to the instructions of trizol.

[0046] 6) The extracted RNA was reverse transcribed, and the amount was 500ng / 10μL, and the detailed operation was performed according to the instructions of the reverse transcription kit.

[0047] 7) Real-time fluorescent quantitative PCR detection was performed on the reverse transcribed sample. The detailed operation was performed according to the instructions of the fluorescent quantitative PCR polymerase, and the information of the fluorescent quantitative primer was 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 shown in Figure 2 The fluorescent quantitative PCR detection showed that the PEDV N RNA in the compound NMI 8739 treatment group was significantly lower than that in the control group 12h after PEDV infection, and showed concentration gradient dependent inhibition of PEDV infection.

[0054] Example 3 Detection of PEDV N RNA when gradient concentration of NMI 8739 is added at 6h of PEDV infection

[0055] 1. Method

[0056] 1) Vero cells were digested and passaged, and inoculated into 12-well cell culture plates at 1×10 5 / mL, 1mL / well. Cultured in a 37℃, 5% CO2 incubator.

[0057] 2) When the cell density was about 70%, the cells were infected with 10 4 TCID 50 of PEDV virions for 6h.

[0058] 3) 6h after adding compound NMI 8739 to a final concentration of 625nM, 1.25μM, 2.5μM, 5μM, 10μM, respectively, collect cells at 12h after virus infection.

[0059] 4) Use trizol lysis solution to lyse and extract RNA in cells, and detailed operation is performed according to the instructions of trizol.

[0060] 5) Reverse transcription is performed on the extracted RNA, and the amount is 500ng / 10μL, and detailed operation is performed according to the instructions of the reverse transcription kit.

[0061] 6) Real-time fluorescent quantitative PCR detection is performed on the sample after reverse transcription. Detailed operation is performed according to the instructions of the fluorescent quantitative PCR polymerase, and the information of the fluorescent quantitative primer 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 shown in the following table: Figure 3 The fluorescent quantitative PCR detection shows that the PEDV N RNA of the group treated with compound NMI 8739 6h after PEDV infection is significantly lower than that of the control group, and shows a concentration gradient-dependent inhibition of PEDV infection.

[0068] Example 4: Detection of the toxicity of compound NMI 8739 treatment on Vero cells.

[0069] 1. Method

[0070] 1) Vero cells are digested and passaged, and inoculated into 96-well cell culture plates at 1×10 5 / mL 0.1mL / well. Incubate in a 37℃, 5% CO2 incubator.

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

[0072] 3) 24h, use CCK8 kit to detect the change of cell proliferation, and detailed operation is performed according to the instructions of CCK8 kit.

[0073] 2. Results

[0074] The results are shown in the following table: Figure 4 As shown in the table, the results of the enzyme-labeled instrument detection showed that 10 μM of NMI 8739 treated cells for 24 h did not affect the activity of the cells.

[0075] The above results show that the compound NMI 8739 of the present application can significantly inhibit the replication and proliferation of PEDV, and can be used for preparing a drug for resisting PEDV infection, for preventing or treating PED, and has a wide application prospect.

[0076] The above description of disclosed embodiments enables a person skilled in the art to implement or use the present application. 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 application. Therefore, the present application will not be limited to these embodiments shown herein, but will conform to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. The use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of an antiviral drug for porcine epidemic diarrhea virus, wherein the structural formula of compound NMI 8739 is shown in formula (Ⅰ) below:

2. The application as described in claim 1, characterized in that, The compound NMI 8739 or a pharmaceutically acceptable salt thereof may be formulated into any pharmaceutically acceptable dosage form by adding pharmaceutically acceptable excipients.

3. The application as described in claim 2, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

4. The use of compound NMI 8739 or a pharmaceutically acceptable salt thereof in the preparation of a drug for the prevention of porcine epidemic diarrhea, wherein the structural formula of compound NMI 8739 is shown in formula (Ⅰ) below:

5. The application as described in claim 4, characterized in that, The compound NMI 8739 or a pharmaceutically acceptable salt thereof may be formulated into any pharmaceutically acceptable dosage form by adding pharmaceutically acceptable excipients.

6. The application as described in claim 5, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

7. The 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 compound NMI 8739 is shown in formula (Ⅰ):

8. The application as described in claim 7, characterized in that, The compound NMI 8739 or a pharmaceutically acceptable salt thereof may be formulated into any pharmaceutically acceptable dosage form by adding pharmaceutically acceptable excipients.

9. The application as described in claim 8, characterized in that, The dosage forms include tablets, sprays, granules, capsules, oral liquids, injections, and suspensions.

Images