New use of compound lomitapide for preventing or treating african swine fever

Abstract

The application belongs to the technical field of African swine fever treatment, and particularly relates to a new use of a compound Lomitapide for preventing or treating African swine fever. It is accidentally found that the compound Lomitapide can inhibit the replication of African swine fever virus and has the effect of inhibiting the infection of African swine fever virus by enabling the transcription level of a structural protein P72 of the African swine fever virus; and the compound Lomitapide has no effect on the replication of FMDV and can specifically inhibit the African swine fever virus, and can be used for preparing a medicine or an adjuvant against the infection of the African swine fever virus and for inhibiting the replication of the African swine fever virus.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biotechnology, and particularly relates to a new use of a compound Lomitapide for preventing or treating African swine fever. BACKGROUND

[0002] African swine fever (ASF) is an acute and severe infectious disease caused by African swine fever virus (ASFV) infection, characterized by fever and hemorrhage in all organs of pigs, and the mortality rate of domestic pigs is as high as 100%. ASFV is the only member of the Asfarviridae family, belongs to the nucleocytoplasmic large DNA virus (NCLDV), and is the only known DNA arbovirus, and the transmission medium is soft tick (Ornithodoros). The ASFV particle is a positive icosahedral structure with a capsid, and the genome is 170 to 190 kb, and according to different strains, more than 150 open reading frames (ORFs) are encoded.

[0003] Since there is no effective vaccine and specific treatment drug so far, once the African swine fever epidemic occurs, it can only be controlled by culling, but this method not only causes economic losses, but also cannot meet the needs of large-scale pig farming in China. Therefore, how to effectively control the ASF epidemic is one of the great challenges facing the world pig industry at present, and it is also a major strategic issue urgently needed to be solved in the prevention and control of ASF in China. Therefore, while developing vaccines, it is also important to find drugs that can prevent or treat African swine fever.

[0004] P30 and p72 are key structural proteins in ASFV, which are the main structural proteins of virus particles and important surface antigens, closely related to host cell tropism, pathogenicity and immunogenicity, and are involved in virus internalization and virus invasion of host cells.

[0005] The compound Lomitapide is a highly effective microsomal triglyceride transfer protein (MTP) inhibitor. The application accidentally found that the compound Lomitapide cannot inhibit the replication of foot-and-mouth disease virus, but can reduce the replication level of African swine fever virus, inhibit the expression of structural proteins of African swine fever virus, has the effect of inhibiting African swine fever virus infection, and can be used as an inhibitor of African swine fever virus for preventing or treating African swine fever. SUMMARY

[0006] In view of the above technical problems, the application provides a new use of a compound Lomitapide for preventing or treating African swine fever, which specifically comprises the following contents:

[0007] In a first aspect, the present application provides a use of a compound Lomitapide or a pharmaceutically acceptable salt thereof in the preparation of a medicine for preventing African swine fever virus infection, wherein the compound Lomitapide has the following structural formula (I):

[0008]

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

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

[0011] In a second aspect, the present application provides a use of a compound Lomitapide or a pharmaceutically acceptable salt thereof in the preparation of a medicine for treating African swine fever virus infection, wherein the compound Lomitapide has the following structural formula (I):

[0012]

[0013] Preferably, the compound Lomitapide or the pharmaceutically acceptable salt thereof is added into 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, and injections.

[0015] In a third aspect, the present application provides a use of a compound Lomitapide in the preparation of a reagent for inhibiting the expression of P72 gene / protein of African swine fever virus, wherein the compound Lomitapide has the following structural formula (I):

[0016]

[0017] The present application has the following beneficial effects: the present application surprisingly finds that the compound Lomitapide can reduce the replication level of African swine fever virus, inhibit the expression of structural proteins of African swine fever virus, and has the effect of inhibiting African swine fever virus infection; and the compound Lomitapide has no effect on FMDV replication, can specifically inhibit African swine fever virus, and can be used for preparing a medicine or an adjuvant for resisting African swine fever virus infection, and for inhibiting the replication of African swine fever virus. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Figure 2 is an experimental result graph of the inhibitory effect of the compound Lomitapide on ASFV-eGFP.

[0019] Figure 2 Figure for the experimental results of the effect of compound Lomitapide on the replication of ASFV-eGFP;

[0020] Figure 3 Figure for the experimental results of the effect of compound Lomitapide on the cell activity of PAM cells;

[0021] Figure 4 Figure for the experimental results of the effect of compound Lomitapide on the replication of FMDV. DETAILED DESCRIPTION

[0022] In order to make the technical means, creative features, purposes and effects achieved by the present application easy to understand, the present application will be further described below in combination with specific embodiments. However, the protection scope of the present application is not limited to the following embodiments.

[0023] The experiments described in the following embodiments obtained biosafety license and African swine fever laboratory activity license:

[0024] According to the requirements of biosafety level 3 laboratory (BSL-3) and African swine fever related biosafety, the Lanzhou Veterinary Research Institute of Chinese Academy of Agricultural Sciences has obtained the license for conducting highly pathogenic ASFV pathogen and animal research from the Ministry of Agriculture and Rural Affairs through the biosafety committee of the Lanzhou Veterinary Research Institute, the experimental animal ethics committee, the biosafety committee of the Chinese Academy of Agricultural Sciences, the experimental animal ethics committee of the Lanzhou Veterinary Research Institute, and the biosafety committee of the Lanzhou Veterinary Research Institute, and has been recorded in the Ministry of Agriculture and Rural Affairs, which meets the requirements of national biosafety level.

[0025] The structural formula of the compound Lomitapide described in the following embodiments is shown in the following formula (I), and the compound is purchased from TargetMol (L2510),

[0026]

[0027] The cells and reagents described in the following embodiments are commercially available without special instructions.

[0028] Example 1 Preliminary screening of the effect of compound Lomitapide on African swine fever virus

[0029] Dilute the compound Lomitapide stock solution (10 mM) to 10 μM with RPMI1640 (10% FBS, 1% trizma) for standby; dilute the ASFV-eGFP fluorescent virus 1:50 with RPMI1640 (10% FBS, 1% trizma) for standby; revive porcine primary alveolar macrophages (PAMs) at 1×10 5Inoculate 1 x 10 cells / well into 96-well plates, discard the culture medium after 6 hours of incubation at 37°C in a 5% CO2 incubator, and add 100 μL of diluted compound Lomitapide and 100 μL of diluted ASFV-eGFP fluorescent virus liquid, and continue to incubate at 37°C in a 5% CO2 incubator for 48 hours. Each compound has 3 repeated wells, and a DMSO group is set as a control. Significant fluorescence changes in 3 repeated wells indicate an effect.

[0030] The results are shown in Table 2. Figure 1 Compared with the DMSO control group, the ASFV fluorescence of the compound Lomitapide group was significantly reduced, indicating that the compound Lomitapide can inhibit the replication of ASFV.

[0031] Example 2 qPCR verification of the effect of compound Lomitapide on African swine fever virus

[0032] The experimental method in Example 1 was repeated, and after 48 hours, the cells and supernatant were repeatedly frozen and thawed at -80°C and 4°C for 3 times to release the virus in the cells, and the transcription level of P72 was detected by qPCR. The qPCR detection system is as follows:

[0033] Upstream primer: 5'-CGTGATGT G / A GC G / A AGAATGAAGAA-3', downstream primer: 5'-C G / T GGAAACGCA C / T GAGCAGTATC-3', probe: FAM-AGCTCCACGA A / G AA A / G GTGTCGAG-BHQ1, detection system 25 μL: 1 μL of upstream and downstream primers, 1 μL of probe, 3 μL of virus liquid, 19 μL of ddH2O; qPCR conditions: 95°C for 2 min; 95°C for 7 s, 60°C for 15 s, 3 cycles; 95°C for 6 s, 57°C for 11 s, 40 cycles; standard curve: Ct value = -3.3652 lg copies + 36.272. After the program ends, the copy number is converted and compared according to the standard curve.

[0034] The results are shown in Table 2. Figure 2 Compound Lomitapide significantly inhibited the P72 transcription level of ASFV.

[0035] Example 3 CCK-8 experiment for detecting the effect of compound Lomitapide on the cell activity of PAM cells

[0036] Recover PAM cells, inoculate 1 x 10 5Cells were seeded into 96-well cell culture plates and cultured at 37°C with 5% CO2 for 6 hours. Cells were then divided into control and experimental groups. Control group cells were supplemented with 0.1% (v / v) dimethyl sulfoxide (DMSO), while experimental group cells were supplemented with RPMI 1640 medium containing 0.5, 1, 2, 5, 10, and 20 μM of the compound Lomitapide, respectively. A blank control group containing the same dose of DMSO as the control group was also included. Cells were incubated for 48 hours. After incubation, 10 μL of CCK-8 reagent was added to each well, the cell plate was gently tapped to mix the reagent, and the cells were incubated at 37°C for another 1 hour. After incubation, the absorbance at 450 nm was measured using a microplate reader. The cell viability at the corresponding compound concentration was calculated using the formula: [(Experimental group absorbance - Blank group absorbance) / (Control group absorbance - Blank group absorbance)] × 100% = Cell viability percentage.

[0037] The results are as follows Figure 3 As shown, the compound Lomitapide had no significant effect on cell viability at 0.5-10 μM, indicating good safety.

[0038] Example 4: Specificity experiment of compound Lomitapide in inhibiting the proliferation of African swine fever virus in PAM cells. 1. Infection and the effect of the compound:

[0039] PK-15 was seeded into 12-well cell plates and cultured at 37°C for 12 hours. Lomitapide (10 mM) was diluted to 10 μM with DMEM containing 2% serum and added to the cells along with FMDV, 500 μL each, for a total volume of 1 mL / well. The FMDV infection dose was 1 MOI. A 0.1% DMSO group was set up as a control group. The cells were cultured at 37°C and 5% CO2 for 16-20 hours. The cells were harvested when significant cytopathic effects were observed.

[0040] 2. Extract total RNA:

[0041] Discard the culture medium and extract total RNA according to the Nucleozol (MACHEREY-NAGEL) procedure. Add 400 μL of RNase-free water, vortex for 15 seconds, incubate at room temperature for 15 minutes, and centrifuge at 12,000 g for 15 minutes at room temperature. Transfer the supernatant to a new centrifuge tube, add isopropanol at a 1:1 ratio, invert to mix, incubate at room temperature for 10 minutes, and centrifuge at 12,000 g for 10 minutes at room temperature. Discard the supernatant, add 500 μL of 75% ethanol to resuspend the precipitate, centrifuge at 8,000 g for 3 minutes at room temperature, discard the ethanol, and dry the precipitate. Dissolve the precipitate in 100-200 μL of RNase-free water.

[0042] 3. Reverse transcription:

[0043] An equal amount of extracted RNA was used for reverse transcription, following the procedure of Rever Tra Ace qPCR RTMaster Mix with gDNA Remover (Takara).

[0044] 4. qPCR:

[0045] Take equal amounts of reverse transcription product and perform qPCR. qPCR enzyme is... Next The qPCR Mix (Takara) was used, with primers 3D-F: 5'-TGGGACCATACAGGAGAAGT-3' and 3D-R: 5'-TTCACCCATCGCAGGTAAAG-3'. The reagent volume was: 10 μL Mix, 0.8 μL 3D-F / R, 2 μL cDNA, and 6.4 μL ddH2O. The qPCR conditions were: 95℃ for 30 s (pre-denaturation), 95℃ for 5 s, and 95℃ for 10 s (40 cycles). The melting curve was: 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s. Each sample was run in triplicate. The results were converted to the logarithm of the genome copy number according to the standard curve.

[0046] 5. Plot the standard curve:

[0047] The standard plasmid pCDNA3.1-3D containing the FMDV 3D gene was diluted to concentrations of 10... 5 10 6 10 7 10 8 10 9 Using the plasmid as a template for the standard curve, perform the same qPCR as in "4", with three replicates for each concentration. Plot the standard curve with the logarithm of the plasmid concentration on the x-axis and the Ct value on the y-axis.

[0048] The results are as follows Figure 4 As shown, the compound Lomitapide has no significant effect on the replication of FMDV in PK-15, which also indicates that the compound Lomitapide described in this application can specifically inhibit ASFV and is used to combat African swine fever virus infection.

[0049] In summary, the Lomitapide described in this invention can inhibit ASFV replication in a dose-dependent manner. It can specifically inhibit ASFV replication and can be used as an inhibitor of African swine fever virus for the prevention or treatment of African swine fever.

Claims

1. Use of compound Lomitapide or its pharmaceutically acceptable salt in the preparation of a drug for treating African swine fever virus infection, the structural formula of the compound Lomitapide is as follows: Formula (I).

2. Use according to claim 1, wherein The compound Lomitapide or its pharmaceutically acceptable salt is added to pharmaceutically acceptable adjuvant to form a pharmaceutically acceptable dosage form.

3. Use according to claim 2, wherein the compound is ###0002### The dosage form includes tablets, sprays, granules, capsules, oral liquids, suspensions, injections.

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