Application of bromophenol-pyrazoline compound in treatment of feline coronavirus diseases

A feline coronavirus and pyrazoline technology, applied in the field of medicine, can solve problems such as easy recurrence, low cure rate, and high price, and achieve the effect of improving survival rate

Pending Publication Date: 2021-11-23
SHANDONG LINGHAI BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although GS-441524 can be used to treat FIP, it is expensive (13,200 per month), the cure rate is not high, and it is easy to relapse. It is of great significance to develop new oral and highly effective targeted FIP drugs

Method used

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  • Application of bromophenol-pyrazoline compound in treatment of feline coronavirus diseases
  • Application of bromophenol-pyrazoline compound in treatment of feline coronavirus diseases
  • Application of bromophenol-pyrazoline compound in treatment of feline coronavirus diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: bromophenol-pyrazoline compound is to feline infectious peritonitis main protease FIPVM pro Inhibitory activity assay

[0040] (1) FIPVM pro Expression and purification of

[0041] FIPVM pro The gene is inserted into the pGEX-6P-1 vector by conventional techniques such as PCR, enzyme digestion and ligation. The constructed vector was transformed into Escherichia coli BL21(DE3) competent cells, and cultured in LB containing 100mg / mL ampicillin at 37°C until the OD600 of the cells reached 0.6. 0.5mM IPTG, 16°C induced protein expression of 16- 18 hours. The collected thalli use suspension buffer (140mM NaCl, 10mM NaCl 2 HPO 4 , 2.7mM KCl, 1.8mM KH 2 PO 4 , 1mM DTT, 10% glycerol, pH 7.3) were resuspended, and the cells were disrupted under high pressure at 4°C. The bacterial fragments were centrifuged to remove, and GST and FIPV M were enriched by GST affinity medium pro The fusion protein, PPase excises FIPV M pro N-terminal GST tag. FIPV M with...

Embodiment 2

[0046] Embodiment 2: Bromophenol-pyrazoline compounds interfere with FIPV virus replication in cells

[0047] In order to further confirm the enzymatic inhibition results in vitro, the present invention evaluated whether bromophenol-pyrazoline compounds could prevent FIPV virus replication in clinical wild-type FIPV-infected cat kidney cells (CRFK) cells.

[0048] Specific operation:

[0049] At 37°C with 5% CO 2 CRFK cells were cultured in DMEM containing 8% fetal bovine serum in a cell culture incubator. When the cells grew to a density of about 80%, they were pretreated with 5 μM bromophenol-pyrazoline compound to be tested for 1 h, and then the FIPV wild strain (MOI: 0.1) was inoculated into CRFK cells. At the same time, the CRFK cells that were inoculated with the drug but not added with the compound were set up as a control. After 24 h, the cell supernatant was collected, and the vRNA in the supernatant was analyzed by qRT-PCR.

[0050] Experimental results:

[0051...

Embodiment 3

[0052] Embodiment 3: the clinical trial of compound 5 treatment feline infectious peritonitis

[0053] (1) Detection of cats with infectious peritonitis

[0054] Feline enteric coronavirus and feline infectious peritonitis virus cannot be distinguished serologically or morphologically. According to the history of disease, epidemiology, clinical symptoms, laboratory diagnosis, imaging examination and histopathological examination and other methods, after excluding other diseases, a comprehensive diagnosis was made on the sick cat. When the sick cat gradually loses weight, loses appetite, increases body temperature, mild non-regenerative anemia, lymphopenia, increased serum globulin concentration, and significantly lower white blood cell ratio compared with normal cats, it can be identified as feline infection peritonitis virus infection.

[0055] (2) Clinical trial grouping and treatment plan

[0056] 30 cats with FIPV infection were divided into wet FIP ​​group (17 cats) an...

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Abstract

The invention belongs to the technical field of medicines, and particularly relates to application of a bromophenol-pyrazoline compound in treatment of feline coronavirus diseases. The research finds that the bromophenol-pyrazoline compound can inhibit the activity of feline infectious peritonitis virus main protease (FIPV Mpro) and interfere replication of Feline Infectious Peritonitis Virus (FIPV) in cells. In clinical experiments, the bromophenol-pyrazoline compound can treat feline infectious peritonitis naturally infected by the FIPV, greatly improves the survival rate of sick cats, and can be used for preparing medicines for treating the feline infectious peritonitis.

Description

technical field [0001] The invention belongs to the technical field of medicine, and relates to a new application of bromophenol-pyrazoline compounds, in particular to the application of bromophenol-pyrazoline compounds in the prevention / treatment of feline coronavirus disease. Background technique [0002] Feline coronavirus (FCV) belongs to the order Coronidoviridae and is an enveloped non-segmented single-stranded positive-sense RNA virus. Feline enteric coronavirus (FECV), the most common FCV, is ubiquitous in cats worldwide and capable of fecal-oral transmission, but is not itself a serious pathogen. Approximately 5% of cats with persistent FECV infection develop highly lethal mutant feline infectious peritonitis virus (FIPV). After FECV is mutated into FIPV, it loses the ability to infect healthy cats, but the pathogenicity and lethality are greatly increased. [0003] FIPV can effectively and sustainably replicate in mononuclear macrophages, and spread to all parts ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K31/415A61K31/427A61P31/14
CPCA61K31/415A61K31/427A61P31/14C07D231/06A61K31/428
Inventor 史大永李祥乾王鑫许凤
Owner SHANDONG LINGHAI BIOTECHNOLOGY CO LTD
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