阅读说明：本技术 以硝唑尼特和咪唑立宾为有效成分的猫杯状病毒抑制剂 (Cat calicivirus inhibitor containing nitazoxanide and mizoribine as effective components ) 是由 王开 崔占鼎 张双 李登亮 裴志花 郭衍冰 赵艳丽 董浩 黄海龙 张茜 陈晓学莹 于 2020-02-26 设计创作，主要内容包括：本发明公开了一种以硝唑尼特与咪唑立宾为有效成分的猫杯状病毒抑制剂,其中硝唑尼特的作用浓度为1.529μmol/L～200μmol/L,在200μmol/L时,细胞活性大于90%。咪唑立宾的作用浓度为1.529μmol/L～200μmol/L,在200μmol/L时,且细胞活性大于90%。硝唑尼特与咪唑立宾对猫杯状病毒的抑制作用发生在病毒感染的复制阶段。0.2μmol/L～2.5μmol/L浓度的硝唑尼特与0.6μmol/L～20μmol/L浓度的咪唑立宾同时使用时,具有协同作用,其中当硝唑尼特0.3μmol/L～1.2μmol/L、咪唑立宾2.5μmol/L～10μmol/L时最为显著。(The invention discloses a feline calicivirus inhibitor with nitazoxanide and mizoribine as effective components, wherein the action concentration of the nitazoxanide is 1.529 mu mol/L-200 mu mol/L, the cell activity is more than 90% at 200 mu mol/L0, the action concentration of the mizoribine is 1.529 mu mol/L1-200 mu mol/L2, at 200 mu mol/L3, the cell activity is more than 90%, the inhibition effect of the nitazoxanide and the mizoribine on feline calicivirus occurs at the replication stage of virus infection, the nitazoxanide with the concentration of 0.2 mu mol/L-2.5 mu mol/L and the mizoribine with the concentration of 0.6 mu mol/L-20 mu mol/L have synergistic effect, and the nitazoxanide with the concentration of 0.3 mu mol/L-1.2 mu mol/L and the mizoribine with the concentration of 0.5 mu mol/L-10 mu mol/L is most obvious.)

1. Application of nitazoxanide and/or mizoribine in preparing anti-feline calicivirus medicines is provided.

2. A feline calicivirus inhibitor characterized by: the molar ratio of nitazoxanide to mizoribine is: 0.2-2.5: 0.6-20.

3. The feline calicivirus inhibitor of claim wherein: the molar ratio is 0.3-1.2: 2.5-10.

Technical Field

The invention relates to the field of medicines, and in particular relates to a feline calicivirus inhibitor taking nitazoxanide and mizoribine as effective components.

Background

Feline Calicivirus (FCV) is a member of the Caliciviridae family (FCaliciviridae) Which is primarily responsible for upper respiratory tract disease in felines. Some virulent strains can cause systemic diseases, mainly manifested by subcutaneous edema, necrosis of multiple organs (liver, spleen, pancreas) and interstitial pneumonia. In the past 30 years, commercial vaccines for feline caliciviruses have been available on the market, but feline caliciviruses are still highly prevalent in the feline flock and none of the currently marketed vaccines can protect cats from all strains of feline caliciviruses. In the case of feline calicivirus, researchers have discovered a variety of drugsThe product has antiviral effect in vitro. However, most of them have been on the level of antiviral studies with a single compound, and it is not known whether these compounds have a synergistic effect. Therefore, there is an urgent need to develop an agent effective against feline calicivirus to inhibit or control transmission of feline calicivirus.

Nitazoxanide (NTZ), the alternative name of 2-acetoxyl-N- (5-nitro-2-thiazolyl) benzamide, is a broad-spectrum antiprotozoal drug, such as treating diarrhea caused by cryptosporidium, giardia, amebiasis. In addition, it has been shown to have antiviral effects against a variety of RNA and DNA viruses. Nitazoxanide is one of the oral drugs approved by the U.S. Food and Drug Administration (FDA) and also found to have antiviral effects against Norovirus (NV), a family of feline caliciviruses. Mizoribine (MZR), the chinese alias 1- [ (2R, 3R,4S, 5R) -3, 4-dihydroxy-5- (hydroxymethyl) oxolan-2-yl ] -5-hydroxy-1H-imidazole-4-carboxamide, is an imidazole nucleoside that has been used as an immunosuppressant in some countries or regions for the treatment of kidney transplantation, autoimmune diseases and steroid resistant nephrotic syndrome due to its inhibitory effect on lymphocytes. In recent years, mizoribine has been found to have antiviral effects against Cytomegalovirus (CMV), Respiratory Syncytial Virus (RSV), Severe acute Respiratory Syndrome (SARS-CoV), Bovine Viral Diarrhea Virus (BVDV), and Foot and Mouth Disease Virus (FMDV).

Disclosure of Invention

The invention aims to provide a feline calicivirus inhibitor taking nitazoxanide and mizoribine as effective components aiming at the problems in the existing feline calicivirus control process. The inhibitor can effectively inhibit the replication of feline calicivirus in vitro, has low cytotoxicity and can obviously reduce the virus titer of the feline calicivirus and the expression level of mRNA thereof.

Application of nitazoxanide and/or mizoribine in preparing anti-feline calicivirus medicines is provided.

A feline calicivirus inhibitor having a molar ratio of nitazoxanide to mizoribine of: 0.2-2.5: 0.6-20;

the molar ratio is 0.3-1.2: 2.5-10.

The invention has the beneficial effects that:

1. the cat calicivirus inhibitor is based on nitazoxanide and mizoribine, and is used for evaluating the inhibition effect on the cat calicivirus, and the inhibitor can effectively inhibit the replication of the cat calicivirus in vitro. The inhibition occurs during the replication phase of feline calicivirus infection. Furthermore, nitazoxanide and mizoribine both have inhibitory effects on the virus at any time during the replication of the virus, more precisely, mainly during the first 8h of the replication phase. In addition, the nitazoxanide and mizoribine have clear and single components, which lays a foundation and provides convenient conditions for applying the nitazoxanide and mizoribine to molecular or cytological research of feline calicivirus and research of antiviral drugs.

2. The feline calicivirus inhibitor provided by the invention can inhibit the replication of feline calicivirus based on nitazoxanide and mizoribine, and is applied to the preparation of the anti-feline calicivirus drug, when the concentration of the nitazoxanide is as high as 200 mu mol/L, the normal cell activity rate can be still ensured to be more than 90%, and when the concentration of the nitazoxanide is 20 mu mol/L, the mRNA expression level of the treated feline calicivirus is reduced by about 88% and the virus titer of the feline calicivirus is reduced by about 25% compared with a negative control group, and when the concentration of the mizoribine is as high as 200 mu mol/L, the normal cell activity rate can be still ensured to be more than 90%, and when the concentration of the mizoribine is 20 mu mol/L, the mRNA expression level of the treated feline calicivirus is reduced by about 92% and the virus titer of the feline calicivirus is reduced by about 24%.

3. The invention relates to a feline calicivirus inhibitor, which is based on the fact that nitazoxanide and mizoribine can synergistically inhibit replication of feline calicivirus, and provides application of the inhibitor in preparation of drugs for resisting feline calicivirus, when the drug concentrations of the nitazoxanide and the mizoribine are respectively not higher than 2.5 mu mol/L and 20 mu mol/L, compared with negative control, the nitazoxanide and the mizoribine have synergistic effect of resisting feline calicivirus, when the nitazoxanide of 0.2 mu mol/L-2.5 mu mol/L and the mizoribine of 0.6 mu mol/L-20 mu mol/L are simultaneously used, the two drugs show synergistic effect of resisting the feline calicivirus virus, wherein when the concentration range of the nitazoxanide is 0.3 mu mol/L-1.2 mu mol/L and the concentration range of the mizoribine is 2.5 mu mol/L-10 mu mol/L, the theoretical basis for preventing and treating feline calicivirus infection by using the nitazoxanide and the drugs related to the feline calicivirus is established.

Drawings

FIG. 1 is a graph of the results of toxicity of nitazoxanide on F81 cells at various concentrations;

FIG. 2 is a graph showing the results of toxicity of mizoribine against F81 cells at various concentrations;

FIG. 3 is a graph of an indirect immunofluorescence OD assay for inhibition of FCV with varying concentrations of nitazoxanide;

FIG. 4 is a graph of the difference in the effect of different concentrations of nitazoxanide on FCV virus titer;

FIG. 5 is a graph of the difference in the effect of varying concentrations of nitazoxanide on FCV mRNA expression levels;

FIG. 6 is a graph of an indirect immunofluorescence OD assay of mizoribine at various concentrations for FCV inhibition;

FIG. 7 is a graph of inhibition concentration analysis of various concentrations of nitazoxanide against FCV;

FIG. 8 is a graph of the difference in the effect of mizoribine at different concentrations on FCV viral titer;

FIG. 9 is a graph of the difference in effect of mizoribine at different concentrations on FCV mRNA expression levels;

FIG. 10 is a graph of the inhibition concentration analysis of FCV by mizoribine at various concentrations;

FIG. 11 is a graph of the difference in the effect of nitazoxanide addition on FCV virus titer at different times;

FIG. 12 is a graph of the difference in the effect of nitazoxanide addition on FCV mRNA expression levels at different times;

FIG. 13 is a graph of the difference in the effect of mizoribine addition on FCV viral titer at different times;

FIG. 14 is a graph of the difference in effect of mizoribine addition on FCV mRNA expression levels at different times;

FIG. 15 is a graph of the analysis of the synergistic inhibition of FCV by nitazoxanide at various concentrations;

FIG. 16 is a graph showing the analysis of the synergistic inhibition of FCV by nitazoxanide and mizoribine at different concentrations.

Detailed Description

Experimental Material

1. The source of the drug is as follows: mizoribine (MZR) was purchased from shanghai alatin biochemical technologies, inc, under the product number M129842. Nitazoxanide (NTZ) was purchased from shanghai alatin biochemical technologies, inc, under the product number N159057.

2. Biochemical reagents: MEM, PBS, FBS were purchased from Gibco. DMSO was purchased from Sigma. CCK8 was purchased from white shark organisms. Simplyp Total RNA extraction kit was purchased from Bioer. RNA reverse transcription reagent, SYBR Prime-script TM RT-PCR Kit purchased from Baosheng organisms. Anti-feline calicivirus IgG (primary antibody) was purchased from VMRD, and secondary antibody FITC-labeled rabbit anti-feline IgG was purchased from boaosen.

3. RT-qPCR primer sequences are shown in Table 1.