阅读说明：本技术 联苯苄唑在制备治疗或预防流感病毒感染的药物中的应用 (Application of bifonazole in preparation of medicine for treating or preventing influenza virus infection ) 是由 陈绪林 刘歌 于 2019-06-28 设计创作，主要内容包括：本发明公开了联苯苄唑(Bifonazole)在制备预防或治疗流感病毒感染药物中的应用。在人单核细胞U937模型上检测了联苯苄唑对细胞的毒性和抗流感活性以及抗炎活性。结果显示联苯苄唑具有显著的抗病毒活性,能够抑制由流感感染诱导的促炎细胞因子释放。在人肺上皮细胞系A549和犬肾细胞系MDCK上,联苯苄唑也具有显著的抗病毒活性。在致死性小鼠流感感染模型中,联苯苄唑可以有效降低感染小鼠肺洗液中的病毒滴度、延缓小鼠体重下降速度、延长小鼠存活时间并提高小鼠存活率。本发明披露的联苯苄唑是一种新型兼具免疫调理活性的抗流感病毒药物,具备安全性好、选择指数高且动物模型实验有效等优点,可以用于开发预防或治疗流感病毒感染的药物。(the invention discloses application of Bifonazole (Bifonazole) in preparing a medicament for preventing or treating influenza virus infection. Toxicity and anti-influenza activity and anti-inflammatory activity of bifonazole on cells were tested on a human monocyte U937 model. The results show that bifonazole has remarkable antiviral activity and can inhibit the release of proinflammatory cytokines induced by influenza infection. Bifonazole also has significant antiviral activity on the human lung epithelial cell line a549 and the canine kidney cell line MDCK. In a lethal mouse influenza infection model, the bifonazole can effectively reduce the virus titer in lung washing liquor of an infected mouse, delay the weight loss speed of the mouse, prolong the survival time of the mouse and improve the survival rate of the mouse. The bifonazole disclosed by the invention is a novel anti-influenza virus medicament with immunoregulation activity, has the advantages of good safety, high selection index, effective animal model experiment and the like, and can be used for developing medicaments for preventing or treating influenza virus infection.)

1. Application of bifonazole in preparing a medicament for treating or preventing influenza virus infection.

2. The use of claim 1, wherein: the influenza virus is influenza A virus.

3. The use of claim 1, wherein: the bifonazole is used as a medicinal active ingredient and is prepared into any pharmaceutically acceptable dosage form.

4. Use according to claim 3, characterized in that: the dosage forms are tablets, capsules, granules, oral liquid and injections.

5. The use according to claim 2, wherein the influenza virus is influenza A virus of subtype H1N1 (A/PuertoRico/8/1934), H3N2 (A/Human/Hubei/3/2005) or H7N8 (A/Duck/Hubei/216/1983).

Technical Field

The invention belongs to the technical field of medicines, and mainly relates to application background technology of bifonazole in preparation of medicines for treating or preventing influenza virus infection

Influenza viruses belong to the family Orthomyxoviridae (Orthomyxoviridae), the genus influenza. Influenza viruses are classified into A, B, C types, also called type A, B and C, according to the antigenic and genetic properties of the viral particle Nucleoprotein (NP) and matrix protein (M). The influenza a virus genome consists of 8 single negative stranded RNAs of different sizes, designated segment 1 to segment 8, respectively. The viral genome is approximately 13.6kb in length and encodes 10 structural proteins (PB2, PB1, PA, HA, NP, NA, M1, M2, PB1-F2 and NS2/NEP) and a non-structural protein (NS 1). Influenza a viruses can be further divided into 17H (H1-H17) and 10N (N1-N10) subtypes, depending on the surface glycoproteins Hemagglutinin (HA) and Neuraminidase (NA) of the virion. Human influenza viruses are predominantly of the H1, H2 and H3 subtypes. Most of the current highly pathogenic avian influenza with serious harm are H5, H7 and H9 subtypes, wherein the lethality rate is highest by using the H5N1 subtype. Influenza B viruses often cause local epidemics of influenza, do not cause major outbreaks of influenza worldwide, and are found only in humans and seals. Influenza C viruses are mostly present in a scattered form, mainly attack infants, generally do not cause influenza epidemics, and can infect humans and pigs.

Influenza viruses have caused five pandemics worldwide since the discovery in the early 20 th century, and a outbreak occurs in about ten years, causing enormous losses worldwide. Influenza epidemics can cause 25 to 50 million deaths per year, with 300 to 500 million cases, and about 5 to 15% of people worldwide are infected. Currently, therapeutic strategies against influenza infection can be largely divided into antiviral therapy and anti-inflammatory immune-opsonic therapy. Antiviral therapy is the primary method of controlling influenza epidemics and plays a central role as the primary prophylactic and therapeutic agent during pandemic outbreaks. To date, only two classes of antiviral drugs are globally approved and available for the treatment of influenza infection, the influenza virus M2 ion channel blocker and the influenza virus Neuraminidase (NA) inhibitor, respectively. Although M2 blockers have had a good antiviral effect in the past, with the emergence of resistant strains, the world health organization has excluded amantadine and rimantadine from the list of anti-influenza drugs recommended for clinical use in 2009. Therefore, NA inhibitors are currently the only influenza antiviral drugs recommended by WHO. Representative neuraminidase inhibitors are oseltamivir and zalamivir, which are effective against all known human influenza viruses and highly pathogenic avian influenza viruses. However, since drug-resistant strains of oseltamivir have been emerging in recent years, it is important to research and develop novel anti-influenza virus drugs.

On the other hand, inflammation induced by severe influenza infection can lead to a variety of pathological conditions, including increased oxidative stress, apoptosis, necrosis, altered adhesion, and migration of immune cells to the lung. In addition, these pathological responses induce the release of additional secondary pro-inflammatory factors and cytokines, resulting in an exaggerated inflammatory response and increased cellular and tissue damage. Therefore, better efficacy may be achieved with anti-inflammatory immunomodulatory drugs alone or in combination with antiviral agents against excessive inflammatory responses induced by influenza and are gaining increasing attention from researchers. Unfortunately, however, no anti-inflammatory drug has yet fully passed clinical trials and confirmed therapeutic efficacy in human patients for the excessive inflammation caused by influenza infection and its resulting lethal complications. Therefore, the need for new anti-influenza immunomodulatory drugs is pressing.

Bifonazole, an imidazole antifungal drug, was patented in 1974 and approved for medical use in 1983. Bifonazole acts by inhibiting the production of ergosterol, an essential component of fungal cell membranes. Inhibition of ergosterol destabilizes fungal cytochrome p450, leading to cell lysis and thus killing of the fungus. To date, no report on bifonazole against influenza virus has been found.

Disclosure of Invention

The invention aims to make up the defects of the prior art and provides the application of the small molecular compound bifonazole in preparing the medicine for treating or preventing influenza virus infection, thereby providing a safe and effective small molecular compound for treating clinical influenza. Bifonazole can effectively inhibit the replication of influenza virus within a non-toxic range, can be further developed into a medicament for treating or preventing influenza virus infection diseases, and has wide application prospect. Bifonazole is known by the english name Bifonazole and is chemically (±)1- (α -biphenyl-4-ylbenzyl) -1H-imidazole, having the structure shown in structural formula i:

In order to achieve the purpose, the invention adopts the technical scheme that:

The application of bifonazole in preparing a medicament for treating or preventing influenza virus infection comprises the following steps:

1 Bifonazole is evaluated for toxicity, antiviral activity and inhibitory activity against the important pro-inflammatory factors CCL2 and CXCL10 in a U937 influenza infection model and its selection index is calculated as follows:

(1) the human monocyte cell line U937 was plated at an appropriate density in 96-well plates and, if antiviral effects were to be tested, was co-infected with an appropriate concentration of influenza virus (A/puerto Rico/8/34(H1N 1)).

(2) Meanwhile, bifonazole diluted by a culture medium into different concentration gradients is added into the holes and cultured for 48 h.

(3) The cell viability of the drug-treated group and the untreated group was examined to examine the cytotoxicity of bifonazole.

(4) Influenza virus neuraminidase activity in the supernatants of the drug-treated group and the untreated group was examined to evaluate bifonazole antiviral activity.

(5) The content of proinflammatory factors CCL2 and CXCL10 in the supernatants of the drug treated group and the untreated group is detected to evaluate the anti-inflammatory activity of the bifonazole.

(6) The antiviral and anti-inflammatory selection index of bifonazole on U937 was calculated.

2 evaluation of Bifonazole toxicity and antiviral efficacy on different cell lines, the procedure was as follows:

(1) The human lung epithelial cell line a549 and the canine kidney cell line MDCK were plated in 96-well plates at appropriate densities, and cultured for 18-24h in 96-well plates at appropriate densities. After the suspension grows into a monolayer, adding bifonazole diluted into different concentration gradients by using a culture medium, and culturing for 48 hours to detect the drug toxicity.

(2) if antiviral effect is detected, the monolayer cells are infected with influenza virus (A/puerto Rico/8/34(H1N1)) at appropriate concentration and added with bifonazole of different concentration gradient and cultured for 48H.

(3) The cell viability of the drug-treated group and the untreated group was examined to examine the cytotoxicity of bifonazole.

(4) Influenza virus neuraminidase activity in the supernatants of the drug-treated group and the untreated group was examined to evaluate bifonazole antiviral activity.

(5) Selection indices of bifonazole on two cell lines were calculated.

Broad-spectrum analysis of antiviral effects of 3-biphenyl benzazoles

(1) Human lung epithelial cell line a549 was plated in 96-well plates at an appropriate density, cultured in a cell incubator for 18-24H and grown into a monolayer, and then infected with different influenza strains at 1MOI (including a/Human/Hubei/1/2009(H1N1), a/Human/Hubei/3/2005(H3N2), a/Duck/Hubei/216/1983(H7N8)) while adding different concentration gradients of drug for 48H of culture.

(2) The activity of influenza virus neuraminidase in the supernatants of the drug-treated group and the untreated group is detected to evaluate the antiviral broad spectrum of the drug.

4 evaluating the antiviral effect of bifonazole in an animal living body in an animal lethal influenza infection model, which comprises the following steps:

(1) Establishing a lethal influenza infection animal model of the mice.

(2) Infected mice were treated with bifonazole by intraperitoneal administration for seven consecutive days, twice a day.

(3) And (3) on the third day after infection, namely the virus replication peak period, taking lung lavage fluid of mice of a control group and a drug treatment group, and detecting the influenza virus titer.

(4) Mouse body weight, mortality, and body weight change curves and mouse survival curves were recorded daily.

(5) Bifonazole effect was assessed as change in body weight, survival rate, and viral titer in lung lavage fluid of mice after infection.

Such influenza viruses include, but are not limited to: influenza A viruses H1N1 subtype (A/puerto Rico/8/1934), H3N2 subtype (A/Human/Hubei/3/2005), H7N8 subtype (A/Duck/Hubei/216/1983).

the protection scope of the invention also includes:

The application of bifonazole in preparing a medicament for inhibiting in vitro influenza virus replication;

The application of bifonazole as the only effective component in preparing the medicine for treating or preventing influenza virus infection;

Application of bifonazole in preparing a medicament for treating mouse influenza virus infection.

Compared with the prior art, the invention has the following advantages and effects:

1 Biphenyl benzazole is a small molecule compound that is CC in U937, A549 and MDCK cells₅₀(half lethal concentrations) are greater than 150. mu.M. It was able to dose-dependently inhibit the replication of the H1N1PR8 influenza virus on three cell lines, its IC in U937 cells₅₀(median inhibitory concentration) was only 1.86. mu.M, with an EC50 of 5.7. mu.M on A549. By calculation, the Selection Index (SI) of the bifonazole is more than 80 in U937 and more than 30 in A549 cells, which indicates that the bifonazole has the characteristics of safety and high efficiency.

the 2-biphenyl benzyl azole can inhibit the replication of influenza A virus subtype H1N1, H3N2 and H7N8 in a dose-dependent manner, and has good broad-spectrum antiviral activity.

the 3-biphenyl benzyl azole has a protection effect in a lethal influenza infection model of a mouse, can obviously reduce the weight reduction degree of the mouse, reduce the virus titer in lung lotion of the mouse, and improve the survival time and the final survival rate of the mouse. This has led to the great potential for clinical treatment of bifonazole.

4-biphenyl benzyl azole has been on the market for many years, has good safety and a large amount of clinical experimental data, and can remarkably reduce the clinical test time and save a large amount of cost if being used as a drug for treating influenza.

Drawings

FIG. 1 toxicity, antiviral and anti-inflammatory effects of Bifonazole in U937

In FIG. 1, A is the activity of U937 cells treated with bifonazole of different concentrations;

in FIG. 1, B is the inhibition rate of bifonazole on U937 to H1N1 influenza virus at different concentrations;

In FIG. 1, C is the inhibition rate of different concentrations of bifonazole on virus-induced proinflammatory factor CCL 2;

In FIG. 1, D is the inhibition rate of different concentrations of bifonazole on the virus-induced pro-inflammatory factor CXCL 10.

FIG. 2 toxicity and antiviral Effect of Bifonazole in A549 and MDCK

In FIG. 2, A is the activity of A549 cells treated by bifonazole with different concentrations;

In FIG. 2, B is the inhibition rate of bifonazole at different concentrations on A549 against H1N1 influenza virus;

in fig. 2, C is the MDCK cell viability after treatment with different concentrations of bifonazole;

In fig. 2D is the inhibition rate of H1N1 influenza virus on MDCK with different concentrations of bifonazole.

FIG. 3 Bifonazole broad-spectrum antiviral Activity assay

In FIG. 3, A is the inhibition rate of different influenza viruses after different concentrations of bifonazole treatment.

FIG. 4 Effect of Bifonazole in mouse lethal influenza infection model

In FIG. 4, A is the weight change curve of the mouse;

In FIG. 4, B is a mouse survival rate curve;

In FIG. 4C is the virus titer in lung wash on the third day after infection of the mice.

Detailed Description

For a better understanding of the present disclosure, the following further description is provided in conjunction with the specific embodiments, but the present disclosure is not limited to the following examples. The technical scheme of the invention is a conventional technology if not particularly specified; the reagents or materials used, if not specifically indicated, are commercially available.

At present, anti-influenza virus drug evaluation models are mainly classified into in vitro models (in vitro models) and in vivo models (in vivo models). The in vitro model mainly uses various influenza sensitive cell lines or influenza pathology-related cell lines to evaluate the medicine, and has the advantages of providing a large number of cells with the same genetic characters as research objects, being convenient to operate, eliminating the influence of other external factors, detecting the toxicity, effective concentration and selection index of the medicine and providing more bases for later mechanism research. In vivo models various models of animal infection are generally used, and the overall effect of the drug in living animals is measured by various phenotypic indicators after drug treatment. The advantage is that the candidate drug can be truly and systematically evaluated for characteristics including toxicity, activity, metabolism, and the like. The invention adopts three cell models to quantitatively analyze the in-vitro anti-influenza activity of the bifonazole. Meanwhile, the antiviral broad spectrum of the bifonazole is detected by using influenza A virus strains of different subtypes. Subsequently, the in vivo anti-influenza effect of bifonazole was evaluated systematically using a mouse lethal influenza infection model.

Experimental materials:

(1) cell line, experimental animal and virus required by experiment

U937, a549, and MDCK cells were purchased from American Type Culture Collection (ATCC);

SPF grade 6 to 8 week old Balb/c mice, purchased from Beijing Wittingle laboratory animal technology, Inc.;

The strains used were: influenza A virus H1N1 subtype (A/puerto Rico/8/1934),

Subtype H3N2 (A/Human/Hubei/3/2005) and subtype H7N8 (A/Duck/Hubei/216/1983).

(2) Medicine required by experiment

Bifonazole was purchased from gangrenon biotechnology; in cell experiments, the drugs are dissolved by DMSO; animal experiments were performed using sterile PBS.

(3) Reagents required for the experiment:

RPMI-1640 medium, Fetal Bovine Serum (FBS) were purchased from GIBCO;

4-methylumbelliferyl- α -N-acetyl-neuraminate (MUNANA) was purchased from Sigma;

The MTS cell proliferation detection kit is purchased from Promega corporation;

AlphaLISA protein detection kits were purchased from Perkin Elmer.

(4) Instruments required for the experiment:

EnSpire multifunctional microplate reader from Perkinelmer;

The CO2 cell culture box was purchased from Thermo.