KR102556061B1 - Antiviral composition for Zika virus comprising lipophilic statins - Google Patents

Abstract

The present invention relates to an antiviral composition against Zika virus comprising a lipophilic statin compound as an active ingredient.
Since the composition containing the lipophilic statin compound of the present invention has very excellent activity to inhibit the proliferation and replication of Zika virus, it can be used as an effective treatment for Zika virus, for which no vaccine or treatment has been developed yet. In addition, the present invention can be usefully used as an efficient and side-effect-free antiviral composition for virus-related diseases that have limitations in vaccine treatment due to antigenic diversity or antigenic mutation.

Description

Antiviral composition for Zika virus comprising lipophilic statins as an active ingredient

The present invention relates to an antiviral composition against Zika virus comprising a lipophilic statin compound as an active ingredient.

Zika Virus (ZIKV) is a mosquito-borne virus discovered in Rhesus monkeys in Uganda in 1947, and also found in humans in Uganda and Tanzania in 1952. Since then, there have been sporadic reports of outbreaks in some countries in Africa and Southeast Asia, but they have not attracted much attention. The viral disease has been largely prevalent in the Americas, centering on Brazil, since 2015, and continues to this day in many countries in Central and South America.

Zika virus has been found to be potentially transmitted from pregnant women to their fetus, and there are reports of a serious birth defect of the brain called microcephaly in actually born infants. This prompted the WHO to declare the Zika virus a public health crisis of international concern.

When Zika virus causes disease in humans, a self-limited febrile illness manifests as low-grade fever, rash, headache, conjunctivitis, myalgia, and arthralgia. Recently, it has been found that Zika virus infection is associated with an increased incidence of microcephaly in fetuses and infants, and with Guillain-Barre syndrome in adults. The incubation period for Zika virus disease is not exactly known, but is expected to be several days to a week, with mild symptoms typically lasting several days to a week. The Zika virus usually stays in the blood of an infected person for about a week, but it may stay for a longer period in some people.

Currently, there is no preventive vaccine or treatment to prevent Zika virus infection. Although symptoms associated with Zika virus infection are generally mild, there is a very deadly threat, such as the development of Guillain-Ballet syndrome, which can cause microcephaly in the fetus when infected in pregnant women, and can also cause paralysis. Therefore, there is a demand for development of substances having antiviral activity against Zika virus.

Under the above-mentioned technical background, the present inventors, while conducting intensive research to develop a new substance having antiviral activity against Zika virus, belong to lipophilic statins among statin compounds widely known as substances for treating cardiovascular diseases through cholesterol reduction. The present invention was completed by confirming that the compounds have the ability to reduce the production of infectious Zika virus particles in Vero cells, to limit the intracellular spread of Zika virus, and to disrupt the replication cycle.

An object of the present invention is to provide an antiviral composition against Zika virus comprising a lipophilic statin compound as an active ingredient.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating Zika virus infectious diseases, comprising a lipophilic statin compound as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or improving Zika virus infectious disease, comprising a lipophilic statin compound as an active ingredient.

The present invention provides an antiviral composition against Zika virus comprising a lipophilic statin compound as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or treating a Zika virus infection disease comprising a lipophilic statin compound as an active ingredient.

The present invention also provides a food composition for the prevention or improvement of Zika virus infectious disease comprising a lipophilic statin compound as an active ingredient.

Since the composition containing the lipophilic statin compound of the present invention has very excellent activity to inhibit the proliferation and replication of Zika virus, it can be used as an effective treatment for Zika virus, for which no vaccine or treatment has been developed yet. In addition, the present invention can be usefully used as an efficient and side-effect-free antiviral composition for virus-related diseases that have limitations in vaccine treatment due to antigenic diversity or antigenic mutation.

Figure 1 shows the anti-ZIKV activity of lipophilic statins in Vero cells monitored using statin chemical structures and time-point studies. Statins used in the present invention can be classified into lipophilic and hydrophilic statins. Each statin has a pharmacophore, either an open ring (eg atorvastatin) or a closed ring (eg lovastatin) linked to a substituent ring system. Lipophilic statins were used to simultaneously treat ZIKV-infected Vero cells (MOI 0.01). Culture supernatants were collected at different time points, and changes in cytopathic effect (CPE) and viral yield (TCID ₅₀ ) were measured after 96 hours (b). Data shown are mean±SEM.
Figure 2 shows the inhibitory effect of infectious ZIKV production by lipophilic statins in Vero cells. Vero cells were infected with ZIKV (MOI 0.01) and treated with a lipophilic statin at a non-cytotoxic concentration for 96 hours. Culture supernatants were collected and plaque assay was performed on Vero cells. The % infectivity was calculated by dividing the statin-treated titer by the untreated titer (PFU/mL), and is shown in (a). In the 10 ⁵ diluted supernatant, plaques formed on Vero cells were compared and shown in (b). Asterisks indicate significant differences (p < 0.01, Student's t-test), data presented are mean ± SEM.
Figure 3 shows the localization of ZIKV in Vero cells by lipophilic statins. Cells were stained for flavivirus E-protein (4G2) using 4G2mAb and counterstained with DAPI as nuclear staining at 48 hpi (MOI of 0.1). ZIKV particles were spread in the perinuclear region and cytoplasm of statin-untreated cells. However, upon statin treatment, ZIKV particles were generally localized to the perinuclear region.
Figure 4 shows the results of dose-dependent evaluation of the effects of (a) pravastatin and (b) rosuvastatin on the viability of ZIKV-infected and uninfected Vero cells. Percent cell viability was calculated as the average absorbance of the statin-treated sample (Absorbance ₄₅₀ ) minus the average absorbance of the non-statin-treated Vero cell control group (Absorbance ₄₅₀ ) divided by the average absorbance of the Vero cell control group (Absorbance ₄₅₀ ). In the case of pravastatin, a minimal effect on the viability of infected cells was observed up to 25 μM. The effect increased at about 50 μM; However, this was equivalent to the viability observed in uninfected Vero cells. The viability values of 50-400 μM pravastatin in Vero cells were similar in both infected and uninfected cells, and this activity of pravastatin may be due to its effect on Vero cells rather than its effect on ZIKV infection. Similar trends were observed in infected and uninfected Vero cells even in the case of rosuvastatin at 25-400 μM. The graph also shows cell viability of controls: uninfected statin untreated cells (Vero) and infected statin untreated cells (ZIKV).
Figure 5 shows the phenotype of ZIKV-infected or non-infected Vero cell monolayer cultures treated with lipophilic statins. To measure the cytopathic effect (CPE), the morphology of Vero cell cultures was observed for 96 hours after treatment with lipophilic statins. The morphology of the uninfected, statin-treated cell cultures was similar to the uninfected, statin-untreated control, indicating that the lipophilic statin had little or no effect on the growth of the cells at the doses evaluated. In ZIKV-infected statin-untreated controls, cytopathic effects such as cell rounding and desquamation were observed. Similar CPE was observed in some of the statinized ZIKV-infected controls (particularly in the LOV setup), but not as markedly as in the ZIKV-infected controls.
Figure 6 shows the layout of a modified checkerboard strategy for testing the synergistic effect of inhibiting ZIKV infection by lipophilic statin combinations. Statin concentration was expressed as a factor of EC ₅₀ . Vero cells are 1×10 ⁴ Seeded at a density of cells/well. Lipophilic statins were used to simultaneously treat ZIKV-infected Vero cells (MOI 0.01). ZIKV-infected Vero cells (MOI 0.01) (infection group) were incubated for 5 days, and % protection was expressed as the average of three experimental values.
Figure 7 shows the synergistic effect of ZIKV infection inhibition by a combination of fluvastatin and mevastatin (FLU-MEV) and a combination of fluvastatin and simvastatin (FLU-SIM) in Vero cells. (A) shows the combined capacity of FLU and MEV showing a protection rate of 90% or more, and (B) shows the combined capacity of FLU and SIM showing a protection rate of 99% (red line: line with FIC of 1.0, ( □): FIC = 1.0; (●): FIC < 1.0 (synergy); (▽): FIC > 1.0 (no synergy, no additivity)).
Figure 8 shows the synergistic effect of inhibiting ZIKV infection by the combination of atorvastatin and mevastatin (ATO-MEV) in Vero cells, showing the combined dose of ATO and MEV showing a protection rate of 75% (red line: FIC Line with 1.0, (□): FIC = 1.0; (●): FIC < 1.0 (synergy); (▽): FIC > 1.0 (no synergy, no additivity)).
Figure 9 shows the synergistic effect of inhibiting ZIKV infection by the combination of atorvastatin and chloroquine (ATO-CLQ) in Vero cells, showing the combined dose of ATO and CLQ showing a protection rate of 60% (red line: FIC of 1.0 In line, (□): FIC = 1.0; (●): FIC < 1.0 (synergy); (▽): FIC > 1.0 (no synergy, no additivity)).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.

As used herein, the term "composition" is intended to include any product made directly or indirectly by combining the specific components, as well as products containing the specific components.

As used herein, the term "anti-viral activity" refers to the virus infection cycle, specifically, virus penetration in a host cell, virus replication, virus assembly, and virus release. means an activity that directly or indirectly interferes with or inhibits one or more stages of the viral infection cycle. This includes any effect that unspecifically inhibits viral titer increase in a virus-infected individual or unspecifically decreases the virus titer level.

As used herein, the term "Zika virus infection disease" refers to any disease or pathologic condition that occurs, progresses, or worsens as a direct or indirect cause of Zika virus infection.

As used herein, the term "treatment" includes (a) inhibition of the development of a disease, condition or condition; (b) alleviation of disease, illness or symptoms; or (c) eliminating the disease, disorder or condition. The composition of the present invention inhibits the effect of viral infection by improving the survival rate of Zika virus-infected cells, or inhibits the development of diseases or symptoms caused by viral infection by inhibiting the generation, proliferation and replication of Zika virus, It serves to eliminate or alleviate it. Therefore, the composition of the present invention may be a treatment composition for Zika virus infection by itself, or may be administered together with other anti-viral compositions to be applied as a treatment adjuvant to inhibit viral activity. The term "treatment" or "therapeutic agent" includes the meaning of "therapeutic adjuvant" or "therapeutic adjuvant".

As used herein, the term “prevention” means inhibiting the occurrence of a disease or condition in a subject who has not been diagnosed with, but is likely to have, the disease or condition.

As used herein, the term "administration" or "administer" refers to directly administering a therapeutically effective amount of the composition of the present invention to a subject so that the same amount or an equivalent amount is formed in the body of the subject.

While conducting intensive research to develop new substances having antiviral activity against Zika virus, the present inventors found that among statin compounds widely known as substances for treating cardiovascular diseases through cholesterol reduction, compounds belonging to the lipophilic statins showed no infectious activity in Vero cells. The present invention was completed by confirming that the present invention has the ability to reduce the production of Zika virus particles, limit the intracellular spread of Zika virus, and disrupt the replication cycle.

Accordingly, the present invention provides an antiviral composition against Zika virus comprising a lipophilic statin compound as an active ingredient.

The antiviral composition is in the form of a pharmaceutical composition administered to a vertebrate, preferably a mammal including a human, or a food composition, tableware, kitchen utensils, household items, and other various cleaning agents, disinfectants, and conventional antiviral agents. It can be applied in all known forms.

In addition, the lipophilic statin compound is not necessarily limited thereto as long as it has lipophilicity among statin compounds, but, for example, atorvastatin (ATO), cerivastatin (CER), fluvastatin (fluvastatin , FLU), lovastatin (LOV), mevastatin (MEV), and simvastatin (SIM)).

In addition, the lipophilic statin compound may be characterized in that it exhibits antiviral activity by reducing the production of Zika virus particles, as can be seen from the results of the following examples.

In addition, the lipophilic statin compound may exhibit antiviral activity by inhibiting the proliferation and replication of Zika virus.

The present invention also provides a pharmaceutical composition for preventing or treating a Zika virus infection disease comprising a lipophilic statin compound as an active ingredient.

The pharmaceutical composition according to the present invention may further include a pharmaceutically acceptable carrier in addition to the active ingredient. The pharmaceutically acceptable carriers are commonly used, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, and polyvinylphy. Rolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition according to the present invention may be administered orally or parenterally. In the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, intranasal administration, etc. may be used. Most preferably, it is administered by nasal administration.

A suitable dosage of the pharmaceutical composition according to the present invention may be prescribed in various ways depending on factors such as formulation method, patient's age, weight, sex, morbid condition, food, administration time, administration route, excretion rate and reaction sensitivity. there is. On the other hand, when the pharmaceutical composition of the present invention is used as a composition for nasal administration, a preferred unit spray amount is 20 to 500 μg, and the most preferable unit spray amount is 50 to 200 μg. In a unit spray amount of less than 20 μg, the amount of the composition is small, making it difficult to obtain the targeted effect, and in a unit spray amount of more than 500 μg, the composition flows down from the nasal cavity when sprayed into the nasal cavity, making it difficult to administer.

The pharmaceutical composition according to the present invention is prepared in unit dosage form by using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by those skilled in the art. can be manufactured.

The present invention also provides a functional food composition for preventing or improving Zika virus infectious disease, comprising a lipophilic statin compound as an active ingredient.

The functional food composition according to the present invention includes not only the above active ingredients, but also ingredients commonly added during food preparation, and may include, for example, proteins, carbohydrates, fats, nutrients, seasonings and flavoring agents. The carbohydrates include monosaccharides such as glucose, fructose, and the like; disaccharides such as maltose, sucrose, oligosaccharides and the like; and polysaccharides, for example, common sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents, natural flavoring agents [thaumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.]) and synthetic flavoring agents (saccharin, aspartame, etc.) can be used. For example, when the food composition of the present invention is prepared as a drink, in addition to the active ingredient of the present invention, citric acid, high fructose corn syrup, sugar, glucose, acetic acid, malic acid, fruit juice, eucalyptus extract, jujube extract, licorice extract, etc. can do.

In addition, the present invention provides a method for preventing or treating a Zika virus infectious disease comprising administering a therapeutically effective amount of a lipophilic statin compound to a mammal.

As used herein, the term "mammal" refers to a mammal that is a subject of treatment, observation or experimentation, and preferably refers to a human.

As used herein, the term "therapeutically effective amount" refers to an amount of an active ingredient or pharmaceutical composition that induces a biological or medical response in a tissue system, animal or human, as thought by a researcher, veterinarian, physician or other clinician, This includes amounts that induce relief of the symptoms of the disease or disorder being treated. It is obvious to those skilled in the art that the therapeutically effective dosage and frequency of administration of the active ingredient of the present invention will vary depending on the desired effect. Therefore, the optimal dose to be administered can be easily determined by those skilled in the art, and the type of disease, the severity of the disease, the content of the active ingredient and other ingredients contained in the composition, the type of formulation, and the patient's age, weight, and general health condition , Gender and diet, administration time, administration route and secretion rate of the composition, treatment period, can be adjusted according to various factors including drugs used simultaneously.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments and the like. However, these examples are intended to explain the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited thereto.

Example One.

(1) Experiment method

cells and viruses

African green monkey (Vero; ATCC® CCL-81TM) cells were grown in growth medium containing Minimal Eagle's Medium (MEM, Gibco) supplemented with 10% fetal calf serum, antibacterial antibiotics (Gibco) and L-glutamine (Gibco). grew up in ZIKV (ATCC ^® VR-1838 ^TM ) was propagated in Vero cells using MEM supplemented with 0.3% bovine serum albumin, antibacterial antibiotics and L-glutamine (infection medium). ZIKV particles were harvested 5 days after infection (dpi), and the culture supernatant was stored at -80 °C until use.

reagent

Atorvastatin calcium salt trihydrate (ATO), cerivastatin sodium salt hydrate (CER), fluvastatin sodium hydrate (FLU), pravastatin sodium salt hydrate hydrate (PRA), rosuvastatin calcium (ROS) and simvastatin (SIM) were purchased from Sigma-Aldrich. Lovastatin sodium salt (LOV) and mevastatin sodium salt (MEV) were purchased from Calbiochem. Statins ATO, FLU, PRA, ROS, MEV, LOV and SIM were reconstituted at 20 mM in DMSO; PRA was reconstituted at 50 mM in DMSO; CER was reconstituted at 20 mM in distilled water. All statins were stored at -20 °C and diluted to working concentrations in infection medium on the day of use. Mouse anti-flavi virus group antigen monoclonal antibody clone D1-4G2-4-15 (4G2 mAb) was purchased from Merck-Millipore (MAB10216) and aliquoted and stored at -20 °C before use. The secondary antibody, FITC-conjugated goat anti-mouse IgG, was purchased from Sigma-Aldrich. EZ-CYTOX, a cytotoxicity assay kit, was purchased from DoGenBio, Co., Ltd. in Korea.

In the dose-response curve, anti- ZIKV Screening for activity

The highest dose ⁽ n = 3) was prepared prior to analysis of 12 dose response curves consisting of half dilutions starting at 50 μM. Prior to any treatment, cells were washed once with phosphate buffered saline (PBS). For cytotoxicity curves, statins were diluted to required concentrations in infection medium (100 μL/well) and added to Vero cells. Except for the CER setup without additives, all Vero cell controls contained 0.05% DMSO. Cells were cultured for 5 days at 37° C., 5% CO ₂ , and after culture, EZ-CYTOX (10 μl/well) was added. Cells were incubated at 37° C., 5% CO ₂ , and absorbance was read at 450 nm after 3-4 hours. Percent viability was calculated by dividing the absorbance of the treated (Abstreated) by the absorbance of the Vero cell control (Absvero) and then multiplying by 100. Percent cytotoxicity was calculated as 100% minus viability. CC50 based on % cytotoxicity was calculated using the 4-parameter logistic curve fitting method of SigmaPlot (Systat Software, San Jose, Calif.) (P<0.0001).

In the activity curves, statins were diluted in infection medium to required concentrations as described above and transduced together with ZIKV (MOI 0.01) in Vero cells. Cells were cultured for 5 days at 37° C., 5% CO ₂ , and after culture, EZ-CYTOX (10 μl/well) was added. Cells were incubated at 37° C., 5% CO ₂ , and absorbance was read at 450 nm after 3-4 hours. The % protectivity was calculated according to Equation 1 below:

[Equation 1]

% Protectivity = (Abstreated-Absinfected)/(Absvero-Absinfected) x 100%

Here, Abstreated represents the absorbance of the treated group, Absinfected represents the absorbance of the infected control group, and Absvero represents the absorbance of the uninfected control group.

EC ₅₀ was calculated using the 4-parameter logistic curve fitting method of SigmaPlot (Systat Software, San Jose, CA) (P<0.0001).

to ZIKV infected Vero cellular statin process

Vero cells (1.5x10 ⁵ cells/well) were prepared in 12-well plates and cultured overnight at 37° C., 5% CO ₂ . Cells were washed once with PBS and infected with ZIKV (MOI 0.01) in infection medium. In treatment groups, ZIKV and lipophilic statins were co-introduced into Vero cultures. Lipophilic statins (ATO: 14.69 μM; CER: 91 μM; FLU: 2.97 μM; LOV: 29.18 μM; MEV: 6.78 μM; SIM : 5.09 μM) was treated with cells for 96 hours.

TCID ₅₀ second Time-point used ZIKV Titer decision

Supernatants of 12-well plate infected and treated Vero cultures were harvested at 6, 24, 48, 72 and 96 hpi (n=3 per time point). The supernatant from each time point was diluted 10-fold (10 ^-1 -10 ^-8 ) and added to the overnight Vero cell culture (1.0x10 ⁴ cells/well, n=4 per dilution). Cells were cultured at 37° C., 5% CO ₂ and cytopathological effects (CPE) were observed at 96 hpi compared to uninfected Vero cell controls. TCID ₅₀ per time-point was determined using the Reed-Muench method.

by plaque assay ZIKV of titre decision

Vero cells (~ 2.5 x 10 ⁵ cells/well) were prepared in a 6-well plate and cultured for two days at 37 °C and 5% CO ₂ (~ 90% confluence). Cells were washed with PBS, and then inoculated with statin-treated ZIKV infection supernatant (n = 3) serially diluted 10-fold in infection medium at 37 °C, 5% CO ₂ for 1.5 hours. The infection supernatant was removed and the cells were covered with MEM infection medium containing bacterial agar (0.9%). The culture was incubated for 96 hours at 37° C., 5% CO ₂ and then the agar medium was removed. Plaques were stained and fixed in 0.1% crystal violet and 10% formaldehyde. Mean PFU/mL for each statin treatment was calculated and the % infectivity of ZIKV generated from each treatment was calculated as the mean titer of the treatment (PFU/mL) divided by the mean titer of the ZIKV-infected untreated control multiplied by 100. Calculated.

Immunofluorescence assay

Vero cells (2x10 ⁴ cells/well) in growth medium were cultured overnight in 96-well plates at 37° C., 5% CO ₂ , and cells were washed with PBS. Statins were introduced together with ZIKV (MOI of 0.1), and cells were cultured for 48 hours at 37 °C and 5% CO ₂ . Cells were washed 3 times with PBS (all subsequent washing steps performed similarly), then fixed and permeabilized using BD Cytofix/Cytoperm (BD Biosciences) for 20 minutes at 4°C. Cells were washed and blocked overnight at 4° C. with 2% bovine serum albumin. Cells were again washed three times with PBS before incubating with 4G2 mAb (1:20 in PBS) for 2 hours at 37°C. Following a washing step, the cells were incubated with FITC-conjugated goat anti-mouse IgG secondary antibody (1:100 in PBS) for 1 hour at 37°C. Cells were washed and counterstained with DAPI (1:10,000) for 1 minute, then washed again and overlaid with 80% glycerol. Cells were observed on a Carl Zeiss Axio Scope. A1 is less than 20x and images were captured with AxioVision 4.8 software. Composite images were merged using ImageJ software.

(2) Results and discussion

in vero cells lipophilicity on statins by ZIKV's reduced infectivity

Eight statins (atorvastatin (ATO), cerivastatin (CER), fluvastatin (FLU), lovastatin (LOV), mevastatin (MEV), pravastatin (PRA), rosuvastatin (ROS) and simvastatin (SIM) )), activity against ZIKV infection was initially investigated at a multiplicity of infection (MOI) of 0.01, and cytotoxicity in Vero cells was evaluated by colorimetric cytotoxicity assay using the EZ Cytox cell viability assay kit. Lipophilic statins (ATO, CER, FLU, MEV, LOV and SIM) inhibited ZIKV in vitro; Their half maximum effective concentration (EC ₅₀ ) and cytotoxic concentration (CC ₅₀ ) are shown in Table 1. Among them, CER showed a difference in pharmacological activity between statins, such as the lowest EC ₅₀ and the highest LOV. However, over the range tested, the hydrophilic statins PRA and ROS showed little or no inhibition of ZIKV at 50 and 25 μM, respectively (FIG. 4).

Through the results shown in Table 1, it was confirmed that the lipophilic statin compound according to the present invention has a very good ability to inhibit ZIKV in vitro.

in vero cells, lipophilicity on statins infectivity by ZIKV reduced particle generation

In this study, we tested the effect of statins on the production of infectious ZIKV particles in vitro through a time-based study. In addition, lipophilic statins and ZIKV (MOI 0.01) at minimal cytotoxic concentrations (see FIG. 5) were added, although they inhibit Vero cell culture. To observe possible effects of drugs on different stages of the ZIKV replication cycle, we did not remove ZIKV and statins during the entire incubation period. Culture supernatants were collected at different time points and ZIKV titers were determined by medium tissue culture transfector (TCID ₅₀ ) based on cytopathic effect ( FIG. 1B ). All statins significantly reduced ZIKV production starting at 48 h post infection (hpi) (p < 0.01), and the reduction was sustained until the end of infection (96 hpi). In particular, FLU and LOV were shown to reduce ZIKV production more effectively than the other statins.

Next, ZIKV (MOI 0.01) and lipophilic statins were co-transduced in Vero cells, and endpoint infectious ZIKV titers were determined in the culture supernatant using a plaque assay at 96 hpi (Figs. 2a and 2b) .

The production of infectious ZIKV particles in the statin-treated environment was significantly lower than that of the non-statin-treated control, and the infectivity was reduced in the range of about 67-98%. Consistent with the results of time-point analysis, FLU and LOV inhibited ZIKV production significantly more than other statins, and showed infectivity of 2.94% and 1.42%, respectively. These results confirm that statins can significantly reduce the production of infectious ZIKV particles in Vero cells.

in vero cells, lipophilicity statin intracellular by treatment ZIKV decrease in production

To confirm the effect of lipophilic statins against ZIKV intracellularly, the same treatment scheme was adopted in ZIKV-infected (0.1 MOI) Vero cells and immunofluorescence assay (IFA) for flavivirus E protein (4G2) 48 hpi was performed (FIG. 3). All statin-treated cultures had fewer foci of infection and a lower percentage of infected cells than non-statin-treated ZIKV-infected cultures. The replication process of flaviviruses, which is also thought to be observed in ZIKV, occurs through invasions of the endomembrane system, especially at the endoplasmic reticulum surface. Thus, replication can be observed around the nucleus of cells. Statin-untreated cultures had numerous ZIKV particles in the intracellular, extranuclear region and throughout the cytoplasm, suggesting replication and trafficking of the virus. However, fewer ZIKV particles were observed intracellularly in statin-treated infected Vero cells. ZIKV particles appeared to be localized to nuclear regions within cells in the statin-treated environment, and some appeared as perinuclear dots. These results suggest inhibition of replication or trafficking in the infected cells. There was also a visual difference in the effect of the statins on the spread of the virus, where CER appeared to have minimal inhibitory action on replication, but a greater localization of the virus and a greater foci of infection than the other statins. has been shown to cause These results indicate that statins can reduce infection by inhibiting entry as the number of infected cells decreases, or by inhibiting replication by infected cells having a restricted ZIKV region.

(3) Conclusion

Statins are selective inhibitors of HMGCR, and as analogs of HMG-CoA, they compete with HMG-CoA for the binding site of the enzyme and interfere with the conversion of HMG-CoA to L-mevalonate. This conversion is the rate limiting step of the mevalonic acid (MVD) pathway; Thus, statins interfere with downstream processes in the pathway, including the synthesis of cholesterol and the production of isoprenoid metabolites (eg, geranylgeranylpyrophosphate and farnesylpyrophosphate). The pharmacophore of statins is a closed or open ring modified hydroxy glutaric acid segment attached to a substituent ring system (Fig. 1a). Variation of these substituents results in differences in the pharmacological properties of statins (eg solubility, bioavailability and stability).

In the present invention, first, the activity of lipophilic statins (ATO, CER, FLU, LOV, MEV, and SIM) and hydrophilic statins (PRA, ROS) against ZIKV was measured in vitro. Results according to the present invention show that the use of lipophilic statins reduces the production of infectious ZIKV particles in Vero cells. Specifically, lipophilic statins have the ability to reduce the production of infectious ZIKV particles released into the supernatant of infected Vero cell cultures over time, with a particularly significant reduction starting at 48 hpi. According to the plaque assay, as seen with the LOV treatment, the reduction was as high as 98% plaque forming units (PFU). Immunofluorescence analysis also showed that statin treatment reduced the ability of ZIKV to infect cells, resulting in a lower percentage of infected cells and fewer foci of infection. Statin-treated cells showed more restricted localization of ZIKV particles, which was confirmed by the fact that ZIKV was more localized to the perinuclear region than non-statin-treated cells, where ZIKV was predominantly present throughout the cell.

Through these results, it was confirmed that the lipophilic statin compound according to the present invention has very excellent antiviral activity against Zika virus. Therefore, the lipophilic statin compound according to the present invention is expected to be used as an effective treatment for Zika virus, for which no vaccine or treatment has been developed yet, and also has limitations in vaccine treatment due to antigenic diversity or antigenic mutation. It is expected to be useful as an antiviral composition that is effective and has no side effects in virus-related diseases.

Example 2.

(1) Experiment method

Through Example 1, it was confirmed that lipophilic statins showed various inhibitory effects on ZIKV in vitro. In particular, at different stages of the ZIKV replication cycle, ATO and FLU act in the early stages of infection and have open-ring pharmacophores coupled to substituent ring systems, whereas SIM and MEV have closed-ring pharmacophores. Of these, SIM acted at the intermediate stage of infection. Therefore, it was tested whether the combination of the lipophilic statins exerts a synergistic effect on inhibiting ZIKV production in Vero cells. In addition, the effect of combination with chloroquine (CLQ), known as an antiviral candidate for various viruses including ZIKV, was also tested.

To this end, first, based on the EC ₅₀ values calculated from previously established dose-response curves, the early stage of infection and opening within the range of 0.125-2 × EC _-50 (50% the maximal effects for inhibiting ZIKV infection) Half-dilutions of early-stage acting statins with ringed pharmacogroups (ATO, FLU) and statins with intermediate-stage acting or closed-ring pharmacogroups (MEV, SIM) were used. In addition, a modified checkerboard strategy was used as shown in Figure 6, cells treated with 0.5% dimethyl sulfoxide (DMSO) were used as a negative control, and 5 days after infection, EZ Cytox reagent (DoGenBio, Seoul, South Korea) was used to measure cell viability. The infection inhibitory effect was expressed as % protection, and the % protection was calculated according to Equation 2 below.

[Equation 2]

%Protectivity = (Absorbance _TREATED - Absorbance _{INFECTEDCONTROL} ) / (Absorbance _CELLCONTROL - Absorbance _{INFECTEDCONTROL} ) Х 100%

The fractional inhibitory concentration index (FIC) was calculated by FIC = (a/a _i ) + (b/b _i ), where a and b are the concentrations of drugs 1 and 2, respectively, and the desired % protection. Combinations _ai and _bi that yield rates are the concentrations of drugs 1 and 2, respectively, that yield the desired percent protection. FIC = 0.5-<1.0 is considered suggestive of synergy, and FIC <0.5 can be considered a strong indicator of synergy.

(2) Results and discussion

Combination of fluvastatin and mevastatin (FLU- MEV ), fluvastatin and of simvastatin Synergistic effect by combination (FLU-SIM)

Combinations of FLU and MEV (FLU-MEV) and FLU and SIM (FLU-SIM) tended to show synergistic effects. The lowest dose of the FLU-MEV combination that showed at least 90% protection in Vero cells against ZIKV infection was the combination of 0.1695 μM MEV and 0.18625 μM FLU ( FIG. 7A ). The FIC calculated for this combination was 0.3, indicating a strong synergistic effect. The combined doses of FLU and SIM that provided 99% protection against ZIKV infection in Vero cells are shown in Figure 7B, and all FLU-MEV and FLU-SIM combinations (FIC < 1.0) showing synergistic effects are shown in Table 2 below. shown in

with atorvastatin Combinations of mevastatin ( ATO - MEV ) synergistic effect

A combination of ATO and MEV (ATO-MEV) also showed a tendency to exhibit a synergistic effect (FIG. 8). Synergistic combinations of ATO and MEV that provided 75% protection against ZIKV infection in Vero cells and their corresponding FIC values are shown in Table 3 below.

with atorvastatin of chloroquine Combination( ATO - CLQ ) synergistic effect

Even when ATO was combined with chloroquine (ATO-CLQ), it tended to exhibit a synergistic effect (FIG. 9). The synergistic combination of ATO and chloroquine providing 75% protection against ZIKV infection in Vero cells and the corresponding FIC values are shown in Table 4 below.

(3) Conclusion

Through Example 2, when a statin (ATO, FLU) having an action in the early stage of infection and an open-loop drug molecule (ATO, FLU) is used together with a statin (MEV, SIM) having an action in the middle of an infection or a closed-ring drug molecule, ZIKV It was confirmed that a synergistic effect of suppression of infection was shown. In particular, when FLU was used as an early-stage-acting statin in infection, a more dramatic effect (90 -99% protection rate) was also confirmed. In addition, even when the lipophilic statin according to the present invention (ATO is used in the examples) is combined with chloroquine (CLQ), which is known as an antiviral candidate for various viruses, it can be confirmed that it exhibits a synergistic effect on inhibiting ZIKV infection. could

Having described specific parts of the present invention in detail above, it is clear that these specific descriptions are only preferred embodiments for those skilled in the art, and the scope of the present invention is not limited thereby. something to do. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (6)

a first active ingredient that is a first lipophilic statin compound having an open-ring drug molecule group; and
A second active ingredient, which is a second lipophilic statin compound or chloroquine having a closed-ring drug molecule group;
The first lipophilic statin compound is atorvastatin (ATO) or fluvastatin (FLU), and the second lipophilic statin compound is mevastatin (MEV) or simvastatin (SIM),
When the first active ingredient is atorvastatin (ATO), the second active ingredient is mevastatin (MEV) or chloroquine,
The antiviral composition for Zika virus, characterized in that when the second active ingredient is fluvastatin (FLU), the second active ingredient is mevastatin (MEV) or simvastatin (SIM). delete According to claim 1,
Wherein the first lipophilic statin compound or the second lipophilic statin compound exhibits antiviral activity by reducing the production of Zika virus particles. According to claim 1,
Wherein the first lipophilic statin compound or the second lipophilic statin compound exhibits antiviral activity by inhibiting the proliferation and replication of Zika virus. a first active ingredient that is a first lipophilic statin compound having an open-ring drug molecule group; and
A second active ingredient, which is a second lipophilic statin compound or chloroquine having a closed-ring drug molecule group;
The first lipophilic statin compound is atorvastatin (ATO) or fluvastatin (FLU), and the second lipophilic statin compound is mevastatin (MEV) or simvastatin (SIM),
When the first active ingredient is atorvastatin (ATO), the second active ingredient is mevastatin (MEV) or chloroquine,
A pharmaceutical composition for preventing or treating Zika virus infectious diseases, characterized in that when the second active ingredient is fluvastatin (FLU), the second active ingredient is mevastatin (MEV) or simvastatin (SIM). a first active ingredient that is a first lipophilic statin compound having an open-ring drug molecule group; and
A second active ingredient, which is a second lipophilic statin compound or chloroquine having a closed-ring drug molecule group;
The first lipophilic statin compound is atorvastatin (ATO) or fluvastatin (FLU), and the second lipophilic statin compound is mevastatin (MEV) or simvastatin (SIM),
When the first active ingredient is atorvastatin (ATO), the second active ingredient is mevastatin (MEV) or chloroquine,
A food composition for preventing or improving Zika virus infectious disease, characterized in that when the second active ingredient is fluvastatin (FLU), the second active ingredient is mevastatin (MEV) or simvastatin (SIM).

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