CN115089591B

CN115089591B - Application of brinib in preparation of medicines for inhibiting enterovirus 71 type neurotropic viruses

Info

Publication number: CN115089591B
Application number: CN202210556766.2A
Authority: CN
Inventors: 王鹏飞; 赵晓宇; 乔芮
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2022-05-21
Filing date: 2022-05-21
Publication date: 2024-04-12
Anticipated expiration: 2042-05-21
Also published as: CN115089591A

Abstract

The invention belongs to the field of biological medicine, and provides an application of brinib in preparing a medicament for inhibiting enterovirus 71 type neurotropic virus. The invention also comprises corresponding medicine combinations, using methods, kits and the like. The study of the invention shows that the brinib can effectively inhibit the activity of enterovirus 71 type neurotropic virus, the virus of RD cells is reduced after brinib treatment, and the EV-A71 inhibition effect in the brinib and the RD cells shows dose-dependent inhibition. The invention screens the medicines with the activity of resisting EV-A71 viruses from the medicines on the market, saves research cost related to medicine metabolism, medicine safety, toxicology and the like in the medicine screening process, reduces the risk of medicine research and development, and provides a new thought and approach for symptomatic treatment of EV-A71 infection diseases and new medicine development.

Description

Application of brinib in preparation of medicines for inhibiting enterovirus 71 type neurotropic viruses

Technical Field

The invention belongs to the field of biological medicine, and relates to a novel inhibitor of enterovirus 71 type neurotropic virus. In particular to application of brinib in preparing a virus-inhibiting medicament.

Background

Enterovirus 71 (EV-a 71) is a neurotropic virus belonging to the genus enterovirus of the family picornaviridae, and is one of the main pathogens causing hand-foot-mouth disease, angina, and herpes. The hand-foot-mouth disease is a global disease, and occurs all the year around in China, the incidence rate is 37.01-205.06 cases for every 10 thousands of people, and the death rate is 6.46-51.00 cases for every 10 thousands of people; in the diagnosis results of the etiology of the hand-foot-and-mouth disease in the laboratory, the positive proportion of EV-A71 accounts for 44%, the positive proportion of EV-A71 accounts for 74% in severe cases, and the positive proportion accounts for 93% in death cases; hand-foot-and-mouth disease caused by EV-A71 infection is common in infants and is an important cause of death for children in China nowadays. The most effective method for preventing EV-A71 infection is to vaccinate the age-appropriate children with EV-A71 vaccine; despite the current market of vaccines, there is no effective immunopotentiation study data for which the efficacy and duration of immunization remains to be further validated.

At present, specific anti-EV-A71 infection medicines are not clinically available, the research of the anti-EV-A71 medicines is in basic experimental stages, and the lack of high-efficiency antiviral medicines is still a problem to be solved urgently. Because the new medicine development has long time consumption, high cost and high risk, the medicine with the activity of resisting the EV-A71 virus is screened from the medicines on the market, the research cost related to medicine metabolism, medicine safety, toxicology and the like in the medicine screening process is saved, the risk of medicine research and development is reduced, and a new thought and approach are provided for symptomatic treatment of EV-A71 infection diseases and new medicine development.

Brinib (Brivanib alaninate) (BMS-582664) is an ATP-competitive VEGFR2 inhibitor, IC ₅₀ A value of 25nM; can moderately inhibit VEGFR1 and FGFR1, and has 240 times of selectivity to VEGFR2 than to PDGFRbeta [1 ]]。

Disclosure of Invention

The invention aims to solve the technical problem of providing an application of brinib in preparing a medicine for inhibiting EV-A71 virus.

In one aspect, the invention provides an application of brinib in preparing a medicament for inhibiting viruses, wherein the viruses are enterovirus 71 type neurotropic viruses EV-A71.

Preferably, the drug is a drug that inhibits EV-A71 virus activity.

Further, the drug is a drug for reducing the activity, viral load or VP1 protein expression of EV-A71 virus.

Brianib (Brivanib) shows remarkable curative effects on liver cancer in clinical trials. Thus, currently, brinib is generally used as an anti-liver cancer drug. The structure is as follows:

the brinib can effectively delay the development process of the hepatocellular carcinoma. In a Global Phase III three (BRISK-PSStudiy) clinical trial of brinib as a second-line drug for the treatment of hepatocellular carcinoma:

the brinib group had an ORR (objective remission rate) of 12% and the placebo group had an ORR of 2% (12% vs 2%);

the brinib group had a DCR (disease control rate) of 71% and the placebo group had a DCR of 49% (71% vs 49%);

of the patients taking brinib, 26 were evaluated for efficacy as PR (partial relief) and only 2 were evaluated for efficacy in the placebo group as PR. In another BRISK-TA study, followed by adjuvant therapy in phase III clinical study TACE, the brinib group had 120 Chinese patients, and was shown after subgroup analysis:

chinese patients have characteristics different from those of foreign patients (older age, HBV infection is dominant);

the treatment is also 800mgQD treatment, so that the drug exposure of Chinese patients is higher and the tolerance is better;

brinib can significantly prolong extrahepatic metastasis or portal infiltration time of Chinese HCC and imaging progression time.

In terms of safety, brinib cloth also has the advantage: the side effects are 1-2 grade, the most common side effects of vascular inhibitor targeting drugs, namely, the incidence of hand-foot syndrome is 15%, and the incidence of sorafenib hand-foot syndrome is 45% (Oriental research).

Brinib acts on VEGFR and FGFR, with phase III clinical trials showing: brivanib first-line treatment for hepatocellular carcinoma, OS is not superior to sorafenib, TPP, ORR and DCR are similar to sorafenib; brivanib second line treatment of hepatocellular carcinoma, OS did not benefit; brivanib is used to assist in the treatment of hepatocellular carcinoma after TACE, and OS does not benefit. OS refers to the time from randomization of patients to death due to any cause in a clinical trial.

In another aspect, the present invention provides a method of inhibiting EV-A71 virus in vitro cells by brinib, said method comprising the steps of:

obtaining in vitro cultured cells containing EV-A71 virus; and/or

Brinib was added to the culture environment of the in vitro cultured cells containing EV-a71 virus and incubated.

Preferably, the brinib is added and distributed uniformly in the culture environment in which the cells are cultured in vitro, and the final concentration of brinib in the culture environment in which the cells are cultured in vitro is not less than 0.5. Mu.M, usually 1 to 25. Mu.M, more preferably 5 to 10. Mu.M, for example, 0.8, 1.0, 1.3, 2.5, 3.0, 5.0, 7.0, 8.0, 10.0, 12.5, 15.0, 18.0, 20.0, 22.5, 25. Mu.M, etc.

Preferably, the brinib is added and uniformly distributed in a culture environment for in-vitro cell culture, and the incubation time is not less than 8 hours; more preferably, the incubation time is not less than 12 hours. In one embodiment of the invention, the time of incubation after the brinib is added to the culture environment in which the cells are cultured in vitro is not less than 24 hours.

In the present invention, the in vitro cultured cells containing the EV-A71 virus can be obtained by a conventional method, for example, by placing virus particles in the culture environment of the cells, or expressing the EV-A71 virus or core components thereof in the cells. In one embodiment of the invention, the cells are RD cells cultured in vitro.

In yet another aspect, the present invention provides a pharmaceutical composition comprising a conjugate comprising brinib and

a) A carrier linked to a label, or

b) An adaptor attached to a solid.

Alternatively, the invention provides a composition comprising an enterovirus type 71 neurotropic virus inhibitor or any pharmaceutically acceptable salt, ester or prodrug thereof. Pharmaceutically acceptable salts, esters or prodrugs include, but are not limited to: sulfate, dimethyl isosorbide,20-80, cyclodextrin (e.g. ] for example>) Squalene, a second propylene glycol, polyethylene glycol (preferably of low molecular weight, such as PEG 400), polysorbate, poloxamer, polyoxyl and combinations thereof.

In still another aspect, the invention provides a kit for inhibiting EV-A71 virus, which comprises an anti-EV-A71 virus drug taking brinib as an active ingredient and being supplemented with pharmaceutically acceptable auxiliary materials, and a container for containing the drug.

In yet another aspect, the present invention provides a method of inhibiting VP1 protein, comprising the steps of:

a) Obtaining VP1 protein or cells producing VP1 protein;

b) Obtaining a medicament containing a brinib component;

c) The agent comprising a brinib component is contacted with a VP1 protein-producing cell or VP1 protein.

In the present invention, the drug to be used for the treatment will vary not only depending on the particular inhibitor selected but also depending on the route of use, which treatment is required and the age, weight and condition of the patient, and will ultimately be decided by the attending physician. Generally, suitable dosages may range from about 0.005 to about 30mg/kg body weight per day, preferably from about 0.05 to about 10 mg/kg/day. The required dose may conveniently be administered as a single dose or in divided doses at appropriate intervals (e.g. twice, 20 times) of three, four or more doses per day. The required dosage may also be, for example, once every two days, once every three days, or even once a week, depending on the therapeutic needs and/or prophylaxis. The composition is conveniently administered in unit dosage form; preferably, the unit dose. For compositions containing 0.5 to 1500mg, conveniently 1 to 1000mg, examples of up to 25 conveniently contain 5 to 700mg of active ingredient per unit dosage form.

The compositions of the present invention will typically be administered orally, parenterally, intravenously, intramuscularly, subcutaneously or by other injection routes, bucally, rectally, vaginally, transdermally and/or nasally and/or by inhalation in pharmaceutically acceptable dosage forms. The compositions may be administered in different dosages depending on the disease to be treated and the patient and the route of administration. Pharmaceutical compositions include, but are not limited to, those suitable for oral, rectal, nasal, topical (including buccal and sublingual), transdermal, vaginal or parenteral (including intramuscular, subcutaneous and intravenous) administration or inhalation or insufflation in a form suitable for administration by the following means.

The composition may conveniently be presented in discrete dosage units, as appropriate, by any of the methods well known in the art of pharmacy. Compositions suitable for oral administration in the pharmaceutical industry are conveniently presented in individually packaged units, such as capsules, cachets or tablets, each containing a predetermined amount of the active agent. Oral tablets and capsules may contain conventional pharmaceutical excipients such as binding agents, fillers, lubricants, disintegrants or wetting agents. The tablets may be coated according to methods known in the art. The compositions may be formulated for parenteral administration (e.g., by injection (e.g., bolus injection or continuous infusion) and may be in unit dosage form in small dosage ampoules or in multi-dose containers with added preservative.

The following examples are provided to illustrate the invention without being limited thereto and should not be construed as limiting the invention. The following are examples of many non-limiting examples of compositions comprising stable amorphous hybrid nanoparticles. The abbreviations have the following meanings:

"I" represents the viral inhibitor brinib; "P" represents a component of the polymer that stabilizes and forms a matrix; "S" represents a solubilizing agent;

"I+P" represents the physical mixture of the brinib inhibitor with the polymer stabilizing and matrix forming components, i.e. without further treatment;

"I+S" represents a physical mixture of a brinib inhibitor and a solubiliser;

"I+P+S" represents a physical mixture of brinib inhibitor including brinib inhibitor, polymer stabilizing and matrix forming components and solubilizing agent;

"I/P" represents a stable, amorphous hybrid nanoparticle with a brinib inhibitor and a polymer stabilizing and matrix forming component;

"I/P+S" represents stable amorphous hybrid nanoparticles with 15 Brinib inhibitor and polymer stabilizing and matrix forming components and addition of a separate solubilizing agent;

"I/P/S" means stable, amorphous, hybrid nanoparticles with brinib inhibitor, polymer stabilizing and matrix forming components and solubilizing agents.

As used herein, the term "composition" is intended to encompass a product comprising the specified ingredients in the specified amounts, as well as any product which results, directly or indirectly, from combination of the specified ingredients in the specified amounts.

The term "treating" refers to reversing, alleviating, inhibiting or slowing the progression of a disease, disorder or condition for which these terms apply or one or more symptoms of such a disease, disorder or condition.

Unless otherwise indicated, all percentages of ingredients are weight per volume (w/v), unless otherwise indicated, w/v percentages refer to weight percentages of the final composition.

As used herein, all values defined as "about" each particular value, relating to amount, weight, etc., are plus or minus 10%. For example, the phrase "about 5% w/v" is understood to mean "4.5% to 5.5% w/v". Accordingly, the scope of the claims encompasses amounts within 10% of the claimed value.

The term "pharmaceutically acceptable" describes materials that are not biologically or otherwise undesirable, i.e., materials that do not cause unacceptable levels of undesirable biological effects or interact in a deleterious manner.

As used herein, the term "effective amount" refers to an amount sufficient to affect a desired biological effect, such as a beneficial result, including, but not limited to, preventing, reducing, alleviating or eliminating a sign or symptom of a disease or disorder. Thus, the total amount of each active ingredient of the pharmaceutical composition or method is sufficient to exhibit a meaningful subject benefit. Thus, an "effective amount" will depend on the environment in which it is to be administered. An effective amount may be administered in one or more prophylactic or therapeutic modes of administration.

The term "prodrug" refers to compounds, including monomers and dimers of the compounds of the invention, which have cleavable groups and become pharmaceutically active in vivo under physiological conditions.

As used herein, "salts" refer to those salts that retain the biological effectiveness and properties of the parent compound and are not biologically or otherwise harmful at the dose administered. Salts of the compounds of the present invention may be prepared from inorganic or organic acids or bases.

The compounds of the present invention may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids or bases. The phrase "pharmaceutically acceptable salt" refers to salts that are, within the scope of sound medical judgment, suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, commensurate with the salt. Reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S.M. Berge et al 1977 describe in detail pharmaceutically acceptable salts in the pharmaceutical science.

These salts may be prepared in situ during the final isolation and purification of the compounds of the present invention or separately by reacting the free base functionality with a suitable organic acid. Representative acid addition salts include, but are not limited to, acetates, adipates, alginates, citrates, aspartate, benzoate, benzenesulfonates, bisulphates, butyrates, camphorates, camphorsulfonates, digluconates, glycerophosphate, hemisulfates, heptanates, caprates, fumarates, hydrochlorides, hydrobromides, 2-hydroxyethanesulfonates (isothiocyanates), lactates, maleates, methanesulfonates, nicotinic acid salts, 2-naphthalenesulfonates, oxalates, palmitate, pectates, persulfates, 3-phenylpropionates, picrates, pivalates, propionates, succinates, tartrates, thiocyanates, bicarbonates, phosphates, glutamate p-toluenesulfonates, and undecanoates. Alternatively, basic nitrogen-containing groups may be quaternized with quaternizing agents such as lower alkyl halides, e.g., methyl, ethyl, propyl and butyl chlorides, bromides and iodides. Dialkyl sulfates such as dimethyl, diethyl, dibutyl and diamyl sulfate; long chain halides, such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides; aralkyl halides such as benzyl and phenethyl bromides and the like. Thus obtaining a water-soluble or oil-soluble or dispersible product. Examples of acids that can be used to form pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric, hydrobromic, malic, sulfuric and phosphoric acids, and organic acids such as oxalic, malic, maleic, methanesulfonic, succinic and citric acids. Preferred acid addition salts are prepared from methanesulfonic acid, malic acid and phosphoric acid.

Basic addition salts may be prepared in situ during the final isolation and purification of the compounds of the invention by contacting the carboxylic acid-containing moiety with a suitable base, such as a hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, or with ammonia or an organic primary, secondary or tertiary amine. Pharmaceutically acceptable salts include, but are not limited to, alkali or alkaline earth metal based cations such as lithium, sodium, potassium, calcium, magnesium, and aluminum salts and the like, as well as non-toxic quaternary ammonium and amine cations including ammonium, tetramethyl ammonium, tetraethyl ammonium methyl ammonium, dimethyl ammonium, trimethyl ammonium, triethyl ammonium, diethyl ammonium, and ethyl ammonium and the like. Other representative organic amines useful in forming base addition salts include ethylenediamine, ethanolamine, diethanolamine, piperidine, piperazine and the like.

As used herein, the term "ester" is represented by the formula-OC (O) a, 1 or-C (O) OA1, wherein A1 may be alkyl, cycloalkyl, alkenyl, cycloalkenyl, alkynyl, cycloalkynyl, aryl, heteroaryl, or other suitable substituents.

As used herein, the term "total survival" (OS) is defined as the time from the onset of randomization to the death of a patient for various reasons, and is calculated as the intended treatment population (ITT). Generally refers to the time from randomization to death of the patient in the clinical trial for any reason.

As used herein, the term "objective remission rate" (ORR) refers to the proportion of patients whose tumor volume is reduced to 30% (in general) and which can sustain minimum time period requirements, as a short efficacy evaluation index, is the sum of the Complete Remission (CR) and Partial Remission (PR) proportions. Colloquially, a higher ORR means more patients with reduced tumors using a therapy than the proportion of patients who are effective after receiving a certain treatment. Objective Remission Rate (ORR) refers to the proportion of patients whose tumor volume is reduced to a predetermined value and which can maintain minimum time period requirements. Remission generally refers to the period of time from the onset of therapeutic effect until the onset of tumor progression is confirmed.

As used herein, the term "disease control rate" (DCR) is calculated by the formula dcr=cr+pr+sd. CR and PR refer to complete remission and partial remission, respectively, SD (stabledisease) refers to stable and substantially controlled disease.

As used herein, the term "CC ₅₀ "means the concentration of compound required to reduce cell viability by 50%.

As used herein, the term "EC ₅₀ "means the concentration of compound required to achieve 50% protection from virus-induced cytopathogenicity.

As used herein, the term "SI" refers to the selectivity index, which is the ratio of CC50/EC 50.

As used herein, the term "patient" refers to, but is not limited to, a human or other animal.

As used herein, the term "drop-wise" refers to any method of incrementally adding one solution to another solution.

The invention provides a new application of brinib, which can effectively inhibit the activity of enterovirus 71 type neurotropic virus, reduce the virus of RD cells after being treated by brinib medicaments, and inhibit the dose dependence of the brinib on EV-A71 in the RD cells. The invention provides a medicament with the anti-EV-A71 virus activity of brinib cloth, which saves research cost related to medicament metabolism, medicament safety, toxicology and the like in the medicament screening process, reduces the risk of medicament research and development, and provides a new thought and approach for symptomatic treatment of EV-A71 infection diseases and development of new medicaments.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present application, and that other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 shows the relationship between cell activity (%) and brinib concentration in RD cells to obtain the CC of brinib against RD cells ₅₀ 23.87. Mu.M;

FIG. 2 viral reduction of RD cells following brinib treatment;

RD cells treated with DMSO or an indicator inhibitor (10. Mu.M) were seeded with EV-A71 at an MOI of 0.1 and after 24 hours, wherein FIG. 2A is the relative viral load (%) after DMSO and brinib treatment in the cell lysate; FIG. 2B is the relative viral load (%) after DMSO and brinib treatment in the supernatant; FIG. 2C by TCID ₅₀ Determining, by determining, the viral titer of the inhibitor-treated sample; FIG. 2D analysis of EV-A71 VP1 protein by Western blot, GAPDH was used as loading control;

FIG. 3 dose-dependent inhibition of EV-A71 by Brivanib in RD cells.

RD cells treated with the indicated concentrations of Brivanib inhibitor are inoculated with EV-A71 at an MOI of 0.1, and after 24 hours, the virus in cell lysates (A) and supernatants (B) are analyzed using RT-qPCR based on copy number and the Brivanib inhibitor is found to exhibit dose-dependent inhibition of EV-A71 infection. Results represent viral load in cells treated with Brivanib inhibitors at the indicated concentrations relative to DMSO-treated cells. At TCID ₅₀ In the assay, the viral titer (C) of the inhibitor treated samples was also determined. (D) EV-A71 VP1 protein was analyzed by Western blotting, and GAPDH was used as a loading control. The data show the mean and SD of three independent experiments. * P (P)<0.05；**P<0.01；***P<0.001。****P<0.0001。

Detailed Description

The following description of the technical solutions in the embodiments of the present application will be clear and complete, and it is obvious that the described embodiments are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by one of ordinary skill in the art without undue burden from the present disclosure, are within the scope of the present disclosure.

Example 1.

Toxicity of brinib on RD cells

To evaluate the antiviral effect of brinib, we measured 50% cytotoxic concentration of brinib (CC ₅₀ ) To exclude the adverse effect of these inhibitors on RD cell viability.

By fingerTreatment of RD cells with defined concentrations of brinib usingDetermination of CC by luminous cell viability reagent ₅₀ The corresponding results are shown in FIG. 1, and the CC of Brivanib (Brivanib) on RD cells is calculated according to the experimental detection results ₅₀ 23.87. Mu.M.

Example 2

Antiviral action of brinib on EV-A71

Based on the cytotoxicity results above, we first selected 10 μm as the concentration of drug-treated RD cells. Next, RD cells treated with DMSO or a specified inhibitor (10. Mu.M) were infected with EV-A71 having an MOI of 0.1, and 24 hours later, the antiviral effect of the Bulinide on EV-A71 was compared by viral load, viral titer and expression of viral structural protein VP1, and the corresponding results are shown in FIG. 2 and Table 1.

TABLE 1 antiviral Effect of brinib on EV-A71

The relative viral load (%) in the cell lysate treated with EV-a71 as 100% as in fig. 2A brinib was 0.575663; the relative viral load (%) in the brinib-treated supernatant in fig. 2B was 3.36421; FIG. 2C at TCID ₅₀ In the assay, TCID in brinib-treated supernatant ₅₀ The volume per ml is 5.01X10 ⁵ Determining the viral titer of the inhibitor-treated sample; FIG. 2D analysis of EV-A71 VP1 protein by Western blot, GAPDH was used as loading control; the data show the mean and SD of three independent experiments. * P (P)<0.05；**P<0.01；***P<0.001。****P<0.0001。

As shown in FIG. 2D, the VP1 protein content in the brinib-treated cells was very low. According to the corresponding experimental detection results, the brinib has a protective effect on RD cells infected by EV-A71 under the non-toxic concentration, namely, the brinib has an inhibitory effect on EV-A71 on the RD cells, which indicates that the brinib has the anti-EV-A71 virus activity.

Example 3

Brivanib dose-dependently inhibits EV-A71 infection and replication

After 24h infection of RD cells treated with DMSO or a specified inhibitor concentration with EV-A71 having an MOI of 0.1, the antiviral effect of Brivanib on EV-A71 was determined by viral load, viral titer and expression of the viral structural protein VP1, corresponding results are shown in FIGS. 3A-3D:

RD cells treated with the indicated concentrations of Brivanib inhibitor are inoculated with EV-A71 at an MOI of 0.1 for 24 hours, and assayed using RT-qPCR with 100% dimethyl sulfoxide (DMSO) as a control. Wherein, the relative viral loads (%) of Brivanib treated RD cells of fig. 3A cell lysates were about 84.9, 36.8, 7.6, 1.8, 0.5, respectively, at 0.5, 1, 5, 10, 25 μΜ, respectively; FIG. 3B shows relative viral loads (%) of 0.5, 1, 5, 10, 25. Mu.M Brivanib-treated RD cells in the supernatant of about 68.1, 52.3, 27.4, 13.9, 2.3, respectively; FIG. 3C at TCID ₅₀ In the assay, the viral titers of inhibitor-treated samples were also determined, as were the progressive decline in viral titers using Brivanib at 0.5, 1, 5, 10, 25 μm, DMSO-treated cells TCID ₅₀ 6.05X10 ⁶ Whereas 25. Mu.M Brivanib-treated cell TCID ₅₀ 1.59X10 ⁴ See table 2 for Brivanib at various concentrations and 100% control of DMSO-treated cells; as shown in FIG. 3D, using GAPDH as a loading control, it was found by Western blot analysis that the EV-A71 VP1 protein was decreased in the cells after 0, 0.5, 1, 5, 10, 25. Mu.M Brivanib treatment, and the VP1 protein was barely detected at a concentration of 5. Mu.M (D). The data show the mean and SD of three independent experiments; * P (P)<0.05；**P<0.01；***P<0.001。****P<0.0001。

TABLE 2 Brivanib at different concentrations 100% control in dimethyl sulfoxide (DMSO) treated cells

Brivanib	Cell lysate (relative%)	Supernatant (relative value%)	TCID ₅₀ /ml
				DMSO	100	100	6.05×10 ⁶
0.5	84.9	68.1	2.82×10 ⁶
				1	36.8	52.3	7.88×10 ⁵
5	7.62	27.4	2.82×10 ⁵
				10	1.84	13.9	1.22×10 ⁵
25	0.467	2.33	1.59×10 ⁴

Experimental results show that, in a certain concentration range, as the concentration of Brivanib gradually increases, the viral load, viral titer and expression of viral structural protein VP1 gradually decrease, indicating that Brivanib can inhibit EV-a71 infection and replication dose-dependently.

Example 4

Antiviral Activity, cytotoxicity and Selectivity index against EV-A71

The invention also evaluates the CC infection of EV-A71 by Brivanib ₅₀ And EC (EC) ₅₀ And a corresponding Selectivity Index (SI).

Experimental results show that Brivanib exhibits higher cytotoxicity (23.87+ -2.23 μM) in RD cells at half maximum Effective Concentration (EC) of 1.94 μM+ -0.33 ₅₀ ) Effective in inhibiting EV-A71 infection according to CC ₅₀ /EC ₅₀ And (5) calculating. According to the experimental detection results, the selectivity index SI (selectivityindex) of the Brivanib (Brivanib) for resisting EV-A71 infection in RD cells is 12.3, and the activity of EV-A71 virus can be effectively inhibited.

The foregoing is merely specific embodiments of the present application, but the scope of the present application is not limited thereto, and any changes or substitutions easily conceivable by those skilled in the art within the technical scope of the present application should be covered in the scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claims.

Claims

1. The application of brinib in preparing medicaments for inhibiting viruses, wherein the viruses are enterovirus 71 type neurotropic viruses EV-A71.

2. The use of brinib according to claim 1 for the preparation of a medicament for inhibiting the viral activity, wherein said medicament is a medicament for inhibiting the EV-a71 viral activity.

3. The use of brinib according to claim 1 for the preparation of a medicament for inhibiting viruses, wherein said medicament is a medicament for reducing EV-a71 viral viability, viral load or VP1 protein expression.