CN109864996A - Pharmaceutical applications of the chitosan oligosaccharide in the medicament for preparing anti-CVA16 type hand-foot-and-mouth disease - Google Patents

Abstract

" pharmaceutical applications of the chitosan oligosaccharide in the medicament for preparing anti-CVA16 type hand-foot-and-mouth disease, belong to technical field of medicine synthesis to the present invention.Antiviral breeding, which is shown, has good anti-CVA16 type virus activity, can be used as the candidate medicine of anti-hand-foot-and-mouth-disease virus.

Description

Pharmaceutical applications of the chitosan oligosaccharide in the medicament for preparing anti-CVA16 type hand-foot-and-mouth disease

Technical field

The present invention relates to field of medicaments, specifically, being related to chitosan oligosaccharide in the medicament for preparing anti-CVA16 type hand-foot-and-mouth disease Pharmaceutical applications.

Technical background

Hand-foot-and-mouth disease (HFMD) is global sexually transmitted disease, is especially widely current in the Asian-Pacific area.In recent years, which is me The highest Class C infectious disease of state's morbidity and mortality falls ill patient based on children.From 1981, in Shanghai, discovery should in China Disease, Beijing, Hebei etc. more than ten of province (city) per having been reported that every year later, and report of infectious disease data is shown in recent years, China's hand-foot-and-mouth disease In sprawling ascendant trend.The prevention and control and treatment of the hand-foot-and-mouth disease public health problem important as one, pay close attention to by people.

Hand-foot-and-mouth disease is the infectious disease being caused by enterovirus, and the enterovirus for causing hand-foot-and-mouth disease has more than 20 kinds (types), It is wherein most commonly seen with coxsackie virus A 16-type (CVA 16) and enterovirns type 71 (EV 71).The common fever of clinical manifestation, Oral cavity and brothers position fash and bleb etc., children with serious disease can cause the mortality such as myocarditis, pulmonary edema, aseptic meningoencephalitis Complication, individual children with serious disease progression of the disease are fast, lead to death.Lack definite effective antiviral drugs at present, it is clinical main Based on symptomatic treatment, mitigates patient symptom, prevent the generation and development of severe complication.

Currently, first 71 vaccine of EV has reached phase III clinical test in China, which feels clinical EV 71 The protective rate of the hand-foot-and-mouth disease of dye is up to 90%, but this vaccine is obvious to the nothing such as other pathogens such as CVA 16 for causing hand-foot-and-mouth disease Protecting effect.It is popular more at home and abroad as the first preponderant genotype for being found pathogen CVA 16 relevant to hand-foot-and-mouth disease Year, but society can not show a candle to EV 71 to its attention rate, and it is also insufficient to the research of its aetology.

The capsid protein of 16 virus of CVA is symmetrical 12 spherical face stereochemical structures, diameter about 30nm.Nucleocapsid is by list Positive chain RNA and protein composition, no protrusion and coating.Genome is made of about 7410 nucleotide.Viral genome is by 5 ' Noncoding region (UTRs), the area P1, P2 and P3 and 3 ' noncoding regions (UTRs) composition.5 ' UTRs include a virion protein (VPg1), related with the synthesis of viral RNA and assembly, 3 ' UTRs have the function of enhanced virus infection ability.Viral genome The open reading frame of albumen, preceding albumen can pass through the hydrolysis of protease before only one poly being made of 2193 amino acid Effect, is cut into tri- kinds of precursor proteins of P1, P2 and P3.P1 can continue to degrade, and ultimately form tetra- kinds of structure eggs of VP1-VP4 White, wherein three kinds are located at viral case surface, i.e. VP1, VP2 and VP3, the overwhelming majority in antigenic determinant is included in it In, and VP4 is embedded on the inside of virus and is connected with viral RNA.Identification of these surface antigen determinants in virus to host It plays a significant role in invasion.P2 and P3 encodes 7 non-structural proteins of 2A, 2B, 2C, 3A, 3B, 3C and 3D, and the region is to virus Duplication and posttranslational modification play an important role, and also play an important role during viral effectively infection host cell.

With the development of drug research new method and new technology, more and more protein three-dimensional structures are confirmed, and are generated More and more drug targets, a large amount of target proteins have been chosen as potential therapeutic targets and have been studied.Molecular docking side Method is widely used to drug research and design, and this main Applied Computer Techniques of method divide to simulate smaller ligand with big Recombination reaction between sub- receptor, to realize area of computer aided drug virtual screening.Virtual screening based on molecular docking technology Method can largely save the research cost of drug and be effectively shortened drug development cycle.Molecular docking technology be according to Receptor structure carries out the important method of drug design, is the important means of innovation research and development drug.

Summary of the invention

In order to solve the problems in the existing technology, the purpose of the present invention is according to small molecule compound and drug targets Between molecular docking operation, a kind of medicament of anti-CVA16 type hand-foot-and-mouth disease of the new high-efficiency with accurate molecular target is provided.

The present invention for target, using molecular docking technology, is screened with coxsackie virus A 16-type (CVA 16) and finds shell widow Sugar has good inhibitory activity to coxsackie virus A 16-type, is based on this, and the claimed scheme of the present invention is as follows:

The purposes of compound shown in Formulas I in medicine preparation, the drug refer to the medicine treated or prevented for hand-foot-and-mouth disease Object, the drug using compound or its salt shown in Formulas I as active pharmaceutical ingredient,

The purposes of compound shown in Formulas I in medicine preparation, the drug refer to the drug for anti-CVA16 type, the medicine Object using compound or its salt shown in Formulas I as antiviral activity ingredient,

It further include adding pharmaceutically acceptable auxiliary material in compound of formula I or its salt in above-mentioned pharmaceutical applications.

In above-mentioned pharmaceutical applications, the drug can be made into injection, creme, oral agents or pill.

For the present invention through experimental study, small molecule agent shown in formula (I) or its salt show obviously 16 type virus of CVA Inhibiting effect.

Specific embodiment

The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

Test material:

Formula ICAS#148411-57-8, chitosan oligosaccharide are purchased from Wuhan City's conjunction Mesophytization Manufacturing Co., Ltd.

Experimental example 1

The antiviral activity of formula (I) compound detects.

For the Anti-viral activity in vitro for detecting test compound, CVA 16 is isolated from brothers mouthful clinical samples, is used Plaque assay carries out the artificial cumulative evidence of extracorporeal antivirus effect to test compound.

By 1 × 10⁶(the pernicious embryo's rhabdomyoma cell of people, Cat NO.:CL-0193, tissue come the RD cell in a/hole Source: muscle, rhabdomyosarcoma) 6 orifice plates are inoculated in, after being incubated overnight, virus liquid to be measured is pressed into 10 times of dilution proportion infection cells, Each dilution repeats 3 holes, and supernatant is removed after 37 DEG C of infection 1h, be added containing 1.2% methylcellulose, 2% serum partly Solid medium (DMEM high sugar (article No.: PM150210)+10%FBS (article No.: 164210-500)+1%P/S (article No.: PB180120)), incubator culture 5-6 days to virus plaques are placed in be formed.

Fixed through paraformaldehyde, the rinsing of violet staining, clear water, dry after make plaque counting, calculate virus titer.With The virus liquid to be measured infection of MOI=0.001/0.01 is inoculated in the RD cell of 6 orifice plates, after infection,

The semisolid culturemedium containing 5 μm of ol/L compounds is added, is placed in 37 DEG C of incubator cultures, and carry out plaque meter Number.

It is control so that the semisolid culturemedium containing solvent DMSO is added, investigates test compound to the shadow of virus titer It rings.

Plaque assay is the results show that compared with DMSO solvent control group, in the presence of test compound, plaque shape It is significantly reduced at number, shows that test compound can inhibit the duplication of 16 virus of CVA, there is antiviral activity.

Experimental example 2

The viral suppression of formula (I) compound measures.

16 suspension of CVA is used the cell maintenance medium for containing 2% serum (DMEM culture solution is added to the fetal calf serum inactivated extremely Final concentration of 2%, and penicillin and streptomysin are separately added into final concentration of 100 μ g/mL, glutamine to final concentration of 2mmol/L) from 10^-1To 10^-10Various concentration gradient dilution is titrated respectively on the adherent RD cell for covering with 96 orifice plate single layers, Continue the CO at 37 DEG C 5% with new cell maintenance medium₂It is cultivated in incubator.

After 96 orifice plate cells of virus infection are incubated for 48h in the incubator, cytopathic effect is estimated under microscope (CPE), there is the titration of virus concentration and lesion hole count of cytopathy in observation, calculates virus liquid according to Reed-Muench formula Cell culture 50 3nfective dose (TCID₅₀), when subsequent experimental, uses virus concentration for 100TCID₅₀。

The cell inoculation of logarithmic growth phase is in 96 orifice plates, in 37 DEG C 5% of CO₂After culture grows up to single layer in incubator, Culture solution is discarded, PBS is washed twice, and the 100TCID of bulk testing compound is added₅₀100 μ l of viral suspension, 37 DEG C 5% CO₂After being incubated for 1.5h in incubator, infection liquid is discarded, PBS is washed three times, is separately added into containing 20 μ g/mL and 10 μ g/mL test The DMEM cell maintenance medium of compound continues to cultivate.

After 48h, when cytopathic effect about 90% occur in virus control wells, the lesion of microscopic visual measurement medicine group cell is imitated It answers.Whole experiment process sets without any processing for cell controls group, and being added without trial drug is virus control group.

Cell CPE judgment criteria: cell-free lesion is denoted as-, 25% or less cytopathy is denoted as+, 25-50% cytopathy It is denoted as ++, 50-75% cytopathy is denoted as +++, 75% or more cytopathy is denoted as ++++.

The tissue culture plate supernatant removal that cytopathy effect observation is finished, PBS is washed twice, with MTT colorimetric method meter Calculate viral suppression:

After measured, the TCID of strain CVA 16 is tested₅₀It is 10^-5.9, the virus concentration that when test uses is 100TCID₅₀.It presses Virus liquid is diluted according to above-mentioned dilution gradient, after infection cell, CPE occur in 48h virus control group cell is ++++when, microscope Drug-treated is estimated for the inhibiting effect of virus, it is 56% that mtt assay, which measures viral suppression,.

Experimental example 3

CC50, EC50 and SI of formula (I) are measured.

CC50, that is, median toxic concentration has guided the drug concentration of 50% poisoning of drug of cell.Test compound DMSO (diformazan Base sulfoxide) it is configured to the mother liquor of 10mg/mL, respective concentration is diluted to cell maintenance medium when experiment.It will have been grown in 96 orifice plates Culture solution is removed at the RD cell of single layer, is replaced respectively with the cell maintenance medium containing various concentration dilution drug.Each concentration 3 A repetition, concurrently setting normal cell is control group.37 DEG C 5% of CO₂48h is cultivated in incubator.It is observed under inverted microscope Cytotoxicity situation after various concentration drug-treated, and with MTT colorimetric method for determining cell survival rate.The Probit of SPSS software The Return Law calculates drug CC50.

EC50, that is, medium effective concentration refers to 50% drug concentration for effectively inhibiting virus.Refer to cytopathy in embodiment 2 Number observation of steps are almost the same, and drug concentration is set as multiple and different concentration gradients.CPE lesion occur to virus control group is ++++ When, the cytopathy situation of microscopically observation various concentration drug-treated group, the respective HIV suppression of MTT colorimetric method for determining Rate.The Probit Return Law of SPSS software calculates drug EC50.

SI, that is, drug therapeutic index=CC50/EC50.SI value is bigger, illustrates that antiviral potentiality are bigger.

The result is as follows:

16 activity of cytotoxicity and anti-CVA of table invention formula (I)

Small molecule agent compound of the invention has passed through specific real as the application of anti-16 type viral agent of CVA Example is described, those skilled in the art can use for reference the content of present invention, and the links such as appropriate feed change, process conditions are realized Corresponding other purposes, correlation change all without departing from the contents of the present invention, this be to those skilled in the art it is aobvious and It is clear to.Therefore, these modifications for being done without departing from theon the basis of the spirit of the present invention improve, and belong to that the present invention claims guarantors The range of shield.

Claims (4)

1. the purposes of compound shown in Formulas I in medicine preparation, the drug refers to the drug treated or prevented for hand-foot-and-mouth disease, The drug using compound or its salt shown in Formulas I as active pharmaceutical ingredient,

2. the purposes of compound shown in Formulas I in medicine preparation, the drug refers to the drug for anti-CVA16 type, the drug Using compound or its salt shown in Formulas I as antiviral activity ingredient,

3. in pharmaceutical applications of any of claims 1 or 2, the drug further includes adding pharmacy in compound of formula I or its salt Upper acceptable auxiliary material.

4. in pharmaceutical applications of any of claims 1 or 2, the dosage form of the drug is injection, creme, oral agents or pill.