CN105456283B - The application of macrolide antibiotics or its pharmaceutical salts in preparing anti-hand-foot-and-mouth-disease drug - Google Patents
The application of macrolide antibiotics or its pharmaceutical salts in preparing anti-hand-foot-and-mouth-disease drug Download PDFInfo
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Abstract
The invention discloses the application of macrolide antibiotics or its pharmaceutical salts in preparing anti-hand-foot-and-mouth-disease drug.Experiment shows have exploitation for the drug for the treatment of hand-foot-and-mouth disease or the potential using value of lead compound with activity of the macrolide antibiotics containing 14 yuan, 15 yuan and 16 membered macrolide parent nucleus with anti-EV71 and CVA16 viruses.
Description
Technical field
The invention belongs to new medical use fields.More particularly, to a series of macrolide antibiotics or its is medicinal
New application of the salt in preparing anti-hand-foot-and-mouth-disease drug.
Background technology
Hand-foot-and-mouth disease (hand-foot-and-mouth disease, HFMD) is the acute infection caused by enterovirus
Disease was mainly fallen ill at 5 years old or less in children.HFMD main clinic symptoms are positions maculopapule, the blebs such as hand, foot, oral cavity, severe
Patient may occur in which serious the nervous system disease, or even dead.The enterovirus for causing HFMD has more than 20 kinds (type), wherein with Ke
Sa Qi virus A 16-types (CVA16) and enterovirns type 71 (EV71) are most commonly seen, the enterovirus of other different shapeds and Coxsack
Virus can also cause a disease.There is EV71 and CAV16 highly infectious, infection to be drawn especially in the Asian-Pacific area in global distribution
The hand-foot-and-mouth disease risen has become a kind of disease seriously endangering public's health.Due to lacking special, efficient antiviral agent
Object mainly takes isolation and symptomatic treatment at present according to hand-foot-and-mouth disease practice guidelines (version in 2013).Therefore, it is badly in need of anti-EV71
With the development and exploitation of the enteropathies cytotoxic drug such as CVA16.
The new drug development time is long, R＆D costs are high, and it is then a kind of quick, warp that antiviral drugs is screened from existing drug
The available strategy of Ji.We have found that tetracycline antibiotics have the work for inhibiting EV71 and CAV16 to replicate in previous studies
Property, there is external curing hand-foot-and-mouth disease or the potential value as lead compound.Infant is the principal patient of hand-foot-and-mouth disease
Group, since tetracycline medication is easy to be deposited in the bone and dental tissue of infant, influence normal development of skeleton, 8
Year old or less children forbid oral and inject and use.Although tetracycline medication external application can reduce the propagation of virus, cannot have
Effect treatment severe hand-foot-and-mouth disease.
Macrolide antibiotic be one kind using a macrolide as the weak base lipophilic compound of parent nucleus, it is carbon containing by parent nucleus
Several differences is divided into 14,15 and 16 membered macrolide antibiotic, and lactonic ring is the active group of such antibiotic, with glycosidic bond and
One or more deoxidation glycosyls are connected, and glycosyl is usually cladinose and desosamine.Macrolide antibiotics is to more
Number gram-positive bacteria, mycoplasma and a small number of Gram-negative bacterias have stronger inhibitory activity, are clinically commonly used to treatment leather
Lan Shi positive bacterias respiratory tract and soft tissue infection as caused by streptococcus pneumonia and haemophilus influenzae.Macrolide antibiotics
Antimicrobial mechanism be by combining ribosomes 50S subunits, blocking displacement at peptide and ribosomes on mRNA, to selectively
Inhibit the synthesis of bacterio protein.
In addition to antibacterial activity, macrolide antibiotics also has anti-inflammatory and immunoregulation effect, in clinical treatment virus
Property respiratory tract infection in show potential huge applications value.Cause the common virus of respiratory tract infection include rhinovirus RV,
Respiratory syncytial virus (RSV) RSV and influenza virus.It can induce the generation of different cytokines, packet after these virus infection respiratory tracts
It includes：IL-1 β, IL-6, IL-8, and TNF-α etc., they are played an important role in Physiopathologic proceeding, respiratory tract infection
High lethality precisely due to excessive inflammatory reaction.Research shows that macrolide antibiotics can not only reduce viral infection
The generation for the proinflammatory factor that respiratory tract is induced can also effectively inhibit the duplication of Respirovirus, such as：By inhibiting intracellular influenza
The proteolysis of hemagglutinin and the duplication for interfering influenza virus pass through the RhoA for reducing RV receptor ICAM-1 and RSV receptor activations
Expression and play anti-RV and RSV activity.
By literature search, it in addition to the virus of infection respiratory tract, does not find that macrolide antibiotics has and resists other viruses
Active report.
Invention content
The object of the present invention is to provide a kind of macrolide antibiotics or the new application of its pharmaceutical salts, i.e., anti-enterovirus
EV71 and Coxsackie virus CAV16 etc. are viral, for preparing the drug for the treatment of hand-foot-and-mouth disease.It is hand-foot-and-mouth disease drug simultaneously
Research and development provide possible target site and lead compound, further to carry out structure of modification to macrolide antibiotics, obtaining
It obtains stronger antiviral compound and working foundation is provided.
In order to achieve the above objectives, the technical solution that the present invention takes is：
Macrolide antibiotics of the present invention refers to using a macrolide as parent nucleus, by hydroxyl, with glycosidic bond
A kind of antibiotics being linked with the sugar of 1-3 molecule is divided into 14,15 and 16 yuan by the difference of the carbon containing parent nucleus of its macrocyclic structure
Ring macrolide antibiotics.So far, three generations's macrolide antibiotics is had been developed that.Further, in these big rings
Esters antibiotic can be with acid at salt, or to its structure of modification, generates derivative, as long as these officinal salts or derivative tool
The ability for having the virus such as anti-enterovirus EV71 and Coxsackie virus CAV16, also belongs to protection scope of the present invention.In the present invention
Specific embodiment in, the macrolide antibiotics includes but not limited to that erythromycin, erythromycin octadecanoate, amber second are red
Mycin, Erythromycin Estolate, erythromycin lactobionate, clarithromycin, Dirithromycin, roxithromycin, azithromycin, josamycin, wheat
Enlightening mycin, spiramvcin, acetyl spiramycin, kitasamycin or meleumycin etc..
Drug of the present invention be using macrolide antibiotics or its pharmaceutical salts as active ingredient with it is pharmaceutically acceptable
Auxiliary material combination is made.The pharmaceutically acceptable auxiliary material includes：Starch, microcrystalline cellulose, sucrose, dextrin, lactose, Icing Sugar, Portugal
Grape sugar, sodium chloride, sodium carboxymethylcellulose, crosslinked polyvinylpyrrolidone, magnesium stearate, superfine silica gel powder, vitamin C, half Guang
Propylhomoserin, citric acid and sodium sulfite, xylitol, maltose, polyethylene glycol, simple syrup, ethyl alcohol, propylene glycol, vegetable oil, mannose
One or more of.
Further, the drug can be oral preparation, ejection preparation or topical formulation.The selection of dosage form and auxiliary
The dosage of material is the conventional selection of this field.It is preferential using injection system for infant patient in order to reach more preferable curative effect
Agent.
Beneficial effects of the present invention：
Clinical data shows that the ratio for occurring adverse reaction after infant taking macrolide antibiotics is very low, is a kind of
Biological safety is good, antibiotic suitable for infant.The present invention passes through reality using macrolide antibiotics as active ingredient
Test whether this kind of antibiotic of research has the function of that EV71 and CVA16 is inhibited to replicate, treats hand-foot-and-mouth disease.The result shows that of the invention
The six kinds of main macrolide antibiotics illustrated have the activity of external anti-EV71 and CVA16, especially anti-with spiramvcin
Virus effectiveness is the most notable.When MOI is 0.01, it is respectively 2.74 μM that spiramvcin, which inhibits the EC50 of EV71 and CVA16 production poison amounts,
With 5.19 μM；Adriamycin and josamycin inhibit the effect of production poison to take second place, and it is respectively 11.9 μM to inhibit the EC50 of EV71 and CVA16
With 44.5 μM, 17.5 μM and 54.3 μM.Further study on mechanism shows conjunction of the spiramvcin by inhibition virus protein
At and play anti-enteropathy toxic action, target site is related to the interpretative function of EV715 ' UTR.Macrolide antibiotics faces
Bed side reaction is few, toxicity is low, safe, and clinic is shown to be children's bacterial-infection resisting medication of a kind of safety, is beta-lactam
The preferred medication of the child patient of class antibiotic allergy selects.It is mainly infant in view of hand-foot-and-mouth disease patient population, therefore, big ring
Lactone antibiotic, especially spiramvcin and josamycin have drug or guide chemical combination of the exploitation for treatment hand-foot-and-mouth disease
The potential using value of object.
Description of the drawings
Fig. 1 flow cytometer showeds spiramvcin, azithromycin and josamycin express reporter gene GFP entrained by EV71-GFP
Influence and antiviral activity.By EV71-GFP by after MOI=1PFU/cell vero cells infections, removal infection liquid, addition contains
There are (A) spiramvcin of various concentration, the culture medium of (B) azithromycin or josamycin.Streaming is used after 37 DEG C of culture 18-20h
The GFP luciferase expression situations of Cytometric Analysis cell are calculated using the GFP positive cell rates of non-dosing object as 100% through drug
Cell GFP corresponding positive rates that treated, numerical value are the average value ± SD of three repeated experiments.Streaming figure is independent three times repeats
It is primary in experiment.
Fig. 2 by produce poison amount reduce (virus titer reduction assay) analyze spiramvcin, azithromycin and
The antiviral activity of josamycin.By EV71-MZ201405 and CVA16-MZ201509 (MOI=0.01PFU/cell) respectively with
Viral supernatants are removed after RD cell incubations 1h, add the culture containing various concentration spiramvcin, azithromycin and josamycin
Base, drug concentration are in doubling dilution；It is control with the infection cell of non-dosing object.Supernatant is collected after infection after 48h, using plaque
Method measures virus titer.The virus titer that control cell supernatant measures is set as 100%, in the cell conditioned medium through drug-treated
Opposite virus titer (%)=(control group virus titer-drug-treated group virus titer)/control group virus titer ×
100%.Opposite virus titer percentage under each drug concentration is the average value ± SD of three repeated experiments.
Influence of the time to production measurement is added in Fig. 3 spiramvcins.By EV71-MZ infection RD cells (MOI=5), exist respectively
100 μM of spiramvcins are added in 0h, 1h, 2h, 4h, 6h and 8h after infecting preceding 1h (- 1h) and infection, and the infection cell of non-dosing object is
Control, 10h collects supernatant and measures virus titer by Plaque Technique Detected after infection, and the virus titer of control group supernatant is set as
100%, calculate the opposite production poison amount of drug-treated group.Opposite virus quantity percentage under each administration timing of drug is to repeat reality three times
Average value ± the SD tested.
The influence that Fig. 4 spiramvcins synthesize viral RNA.EV71-MZ201405 infection RD cells (MOI=5) are stood afterwards
100 μM of spiramvcins are added, as a contrast with the cell of non-dosing object, 2h, 4h, 6h and 8h extraction are total after infection
RNA measures viral RNA levels by reverse transcription-quantitative PCR (RT-QPCR).Using GAPDH mRNA level in-sites as internal reference, when by 2h
The viral RNA relative abundance of control cell is set as 1.Experiment is in triplicate.
Fig. 5 analyzes the influence of spiramvcin viral protein translation by EV71-GFP RNA transfections.By the disease of in-vitro transcription
Malicious EV71-GFP geneome RNAs transfect 293A cells, cell are handled with 20 μM and 100 μM of spiramvcins respectively, with without drug
The cell of processing is control, and the expression of 12h fluorescence microscope GFP after transfection, each cell count 100 regards
Open country calculates the GFP positives (GFP in average each visual field+) cell number.
Fig. 6 spiramvcins play EV715 ' UTR the influence of interpretative function.(A) double Reporter gene vector structural schematic diagrams,
Upstream reporter gene firefly luciferase (F-luc) are by the compound-mediated translation of host cell cap binding protein；Downstream
Reporter gene Renilla luciferase (R-luc) mediate translation by EV71-5 ' UTR.(B) double reporter plasmids are transfected
293A cells measure the activity of F-luc and R-luc after being separately added into 100 μM of spiramvcins and tetracycline effect 36h and calculate phase
Reduced value, the R-luc/F-luc in control cell are set as 1.The ratio of drug-treated group is the flat of independent repetition experiment three times
Mean value ± SD.
Specific implementation mode
In order to illustrate more clearly of the present invention, with reference to preferred embodiment, the present invention is described further.Ability
Field technique personnel should be appreciated that following specifically described content is illustrative and be not restrictive, this should not be limited with this
The protection domain of invention.
Drug and reagent
The Macrocyclolactone lactone kind medicine that the present invention is tested is standard items, is purchased from Dalian U.S. logical sequence Technology Co., Ltd.；
The medicine storage liquid of a concentration of 25.6mM is configured to DMSO；When use respectively with containing 10% serum DMEM culture mediums successively into
2 times of dilutions of row, are respectively 256,128,64,32,16,8,4,2,1,0.5 and 0.25 μM using final concentration.Reagent includes：1×
PBS (NaCl 8g/L, KCl 0.2g/L, Na2HPO42.72g/L, NaH2PO424g/L, pH 7.2), DMEM (Gibco), tire ox
Serum (Sigma), 0.05%Trypsin-EDTA (GIbco), paraformaldehyde (raw work biology), crystal violet (Zhuhai shellfish rope biology
Technology Co., Ltd.), MTT (Sigma), DMSO (Sigma), Xba I (Takara), T7RiboMAXTM Large Scale RNA
Production System (Promega), Plasmid Midi Kit (QIAGEN), Trizol reagent
(invitrogen), PrimeScript RT reagent Kit (Takara), TransStart Tip Green qPCR
SuperMix (Quan Shijin), Lipo2000 (Invitrogen), Dual-Glo Luciferase Assay System
(Promega)。
The macrolide antibiotics structure that the present invention uses is as follows：
Cell and Strain
Natural receptor SCARB2 (the human scavenger of 293A-SCARB2 cytotostatic constitutive expressions EV71
Receptor class B, member 2, scavenger receptor B2)；Rhabdomyosarcoma RD Cells and African green monkey kidney Vero cells
It is highly sensitive to EV71, it is widely used for separation, amplification EV71 and CVA16.EV71-GFP is reporter gene GFP from Beijing
The strain of area separation；EV71-MZ210405 is from the isolated highly pathogenic intestines of In Guangdong Province severe hand-foot-and-mouth disease infant
Viral EV71 strains；CVA16-MZ210509 is from the isolated highly pathogenic Ke's Sa of In Guangdong Province severe hand-foot-and-mouth disease infant
Strange virus A 16 strain；293A cells are used for cell transfecting.
Instrument
Cell incubator (Thermo), inverted microscope (Leica), inverted fluorescence microscope (Leica), flow cytometer
(BD AccuriTMC6), quantitative PCR apparatus (BioRad), chemiluminescence detector (Promega)
Embodiment 1
EV71-MZ201405 and CVA16-MZ201509 are infected into Vero or RD cells respectively, under different pharmaceutical concentration,
The expression for the reporter gene GFP that inventor is carried by flow cytometer showed viral genome, observation virus infection are caused thin
The production of born of the same parents' lesion effect (CPE) and plaque analysis progeny virus, has rated above-mentioned six kinds of macrolide antibiotics
It is whether inhibited to the duplication of viral EV71 and CAV16, to host cell whether have protective effect.
The cellulotoxic effect of 1.1 drugs：293A-SCARB, RD and Vero cell are pressed into 1x104/ be inoculated in respectively per hole
In 96 holes, in 5%CO2, overnight incubation under the conditions of 37 DEG C, a series of Macrocyclolactone lactone kind medicine through doubling dilutions handles cell
After 48h, toxicity of the drug to cell is detected with mtt assay；Cell to be cultivated under the same terms, without drug-treated is control；
Three repeating holes of every group of cell calculate the growth inhibition ratio of each group cell.Growth inhibition ratio=[(control group mean OD value-experiment
Group mean OD value)/control group is averaged 0D values] × 100%.The results show that the six kinds of Macrocyclolactone lactone kind medicines surveyed are to 293A-
The CC50 of SCARB2, RD and Vero cell is all higher than 256 μM, and cytotoxicity is not detected in when cell concentration is 256 μM.
1.2, by micro- sem observation cytopathic effect (cytopathic effect, CPE), detect macrolides medicine
Object anti-EV71 and CVA16, the effect for protecting cell：Virus-mediated CPE refers to cell caused by virus is proliferated in the cell and moves back shape
Venereal disease become, show as cell shrinkage, be rounded, vacuole, death occur and fall off, be judge virus multiplication the most frequently used index it
One.By RD cell inoculations in 96 holes, in 5%CO2, under the conditions of 37 DEG C after culture 18-20h, respectively by 1000TCID50's
EV71-MZ201405 and CVA16-MZ201509 viruses infect 1h, replace and contain various concentration and variety classes macrolides
The culture medium of antibiotic is placed in 37 DEG C, 5%CO2It is cultivated in incubator, observes cytopathy state under the microscope daily, when
When 75% or more lesion occurs in the control cell of non-dosing object, experiment is terminated, the CPE degree of dosing group is observed and recorded, CPE's
Recording method is:No CPE is "-", and it is "+" that lesion, which occurs, in 25% cell；25%~50% cytopathy is " ++ "；50%~
75% cytopathy is " +++ "；75%~100% cytopathy is " ++++", the results are shown in Table 1 and table 2.Spiramvcin, Ah
Miramycin, josamycin and clarithromycin have CPE depression effects, gradually mitigate as drug concentration increases CPE effects, cell
State improves, especially spiramvcin significant effect；Only there are weak CPE suppressions in high drug concentration in erythromycin and medecamycin
Effect processed.
Antagonism of 1. Macrocyclolactone lactone kind medicine of table to CPE caused by EV71 (MZ201405)
| 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | |
| Spiramvcin | ++++ | ++++ | +++ | ++ | ++ | + | + | ||||
| Azithromycin | ++++ | ++++ | ++++ | +++ | +++ | ++ | ++ | + | + | ||
| Josamycin | ++++ | ++++ | ++++ | +++ | +++ | +++ | ++ | ++ | + | ||
| Clarithromycin | ++++ | ++++ | ++++ | ++++ | +++ | +++ | +++ | ++ | ++ | + | + |
| Erythromycin | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | +++ | +++ |
| Medecamycin | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | +++ | +++ |
Antagonism of 2. Macrocyclolactone lactone kind medicine of table to CPE caused by CVA16 (MZ201509)
| 0.25 | 0.5 | 1 | 2 | 4 | 8 | 16 | 32 | 64 | 128 | 256 | |
| Spiramvcin | ++++ | ++++ | +++ | +++ | ++ | ++ | + | + | |||
| Azithromycin | ++++ | ++++ | ++++ | +++ | +++ | +++ | ++ | ++ | + | + | |
| Josamycin | ++++ | ++++ | ++++ | ++++ | +++ | +++ | +++ | ++ | ++ | ++ | + |
| Clarithromycin | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | +++ | +++ | ++ | ++ | + |
| Erythromycin | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | +++ | +++ |
| Medecamycin | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | ++++ | +++ | +++ |
Antiviral effect of the 1.3 flow cytometer showed GFP detection Macrocyclolactone lactone kind medicines to EV71-GFP：By Vero cell inoculations
It is incubated overnight in 96 orifice plates, by MOI=1PFU/cell inoculation EV71-GFP viruses, is changed to containing height (100 μ after infecting 1h
The different macrolide antibiotics culture mediums of (40 or 50 μM) in M), low (1 μM) three kinds of concentration, in 5%CO2, 37 DEG C of cultures
After 18-20h, fixed through pancreatin digestion, PBS cleanings, paraformaldehyde, with the GFP luciferase expression feelings of flow cytometry analysis cell
Condition calculates the GFP positive cell relative percentages after drug-treated using the GFP positive cell rates of non-dosing object as 100%,
Numerical value is the average value ± SD of three repeated experiments.Spiramvcin inhibit EV71-GFP expression most pronounced effects, secondly for Ah
Miramycin and josamycin, the result is shown in Figure 1.
1.4 production poison amounts reduce antiviral activity of the analysis Macrocyclolactone lactone kind medicine to EV71 and CVA16：By EV71-
MZ201405 and CVA16-MZ201509 (MOI=0.01PFU/cell) remove viral supernatants with after RD cell incubations 1h respectively,
The culture medium containing various concentration and variety classes macrolide antibiotics is added respectively, drug concentration is in doubling dilution, point
It Wei not be 0.25,0.5,1,2,4,8,16,32,64,128 and 256 μM；It is control with the infection cell of non-dosing object.48h after infection
After collect supernatant, using Plaque Technique Detected measure virus titer.The virus titer that control cell supernatant measures is set as 100%, warp
Opposite virus titer (%)=(control group virus titer-drug-treated group virus titer) in the cell conditioned medium of drug-treated/
Control group virus titer × 100%.Opposite virus titer percentage under each drug concentration is being averaged for three repeated experiments
Value ± SD.Abscissa, opposite virus titer are done as ordinate using the logarithm of drug concentration, are used by 20 softwares of SPSS
The Probit Returns Law calculate the EC50 (table 3) of Drug inhibition plaque test, no matter EV71 or CVA16, spiramvcin shows
Go out apparent antiviral activity, followed by azithromycin and josamycin.It is mapped, is fitted using Graphpad softwares simultaneously
Amount effect curve (Fig. 2), what is listed in Fig. 2 is the amount of the apparent spiramvcin of antiviral effect, azithromycin and josamycin
Imitate curve graph.
1.5 Plaque Technique Detected measures virus titer：By RD cell inoculations in 96 orifice plates, 1x104Cell/ml after being incubated overnight, connects
For kind by the viral supernatants of 10 doubling dilutions, 10 holes of each dilution inoculation measure TCID50.By Vero cell inoculations in 6 holes
In plate, in 5%CO2, under the conditions of 37 DEG C after culture 18-20h, by the EV71-MZ201405 or CVA16- of 100TCID50
MZ201509 viruses infect 1h, discard supernatant, and the Macrocyclolactone lactone kind medicine containing 1.2% methylcellulose and various concentration is added
Medium culture 4 days or so, fixed through paraformaldehyde, violet staining, clear water rinsing, dry after make plaque counting, calculate
Virus titer.
The antiviral concentration of half of 3. macrolide antibiotics of table
In conclusion all there is the six kinds of macrolide antibiotics detected external anti-enterovirus EV71 and CVA16 to live
Property, wherein it is most notable with spiramvcin inhibiting effect, it is secondly azithromycin and josamycin and clarithromycin.
Embodiment 2
Enterovirus is single stranded positive-sense RNA virus, and genome length is about 7408nt, containing there are one open reading frame, codings
Generate a polyprotein, both sides 5 ' and 3 ' noncoding regions (untranslated region, UTR).Polyprotein digests
Virus structural protein and non-structural protein are generated afterwards；5 ' UTR contain the initial signal IRES of rna replicon signal and polypeptide translation,
By and the combination of the host protein factor play a significant role in the synthesis of virus genome RNA and translation process.The present invention
It is anti-to macrolides on a cellular level by biochemical and molecular biology research means by taking the anti-EV71 of spiramvcin as an example
The possibility mechanism of action that raw element inhibits enterovirus to replicate is analyzed.
The time is added on the active influence of Antiviral Effect in 2.1 detection drugs, and EV71 infection is blocked for analysis spiramvcin
Link give a clue：By 5x104A RD cell inoculations to 24 orifice plate cultures 18~use afterwards for 24 hours MOI=5 EV71-
MZ201405 infects, and 100 μM of spiramvcin, infection is added in 0h, 1h, 2h, 4h, 6h, 8h after 1h (- 1) and infection before infection
10h collects supernatant afterwards, is analyzed by plaque and measures virus titer.As a contrast, by after virus infected cell, in different time points
Single plus DMSO, 10h collects supernatant and measures virus titer after infection, excludes the influence that solvent DMSO generates virus.Such as Fig. 3
Shown, there is 2h additions drug similar inhibition virus to generate activity to after infecting before infection, and it is mould to add spiral after infection 4h
It is plain then the depression effect of virus titer is obviously weakened, illustrate spiramvcin virus replication early stage (including absorption, into
Enter and shell of undressing) or late stage (assembling and release) do not play a role, this is the result shows that spiramvcin blocks answering for EV71
System very likely takes place in rna replicon or protein translation.
The influence that 2.2RT-QPCR detection spiramvcins synthesize viral RNA：By 5x104Vero cell inoculations are in 24 holes
Plate, culture use the EV71-MZ201405 of MOI=10 to infect 1h after 24 hours, remove viral supernatants and be added and contain DMSO or 100
The culture medium of μM spiramvcin.The 2h after infection, 4h, 6h, 8h extract cell total rna with Trizol respectively.It is digested through DNase
Afterwards, reverse transcription is carried out according to PrimeScript RT reagent Kit specifications；Then QPCR is carried out by template of cDNA；Needle
Primer to EV71 is 5 '-TGTATGTCTCATTATCAGGGG-3 ' (SEQ ID No.1) and 5 '-
CCACCTGTTGCTTGTAACCGT-3 ' (SEQ ID No.2), amplicon are EV712C segments；Using the GAPDH of cell as internal reference
It is corrected, primer 5 '-GAAGGTGAAGGTCGGAGT-3 ' (SEQ ID No.3), 5 '-GAAGATGGTGATGGGATTTC-
3’(SEQ ID No.4).PCR uses two-step method：94 DEG C of 30sec, 94 DEG C of 5sec, 60 DEG C of 30sec, 42cycles.It is compareed when 2h
The viral RNA relative abundance of cell is set as 1, and experiment is in triplicate.As shown in figure 4, the presence of spiramvcin obviously inhibits
The synthesis of viral RNA, blocking mechanism are likely to occur in rna replicon or virus protein synthesis link.
2.3RNA transfections merge fluorescence microscope and investigate the mechanism that spiramvcin blocks EV71 to replicate：It is obtained through in-vitro transcription
Carry the EV71 full length genomic rnas (EV71-GFP RNA) of fluorescent reporter gene GFP.293A is reached into 24 orifice plates, 5x104Carefully
Born of the same parents/hole, after cultivating 18h, per hole with being changed to after 1.2 2 μ g EV71-GFP RNA, 4h of μ l Lipo2000 transfection containing 20 or
The culture medium of 100 μM of spiramvcins is control with the culture medium of non-dosing object.Luciferase expression situation when observing 12h, random counter
100 visuals field and the average GFP+ cell numbers for calculating each visual field, it can be seen that 100 μM of spiramvcins obviously inhibit GFP's
It expresses (Fig. 5), illustrates that spiramvcin can block the polyprotein of EV71 viruses to synthesize or process.
Influence of 2.4 pairs of reporter gene detection spiramvcins to EV715 ' UTR IRES functions：It is to set out with pGL3-luc
Plasmid will be inserted into pGL3-luc plasmid reporter genes firefly after Renilla luc (R-luc) reporter gene PCR amplification
The downstream of luc (F-luc) is connected by one section of multiple cloning sites sequence between two reporter genes；Then by PCR amplification
EV715 ' UTR are inserted between F-luc and R-luc, close to the upstream of R-luc initiation codons；Generated plasmid is named as
pGL3-dual luc-EV71IRES.293A cells are passaged in six orifice plates, 5x105Cells/well transfects after cultivating 18h
PGL3-dual luc-EV71IRES measure F-luc and R-luc after being separately added into 100 μM of spiramvcins and tetracycline effect 36h
Activity and calculate relative ratio, the R-luc/F-luc in control cell is set as 1.Data show that spiramvcin can specificity
Inhibit the interpretative function of EV715 ' UTR, the mechanism that tetracycline inhibits EV71 to replicate then unrelated with the interpretative function of EV715 ' UTR
(Fig. 6).The result further demonstrates that spiramvcin inhibits the IRES functions of EV715 ' UTR by participation by played antiviral work
Property.
Obviously, the above embodiment of the present invention be only to clearly illustrate example of the present invention, and not be pair
The restriction of embodiments of the present invention may be used also on the basis of the above description for those of ordinary skill in the art
To make other variations or changes in different ways, all embodiments can not be exhaustive here, it is every to belong to this hair
Row of the obvious changes or variations that bright technical solution is extended out still in protection scope of the present invention.
Claims (3)
1. the application of macrolide antibiotics or its pharmaceutical salts in preparing anti-hand-foot-and-mouth-disease drug, which is characterized in that described
Macrolide antibiotics is erythromycin, clarithromycin, azithromycin, josamycin, medecamycin or spiramvcin.
2. macrolide antibiotics according to claim 1 or its pharmaceutical salts answering in preparing anti-hand-foot-and-mouth-disease drug
With, which is characterized in that the drug be using macrolide antibiotics or its pharmaceutical salts as active ingredient with it is pharmaceutically acceptable
Auxiliary material combination is made.
3. macrolide antibiotics according to claim 2 or its pharmaceutical salts answering in preparing anti-hand-foot-and-mouth-disease drug
With, which is characterized in that the drug is oral preparation, ejection preparation or topical formulation.
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| EP3932409A1 (en) * | 2020-06-29 | 2022-01-05 | Consejo Superior de Investigaciones Científicas (CSIC) | Compounds for the treatment and prevention of viral infections caused by coronaviruses |
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| CN109833326A (en) * | 2017-11-24 | 2019-06-04 | 苏州系统医学研究所 | Macrolide antibiotics is blocking the application in influenza infection |
| CN108794552B (en) * | 2018-04-08 | 2021-12-07 | 天津大学 | Erythromycin ethylsuccinate spherical crystal and preparation method thereof |
| CN110812357B (en) * | 2019-11-06 | 2022-09-23 | 山东省农业科学院奶牛研究中心 | Application of biapenem in preparation of medicine for preventing and treating bovine enterovirus infection |
| CN113197912B (en) * | 2020-01-30 | 2023-02-03 | 沈阳福洋医药科技有限公司 | Isovaleryl spiramycin compound and application of isovaleryl spiramycin compound composition in preparation of antiviral drugs |
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| GB1540397A (en) * | 1976-12-08 | 1979-02-14 | May & Baker Ltd | Spiramycin esters |
| CN104721201B (en) * | 2015-03-26 | 2017-09-15 | 中山大学 | The application of tetracycline antibiotics or its pharmaceutical salts in anti-enteropathy cytotoxic drug is prepared |
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| EP3932409A1 (en) * | 2020-06-29 | 2022-01-05 | Consejo Superior de Investigaciones Científicas (CSIC) | Compounds for the treatment and prevention of viral infections caused by coronaviruses |
| WO2022002789A1 (en) * | 2020-06-29 | 2022-01-06 | Consejo Superior De Investigaciones Cientificas (Csic) | Compounds selected from clarithromycin and lexithromycin for the treatment and prevention of viral infections caused by coronaviruses |
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