CN103251591B

CN103251591B - Application of mepyramine maleate in preparing drug for treating or preventing influenza virus

Info

Publication number: CN103251591B
Application number: CN201310176428.7A
Authority: CN
Inventors: 陈绪林; 廖庆姣
Original assignee: Wuhan Institute of Virology of CAS
Current assignee: Wuhan Institute of Virology of CAS
Priority date: 2013-05-13
Filing date: 2013-05-13
Publication date: 2014-12-17
Anticipated expiration: 2033-05-13
Also published as: CN103251591A

Abstract

The invention discloses application of mepyramine maleate in preparing a drug for treating or preventing influenza virus. The application comprises the following steps of: selecting the mepyramine maleate with complete non-toxic concentration for carrying out an antiviral experiment, wherein the result shows that the compound has remarkable antiviral activity and is dose-dependent; analyzing an anti-influenza virus action period of the mepyramine maleate, wherein the result indicates that the mepyramine maleate can be mainly used for inhibiting virus infection early events including virus adsorption, virus entry and the like; and detecting the antiviral activity of the mepyramine maleate to different types and subtypes of influenza viruses, wherein the result shows that the mepyramine maleate can be developed as a novel anti-influenza virus drug, so that a novel way and means is provided for treating the influenza.

Description

The application of a kind of pyrilamine maleate in preparation treatment or flu-prevention virus drugs

Technical field

The invention belongs to medical art, more specifically relate to the application of a kind of pyrilamine maleate in preparation treatment or flu-prevention virus drugs.

Background technology

Influenza virus (influenza virus) has a strong impact on human health, can cause grippal pathogen.According to virion nucleoprotein (NP) and the antigenic characteristic of stromatin (M) and the difference of genetic characteristics, influenza virus is divided into A, B, C tri-type, also claims first, second, the third three types.All there is significant difference in the aspects such as genome structure, the polypeptide of these three kinds of veriform viruses form, infectious and pathogenic.Influenza A full-length genome is made up of the sub-thread strand RNA that 8 differ in size, and names respectively with sections 1 to sections 8.Full length viral genome is about 13.6kb, encode respectively 10 kinds of structural protein (PB2, PB1, PA, HA, NP, NA, M1, M2, PB1-F2 and NS2/NEP) and non-structural protein (NS1).According to the difference of virion surface glycoprotein hemagglutinin (HA) and neuraminidase (NA), influenza A can be further divided into 16 H (H1-H16) and 9 N (N1-N9) hypotypes.Human influenza virus is H1, H2 and H3 hypotype mainly, and endangers serious high pathogenic avian influenza at present and mostly be H5, H7 and H9 hypotype, wherein the highest with the fatality rate of H5N1 hypotype.Type B influenza virus often causes influenza localized epidemics, does not cause worldwide influenza great outburst, and in other animal except people, does not find that it exists so far.C type influenza virus exists mainly with being dispersed in form, and primary attack infant, does not generally cause influenza pandemic, can infect the mankind and pig.

Influenza virus in viral taxonomy Shang Shu orthomyxoviridae family (Orthomyxoviridae), Influenza Virus.Influenza virus is segmented minus-stranded rna virus, so viral RNA does not have infectivity, each RNA sections forms vRNPs after must combining with RNA polymerase (PB2, PB1 and PA) and nucleoprotein (NP) just has activity.Infect initial be surfaces of viral particles the identification of HA furcella and in conjunction with host cell surface contain sialic receptor.The connecting key of sialic acid and time terminal galactose, determine the host specificity of virus, this connecting key is α (2-3) in birds, is α (2-6) in the mankind.In Influenza virus HA protein molecule, the replacement of Individual amino acids and the different glycosylation modified specificitys that can change its receptors bind.After virus absorption onto cell, cell takes in virus by the receptor-mediated pinocytosis of clathrin.In cell, clathrin molecular dissociation, virus and endosome merge and form phagosome, make the pH value around virion drop to about 5.0.Under this pH condition, the conformation of viral HA protein changes, and the fusogenic peptide being positioned at light chain (HA2) N end is exposed, thus causes virus envelope and cell membrane fusion.Low pH environment also causes a large amount of H ⁺enter virion inside via M2 ion channel, cause dissociating of M1 albumen and vRNPs.Both common results cause the vRNPs of virion to be discharged into the endochylema of infected cell.Different from other RNA viruses, copying and transcribing of influenza virus gene group all occurs in the nucleus of host cell.MRNA transfers to endochylema after synthesizing in core, the structural protein of synthesis virus and non-structural protein.Then start to assemble progeny virus, the glycoprotein of neuraminidase hydrolyzable cell surface, release N-acetyl-neuraminate, impels virion to discharge from site of sprouting.The final step of virus maturation is that HA is cracked into HA1 and HA2 polypeptide under the effect of host protein enzyme, and such virion just has infectivity, thus starts copying of new round virus.

Influenza virus is popular in the mankind have propagated more than 300 year, and several years i.e. once outbreak of epidemic, global flu outbreak then many decades breaks out once.Influenza pandemic can cause 250,000 ~ 500,000 examples dead every year, 3,000,000 ~ 5,000,000 severe disease examples, the whole world about have 5 ~ 15% people infected.For global influenza pandemic, " in epidemic period, vaccine and antiviral drugs reduce the most important counter-measure of M & M ".But at present the whole world can produce the use of 300,000,000 person-portion trivalent flu vaccine-this only enough Hesperian influenza prevention every year, and the demand of global influenza pandemic can not be met, and due to the antigenic variation ability of influenza virus strong, can not develop vaccine before novel strain occurs.Extensive research and development and the manufacture of a usual vaccine at least need six months, even if till that time, because worldwide production is limited in one's ability and production facility concentrates on developed country, many do not have the country of production facility will use less than vaccine at the 1st ripple epidemic period.So the protective rate that current vaccines can provide is not high.Anti-influenza virus medicament not only comes out more late, and medicine quick-acting, effective is clinically also less, belongs to the little kind in anti-infection drug.At present, the anti-influenza virus medicament ratifying official listing through food and drug administration (FDA) has two classes: the M2 ion channel blocking agent that (1) is representative with amantadine and rimantadine.This type of medicine only has preventive and therapeutic action to influenza A virus, and research shows that medicine has the toxic and side effects such as neurotoxicity, and makes persister ubiquity owing to widely using, so CDC advises that this type of medicine is not used further to flu-prevention viral infection.(2) neuraminidase inhibitor, the representative of this type of medicine is oseltamivir and Zha La meter Wei.This type of medicine to all known human influenza viruses and high pathogenic avian influenza virus all effective.But the persister in recent years about oseltamivir but constantly has report.

Pyrilamine maleate is histamine I class inhibitor, is the micromolecular compound of synthetic, belongs to ethylenediamines antihistaminic, be mainly used in various allergic diseases.There is no the application of pyrilamine maleate in resisiting influenza virus at present.

Summary of the invention

The object of the invention is to make up the deficiencies in the prior art, be there are provided the application of a kind of micromolecular compound pyrilamine maleate in preparation treatment or flu-prevention medicine, thus provide the micromolecular compound that a kind of safe and efficient toxic and side effects is little for the treatment of influenza clinically.Pyrilamine maleate can effectively suppress entering of influenza virus within the scope of avirulence, can be developed as the medicine for the treatment of influenza infection disease further, be with a wide range of applications.

The English of pyrilamine maleate is called pyrilamine maleate salt, have another name called Mepyramine maleate salt, chemical formula is N-[(4-anisyl) methyl]-N-(2-pyridine radicals)-N', N'-dimethyl-ethylenediamine maleate (N-(4-Methoxyphenyl) methyl-N ', N '-dimethyl-N-(2-pyridinyl)-1,2-ethanediamine maleate salt), there is the structural formula shown in structural formula I:

Structural formula I

In order to realize above-mentioned object, the present invention by the following technical solutions:

The application of a kind of pyrilamine maleate in preparation treatment or flu-prevention virus (A type or B-mode) medicine, the steps include:

A. pyrilamine maleate is to the cytotoxicity experiment of mdck cell: mdck cell is inoculated in 96 porocyte culture plates by 5000 cells/well (100 μ l), after cell attachment, take 1mM as the continuous 2 times of gradient dilutions of initial concentration 6 gradients with cell maintenance medium (DMEM+2% serum) by medicine, the multiple hole of every gradient 2, is placed in 37 DEG C, 5%CO ₂cultivate in incubator after 48 hours, detect the survival rate of cell with 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide bromine salt (tetrazolium bromide, MTT) method.Result display pyrilamine maleate is to the CC50(median lethal concentration of mdck cell) be 928.6 μMs.

B. the evaluation of pyrilamine maleate anti-influenza virus activity: mdck cell is pressed 2*10 ⁴cells/well is inoculated in 96 porocyte culture plates, cultivates 14 ~ 18h and treats that cell grows up to monolayer, then add 100TCID in 37 DEG C of cell culture incubators ₅₀/ hole virus liquid infection cell, gets each experimental port supernatant after adding each Concentraton gradient medicine simultaneously totally 200 μ l maintain liquid (DMEM+0.3%BSA+2.5ug/L pancreatin) cultivating 48h in 37 DEG C of cell culture incubators and carries out neuraminidase activity detection.Result shows: pyrilamine maleate obviously inhibits influenza virus to copy, and its EC50 is 5.42 μMs.

C. the pyrilamine maleate resisiting influenza virus action period is analyzed: cell presses 5*10 ⁴being inoculated in 24 porocyte culture plates, is after monolayer until cell attachment, and give same concentrations medicine at viral infection-2 ~ 0h, 0 ~ 2h, 2 ~ 4h, 4 ~ 6h respectively and cultivate, 6h fixed cell after infection, is expressed by indirect immunofluorescene assay viral NP protein.Result shows: pyrilamine maleate administration in 0 ~ 2h can suppress viral infection, mainly suppresses viruses adsorption and enters.

D. pyrilamine maleate anti-influenza virus activity broad spectrum activity is analyzed: mdck cell is pressed 2*10 ⁴cells/well is inoculated in 96 porocyte culture plates, cultivates 14 ~ 18h and treats that cell grows up to monolayer, then add 100TCID in 37 DEG C of cell culture incubators ₅₀/ hole virus liquid (A/human/Hubei/1/2009(H1N1), A/human/Hubei/3/2005(H3N2), Influenza B virus) infection cell, get each experimental port supernatant after adding each Concentraton gradient medicine simultaneously totally 200 μ l maintain liquid (DMEM+0.3%BSA+2.5ug/L pancreatin) cultivating 48h in 37 DEG C of cell culture incubators and carry out neuraminidase activity detection.Result shows: pyrilamine maleate obviously can suppress subtypes of influenza A virus H1N1 strain, the copying of influenza A virus H3N2 strain and influenza B virus strain, and has dose-dependent effect.

The present invention compared with prior art has the following advantages and effect:

1. pyrilamine maleate is micromolecular compound, and its CC50 is 928.6 μMs.Pyrilamine maleate dose-dependant ground suppresses influenza virus to be copied, and its EC50 is 5.42 μMs, and therapeutic index is about 171.This illustrates that pyrilamine maleate is the medicine of the efficient resisiting influenza virus of low toxicity.

2. pyrilamine maleate acts on influenza virus and copies in early days, mainly suppresses viruses adsorption and the earliest events such as to enter.

3. pyrilamine maleate can suppress subtypes of influenza A virus H1N1 strain, the copying of influenza A virus H3N2 strain and influenza B virus strain, and has the antiviral activity of wide spectrum.

4. utilize modern common drug preparation means, pyrilamine maleate can be made the pharmaceutically acceptable dosage forms of any one such as tablet, capsule, granule, oral liquid, slow releasing preparation, controlled release preparation, nanometer formulation, injection as active components.

Accompanying drawing explanation

Fig. 1 is a kind of pyrilamine maleate chemical structural formula.

Fig. 2 is that a kind of cytotoxicity of pyrilamine maleate detects schematic diagram.

Fig. 3 is the neuraminidase schematic diagram alive in a kind of culture fluid supernatant of pyrilamine maleate process influenza infection cell.

Fig. 4 is that a kind of pyrilamine maleate that detects affects result schematic diagram to viral single-wheel replicative cycle different phase.

Fig. 5 is that a kind of pyrilamine maleate anti-influenza virus activity broad spectrum activity that detects detects schematic diagram.

Fig. 5 A is subtypes of influenza A virus H1N1 strain (A/human/Hubei/1/2009(H1N1);

Fig. 5 B is influenza A virus H3N2 strain (A/human/Hubei/3/2005(H3N2);

Fig. 5 C is influenza B virus strain (Influenza B virus).

Detailed description of the invention

In order to understand content of the present invention better, below in conjunction with specific implementation method, content of the present invention is described further, but protection content of the present invention is not limited to following examples.

At present, anti-influenza virus medicament in-vitro screening model is divided into the cell free system screening model of cell culture model and viral enzyme.Enzyme reaction screening model has high-throughout feature, but the compound of screening still needs to carry out more cytology, histology and toxicity in vivo, effect experiment etc. to determine its effect.Cell culture model is the most frequently used screening model, its advantage is: the cell that a large amount of hereditary character can be provided identical is object of study, easy to operate, can eliminate the impact of other extraneous factor, and can the valid density of detection of drugs and therapeutic index, for later stage study mechanism provides more how basis.The present invention adopts cell culture screening method to detect pyrilamine maleate infected by influenza infection mdck cell, detects the content of virion in viral supernatants, quantitative analysis pyrilamine maleate anti-influenza virus activity.

Embodiment 1: the evaluation of pyrilamine maleate anti-influenza virus activity

1. experiment material and method

1.1 cells, virus and medicine

Mdck cell is purchased from ATCC, and viral A/PR8/34H1N1 is increased by chick embryo culture and obtains, and pyrilamine maleate is purchased from Sigma company;

1.2 experimental apparatus

Multi-tester PerkinElmer, inverted microscope Costar

2. experimental technique:

2.1 cell culture:

37 DEG C, 5%(volume ratio, identical below) CO ₂cultivate in humidified incubator.Use containing 10%(volume ratio, identical below) penicillin of FBS, 100U/mL and the DMEM culture medium of streptomycin.Go down to posterity after cell to 90% degree of converging, the ratio that goes down to posterity 1/3 – 1/4.

2.2 Virus culture:

Get 9 ~ 11 age in days SPF Embryo Gallus domesticus, before virus inoculation, use Egg checker inspection, and at the position mark away from embryo, sterilize and punch rear by 0.2ml/ piece of inoculation 2 ⁴the virus liquid of hemagglutinative titer, with nial polish sealing, puts 37 DEG C of calorstats and hatches 48h(and inoculate Embryo Gallus domesticus dead after 24h to be generally misoperation lethal, abandon it).Take out Embryo Gallus domesticus and put 4 DEG C of cold embryo 12h.75% alcohol disinfecting chick embryo air sac, sterile working cuts off air chamber shell, and capillary pipette draws chick embryo allantoic liquid and amniotic fluid, 3000rpm4 DEG C of centrifugal 30min, and detect hemagglutinative titer, 200 μ l/ pipe subpackage juxtapositions-70 DEG C are frozen for subsequent use.

(1) drug cytotoxicity detects:

1.MDCK cell is inoculated in 96 porocyte culture plates by 5000 cells/well (100 μ l), for subsequent use after cell attachment;

2. use cell maintenance medium (DMEM+2% serum) to be the continuous 2 times of gradient dilutions of initial concentration 6 gradients with 1mM by medicine, the multiple hole of every gradient 2.In every hole, add 5mg/ml MTT20 μ l after cultivating 48h, put in cell culture incubator and continue to cultivate;

3. after cultivating 4h, abandon culture fluid supernatant, every hole adds 100 μ l/ hole three lysates, and (lysate is by SDS10g, isobutanol 5ml, 10M HCl0.1ml, dissolves with distilled water and is made into 100ml), more than 4h is dissolved or enzyme connection detector detection 570nm wavelength place light absorption value after spending the night in 37 DEG C of incubators, tuning wavelength is 630nm, and calculates each drug level cell survival rate, the results are shown in Figure 2.

Result shows: pyrilamine maleate does not have obvious cytotoxicity to mdck cell within the scope of concentration <250 μM, illustrates that pyrilamine maleate has the safer scope of application.

(2) Activity determination of the anti-A/PR8/34H1N1 Strain of pyrilamine maleate:

A. the effect of pyrilamine maleate resisiting influenza virus is detected based on neuraminidase activity:

1. experimental principle: MUNANA(4-methylumbelliferyl-α-N-acetyl-neuraminate) be the specific substrate of influenza neuraminidase, the catalysate produced under neuraminidase effect is under 355nm excitation light irradiation, 460nm fluorescence can be produced, the change of fluorescence intensity, can sensitive reaction neuraminidase activity, thus can viral load in reacting cells supernatant.

2. mdck cell is pressed 2*10 ⁴cells/well is inoculated in 96 porocyte culture plates, after cultivating 14 ~ 18h, grows up to after monolayer for subsequent use until cell in 37 DEG C of cell culture incubators.Culture medium in orifice plate is discarded, PBS liquid washes twice, add 100TCID50/ hole virus liquid infection cell, add each Concentraton gradient medicine (with 100 μMs for initial concentration simultaneously, continuous 2 times of gradient dilutions 6 gradients, the multiple hole of every gradient 2) get each experimental port supernatant after totally 200 μ l maintain liquid (DMEM+0.3%BSA+2.5ug/L pancreatin) cultivate 48h in 37 DEG C of cell culture incubators and carry out neuraminidase activity detection.

3. in the micro plate of black 96 hole, add 40 μ l buffer (32.5mmol/L MES, pH6.5,4mmol/L CaCl ₂) the substrate 20umol/L MUNANA for preparing, add each experimental port culture supernatant 20 μ l again, 37 DEG C of lucifuges hatch 30min, add reaction terminating liquid (0.014 μM of NaOH, 83% ethanol) 100 μ l/ holes, multi-tester measures fluorescent value (excitation wavelength 355nm, wavelength of transmitted light 465nm).

4. calculate the suppressed rate of NA in each detect aperture.Suppression ratio (%)=100-(sample well-blank)/(enzyme contrast-blank alive) * 100%.

Result shows: pyrilamine maleate obviously inhibits influenza virus to copy, and in dose-dependence (see figure 3)

Variable concentrations pyrilamine maleate infected by influenza inhibition of DNA replication rate is as follows:

Concentration (μM)	Suppression ratio (%)
		100	97.03±2.2
50	95.99±2.98
		25	84.22±0.13
12.5	77.77±8.67

6.25	55.89±0.79
		3.125	30.84±14.08

Embodiment 2: pyrilamine maleate is to the inhibitory action of virus replicative cycle different phase

1. cell presses 5*10 ⁴being inoculated in 24 porocyte culture plates, is for subsequent use after monolayer until cell attachment.Influenza virus biocycle is about 6h, so we are respectively at viral infection-2 ~ 0h, 0 ~ 2h, 2 ~ 4h, the medicine that 4 ~ 6h gives 50 μMs is cultivated, and 6h fixed cell after infection, carries out the expression of virus protein after indirect immunofluorescene assay drug treating according to the following step.

2. abandon culture fluid supernatant, with fixative, cell is fixed 15-20min in room temperature; 3 times are washed, each 5min with PBS; 1h is closed by confining liquid room temperature; Abandon confining liquid, add primary antibodie (Mus source NP monoclonal antibody) the incubated at room 1h in conjunction with liquid dilution; 3 times are washed, each 5min with PBS; Add the fluorescently-labeled two anti-and DAPI dye liquor incubated at room 1h in conjunction with liquid dilution; 3 times are washed, each 5min with PBS; Observe under inverted fluorescence microscope.

Test solution used: fixative: 4%(mass volume ratio) paraformaldehyde is dissolved in PBS (pH7.4); In conjunction with liquid: 3%(mass volume ratio) BSA, 0.3%(mass volume ratio) TritonX-100 is dissolved in PBS (pH7.4); Confining liquid: containing 10%FCS in conjunction with liquid.

Result shows: pyrilamine maleate administration in 0 ~ 2h can suppress viral infection, infers main suppress viruses adsorption and the viral infection earliest events (see figure 4) such as to enter.

Embodiment 3: pyrilamine maleate anti-influenza virus activity broad spectrum activity is studied

This experiment detects the antiviral activity of pyrilamine maleate to subtypes of influenza A virus H1N1 strain, influenza A virus H3N2 strain and influenza B virus strain.

1. experiment uses Strain to comprise: A/human/Hubei/1/2009(H1N1), A/human/Hubei/3/2005(H3N2), Influenza B virus

2. mdck cell is pressed 2*10 ⁴cells/well is inoculated in 96 porocyte culture plates, after cultivating 14 ~ 18h, grows up to after monolayer for subsequent use until cell in 37 DEG C of cell culture incubators.Culture medium in orifice plate is discarded, PBS liquid washes twice, add 100TCID50/ hole virus liquid infection cell, add each Concentraton gradient medicine (with 200 μMs for initial concentration simultaneously, continuous 2 times of gradient dilutions 6 gradients, the multiple hole of every gradient 2) get each experimental port supernatant after totally 200 μ l maintain liquid (DMEM+0.3%BSA+2.5ug/L pancreatin) cultivate 48h in 37 DEG C of cell culture incubators and carry out neuraminidase activity detection.

4. calculate the suppressed rate of NA in each detect aperture.Suppression ratio (%)=100-(sample well-blank/(enzyme contrast-blank alive) * 100%

Result shows: pyrilamine maleate obviously inhibits other strains of influenza viruses to copy, and in dose-dependence (see figure 5).

Variable concentrations pyrilamine maleate is as follows to other strains of influenza viruses inhibition of DNA replication rate:

Claims

1. pyrilamine maleate is as the application of sole active ingredient in preparation treatment or flu-prevention virus drugs; Described influenza virus is influenza A virus or Influenza B virus.