CN106074498B - Calcein and Oseltamivir, which are combined, is preparing the application in preventing H7N9 type flu pharmaceuticals - Google Patents
Calcein and Oseltamivir, which are combined, is preparing the application in preventing H7N9 type flu pharmaceuticals Download PDFInfo
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- 229960003752 oseltamivir Drugs 0.000 title claims abstract description 49
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 title claims abstract description 48
- 229960002378 oftasceine Drugs 0.000 title claims abstract description 46
- 239000003814 drug Substances 0.000 title claims abstract description 19
- VSZGPKBBMSAYNT-RRFJBIMHSA-N oseltamivir Chemical compound CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 VSZGPKBBMSAYNT-RRFJBIMHSA-N 0.000 title claims abstract 8
- 238000002347 injection Methods 0.000 claims 1
- 239000007924 injection Substances 0.000 claims 1
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- NENPYTRHICXVCS-YNEHKIRRSA-N oseltamivir acid Chemical compound CCC(CC)O[C@@H]1C=C(C(O)=O)C[C@H](N)[C@H]1NC(C)=O NENPYTRHICXVCS-YNEHKIRRSA-N 0.000 description 43
- 150000001732 carboxylic acid derivatives Chemical class 0.000 description 23
- 229940079593 drug Drugs 0.000 description 10
- 239000000243 solution Substances 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 7
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- 230000000694 effects Effects 0.000 description 7
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- 238000001514 detection method Methods 0.000 description 6
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 4
- 239000007853 buffer solution Substances 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- 230000004083 survival effect Effects 0.000 description 4
- 230000002195 synergetic effect Effects 0.000 description 4
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 206010059866 Drug resistance Diseases 0.000 description 3
- 229910052791 calcium Inorganic materials 0.000 description 3
- 239000011575 calcium Substances 0.000 description 3
- 239000003112 inhibitor Substances 0.000 description 3
- PGZUMBJQJWIWGJ-ONAKXNSWSA-N oseltamivir phosphate Chemical compound OP(O)(O)=O.CCOC(=O)C1=C[C@@H](OC(CC)CC)[C@H](NC(C)=O)[C@@H](N)C1 PGZUMBJQJWIWGJ-ONAKXNSWSA-N 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 229940061367 tamiflu Drugs 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 description 2
- CERZMXAJYMMUDR-QBTAGHCHSA-N 5-amino-3,5-dideoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid Chemical compound N[C@@H]1[C@@H](O)CC(O)(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO CERZMXAJYMMUDR-QBTAGHCHSA-N 0.000 description 2
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- DKNWSYNQZKUICI-UHFFFAOYSA-N amantadine Chemical compound C1C(C2)CC3CC2CC1(N)C3 DKNWSYNQZKUICI-UHFFFAOYSA-N 0.000 description 2
- 229960003805 amantadine Drugs 0.000 description 2
- 229910021529 ammonia Inorganic materials 0.000 description 2
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- CERZMXAJYMMUDR-UHFFFAOYSA-N neuraminic acid Natural products NC1C(O)CC(O)(C(O)=O)OC1C(O)C(O)CO CERZMXAJYMMUDR-UHFFFAOYSA-N 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-N sodium;5-ethyl-5-pentan-2-yl-1,3-diazinane-2,4,6-trione Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)NC1=O QGMRQYFBGABWDR-UHFFFAOYSA-N 0.000 description 2
- UBCHPRBFMUDMNC-UHFFFAOYSA-N 1-(1-adamantyl)ethanamine Chemical compound C1C(C2)CC3CC2CC1(C(N)C)C3 UBCHPRBFMUDMNC-UHFFFAOYSA-N 0.000 description 1
- XIAYFENBYCWHGY-UHFFFAOYSA-N 2-[2,7-bis[[bis(carboxymethyl)amino]methyl]-3-hydroxy-6-oxoxanthen-9-yl]benzoic acid Chemical compound C=12C=C(CN(CC(O)=O)CC(O)=O)C(=O)C=C2OC=2C=C(O)C(CN(CC(O)=O)CC(=O)O)=CC=2C=1C1=CC=CC=C1C(O)=O XIAYFENBYCWHGY-UHFFFAOYSA-N 0.000 description 1
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- PWHULOQIROXLJO-UHFFFAOYSA-N Manganese Chemical compound [Mn] PWHULOQIROXLJO-UHFFFAOYSA-N 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- IWUCXVSUMQZMFG-AFCXAGJDSA-N Ribavirin Chemical compound N1=C(C(=O)N)N=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](CO)O1 IWUCXVSUMQZMFG-AFCXAGJDSA-N 0.000 description 1
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- 230000001154 acute effect Effects 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 206010064097 avian influenza Diseases 0.000 description 1
- 229910052788 barium Inorganic materials 0.000 description 1
- DSAJWYNOEDNPEQ-UHFFFAOYSA-N barium atom Chemical compound [Ba] DSAJWYNOEDNPEQ-UHFFFAOYSA-N 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
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- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- NJDNXYGOVLYJHP-UHFFFAOYSA-L disodium;2-(3-oxido-6-oxoxanthen-9-yl)benzoate Chemical compound [Na+].[Na+].[O-]C(=O)C1=CC=CC=C1C1=C2C=CC(=O)C=C2OC2=CC([O-])=CC=C21 NJDNXYGOVLYJHP-UHFFFAOYSA-L 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
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- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- -1 inscription Chemical compound 0.000 description 1
- 231100000225 lethality Toxicity 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
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- 229910052748 manganese Inorganic materials 0.000 description 1
- 239000011572 manganese Substances 0.000 description 1
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- 239000011535 reaction buffer Substances 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 229960000329 ribavirin Drugs 0.000 description 1
- HZCAHMRRMINHDJ-DBRKOABJSA-N ribavirin Natural products O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1N=CN=C1 HZCAHMRRMINHDJ-DBRKOABJSA-N 0.000 description 1
- 229960000888 rimantadine Drugs 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
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- 230000009385 viral infection Effects 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- ARAIBEBZBOPLMB-UFGQHTETSA-N zanamivir Chemical compound CC(=O)N[C@@H]1[C@@H](N=C(N)N)C=C(C(O)=O)O[C@H]1[C@H](O)[C@H](O)CO ARAIBEBZBOPLMB-UFGQHTETSA-N 0.000 description 1
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Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/365—Lactones
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Emergency Medicine (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
Abstract
Calcein and Oseltamivir combine the application in preventing H7N9 type flu pharmaceuticals, implement by the following technical programs：H7N9 wild types or saltant type influenza neuraminidase liquid and calcein and Oseltamivir mixed liquor is obtained first to be incubated 30 minutes, substrate is added to be detected, the experimental results showed that two kinds of Drug combinations are to H7N9 wild types and saltant type influenza neuraminidase is active inhibiting effect, the anti influenza that calcein is used in combination on a cellular level with Oseltamivir is then had detected to act on, it was found that the two, which is used in combination, to play therapeutic effect to the cell of infection H7N9 wild types and saltant type influenza virus, finally, both have detected the influence being used in combination to mouse influenza models, it was found that after calcein and Oseltamivir mixture is added, the mitigation of influenza mouse weight can be relieved, mouse is set to be gradually restored to the general level of the health, and it can significantly reduce the Lung Exponent of influenza mouse.
Description
Technical field
Present invention relates particularly to calceins and Oseltamivir to combine the application in preventing H7N9 type influenza virus drugs.
Background technology
Influenza is a kind of acute viral respiratory infectious disease caused by influenza virus, and infectiousness is strong, is propagated fast
Speed all brings serious threat and economic massive losses to global human health every year, is that the mankind so far still cannot be effective
The pandemic infection disease of control.The randomness of flu outbreak, it is not regular to say that it is even more impossible to predict next row virus.So
Up to the present, the generation of unknown influenza can not effectively be prevented by way of vaccine, and anti-influenza virus medicament becomes to control
Treat the most effective means that influenza is opened up.The inhibitor medicaments of targeting research in recent years have remarkable break-throughs, wherein mainly there is matrix
(M2) the inhibitors of ion channels amantadine of albumen 2 and Rimantadine, neuraminidase (NA) inhibitor oseltamivir (Tamiflu)
With the three classes drug such as zanamivir and broad-spectrum antiviral drug Ribavirin.Amantadine clinically applies for many years, a variety of hypotypes
Influenza strain has generated it serious drug resistance, and Tamiflu is expensive, and also day is cumulative for report of the virus to its drug resistance
It is more.2013, in the novel H7N9 type A avian influenza virus that can infect the mankind of China's outburst, neuraminidase saltant type
Strain is to more than 10000 times that the tolerance of Oseltamivir is wild-type strain.To improve curative effect of medication, slow down influenza strain
The generation of drug resistance, Tamiflu are used in combination as the research hotspot of international the world of medicine in recent years.
Calcein (Calcein) is a kind of micromolecular water soluble fluorescein, have complexometric indicator, fluorescence indicator and
Measure the purposes such as calcium, inscription, barium and manganese.Calcein is passed through carries medicine model frequently as liposome, evaluates drug leak case.At present
There is no the reports that it has resisiting influenza virus aspect effect.
Invention content
The purpose of the present invention is to provide calceins and Oseltamivir to be used in combination in preventing H7N9 type flu pharmaceuticals
Application.Calcein is used in combination with carboxylic acid oseltamivir can effectively inhibit H7N9 wild types and saltant type influenza virus
The activity of neuraminidase to inhibit the breeding of influenza virus in vivo, and then mitigates or cures flu victims.The present invention couple
Calcein and carboxylic acid oseltamivir have carried out external zymetology inhibition to H7N9 wild types and saltant type neuraminidase
Detection finds half-inhibition concentration (IC50) difference of the carboxylic acid oseltamivir to H7N9 wild types and saltant type neuraminidase
For 1.75 ± 0.12nmol/L and 26.52 ± 1.06 μm of ol/L；Calcein is to H7N9 wild types and saltant type neuraminic acid
The IC50 of enzyme is respectively 12.68 ± 2.26 μm of ol/L and 30.19 ± 1.61 μm of ol/L.It is difficult to understand with carboxylic acid calcein has then been carried out
The detection of external zymetology inhibition is used in combination in Si Tawei, and the function and effect of drug combination are indicated with association index value：Combine and refers to
Numerical value shows that two kinds of Drug combinations play synergistic effect less than 1.The experimental results showed that two kinds of drugs are to H7N9 wild type god
Synergistic effect is played in inhibition through propylhomoserin enzyme, and more obvious to the synergy of H7N9 saltant type neuraminidases inhibition.I
To both H7N9 influenza infections mdck cells and calcein and carboxylic acid Oseltamivir mixture be added carry out again
The measurement of cell survival rate finds that calcein and carboxylic acid Oseltamivir mixture can improve H7N9 cells following viral infections
Survival rate.Finally, we to calcein and Oseltamivir mixture to H7N9 wild types and saltant type influenza virus to mouse
Changes of weight, lethality and Lung Exponent be determined, find calcein can be relieved with Oseltamivir mixture
Symptom of the H7N9 types influenza virus to mouse.The present invention provides basis to develop by the new drug of target spot of neuraminidase, is calcium
Yellowish green element is used in combination with Oseltamivir provides reference applied to the treatment of Human Influenza.
Description of the drawings
Fig. 1 is that various concentration calcein is used in combination with carboxylic acid Oseltamivir to H7N9 wild type influenza virus nerve ammonia
The inhibiting effect of sour enzyme.
Fig. 2 is that various concentration calcein is used in combination with carboxylic acid Oseltamivir to H7N9 saltant type influenza virus nerve ammonia
The inhibiting effect of sour enzyme.
Fig. 3 is that various concentration calcein is used in combination with carboxylic acid Oseltamivir to infecting H7N9 wild type influenza virus
The therapeutic effect of mdck cell.
Fig. 4 is that various concentration calcein is used in combination with carboxylic acid Oseltamivir to infecting H7N9 saltant type influenza viruses
The therapeutic effect of mdck cell.
Fig. 5 is the shadow for the mouse weight that calcein is used in combination with Oseltamivir to infecting H7N9 wild type influenza virus
It rings.
Fig. 6 is the shadow for the mouse weight that calcein is used in combination with Oseltamivir to infecting H7N9 saltant type influenza viruses
It rings.
Specific implementation mode
Below by way of the specific implementation modes of example forms, the present invention is described in further detail.
Embodiment 1
H7N9 wild types or saltant type neuraminidase solution are prepared with pH 6.5MES buffer solutions (32.5mM), will
A certain amount of neuraminidase is added in reaction buffer so that total volume is 40 μ L, then is separately added into 10 μ L various concentrations
Calcein or carboxylic acid oseltamivir.30min is placed in 37 DEG C of incubators, and 50 μ L, 20 μM of MU-NANA substrates are then added
Buffer solution, end reaction system are 100 μ L, and reaction solution is placed in black ELISA Plate, is detected in microplate reader, and excitation wavelength is
360nm, launch wavelength 450nm, detection duration 8min.The fluorescence intensity change of detection just represents the strong of enzyme degradation substrate vigor
It is weak.Testing result is：Carboxylic acid oseltamivir to the IC50 of H7N9 wild types and saltant type neuraminidase be respectively 1.75 ±
0.12nmol/L and 26.52 ± 1.06 μm of ol/L；Calcein is respectively 12.68 to the IC50 of H7N9 wild types and saltant type
± 2.26 μm/L and 30.19 ± 1.61 μm ol/L.
Embodiment 2
According to the calcein of measurement with carboxylic acid Oseltamivir to the IC50 of H7N9 wild types and saltant type neuraminidase
To determine the addition concentration of calcein and carboxylic acid Oseltamivir.The concentration of calcein and carboxylic acid Oseltamivir is selected as respectively
0.25,0.5,1,2 and 4 times of the IC50 concentration relative to H7N9 wild types and saltant type neuraminidase.And it is added
The ratio of the concentration of calcein and the concentration of carboxylic acid Oseltamivir is to remain consistent, is the ratio of its IC50 value.Exist first
The H7N9 wild types after the dilution of 30 μ L or saltant type neuraminic acid enzyme solution are added in 96 hole elisa Plates, then in each hole
The carboxylic acid Oseltamivir of the calcein and 10 μ L corresponding concentrations of 10 μ l various concentrations is added, each concentration does 4 repetitions.37
After being incubated 30min at DEG C, the NA specific substrate MU-NANA of 20 μM of 50 μ l, detection H7N9 wild types and saltant type god are added
Fluorescence intensity through propylhomoserin enzyme.Data are recorded, the association index (CI of both drugs is then calculated with CalcuSyn softwares
Value), it is as a result as follows：
<Note>：ED50, ED75 and ED90 indicate that enzymatic activity is suppressed 50%, 75% and 90% respectively.
CI values are medication combined less than 1 two kinds of explanation to play synergistic effect, and the smaller synergy of CI values is better.By number in table
According to understanding that two kinds of Drug combinations play synergistic effect to the inhibition of H7N9 neuraminidases.
Embodiment 3
In order to detect calcein and carboxylic acid Oseltamivir mixture anti-H7N9 wild types and saltant type on mdck cell
The effect of influenza virus chooses exponential phase mdck cell.It spreads 96 orifice plates, 3000/hole, 37 DEG C, trains in 5%CO2 incubators
It supports for 24 hours, until cell monolayer degrees of fusion reaches 50-60%.Next day abandons culture solution, and washing 2 times with serum-free DMEM removes residual serum.
It is dense with the H7N9 wild types of 100 μ L or saltant type influenza virus allantoic fluid (a concentration of 100 times of TCID50 concentration) and difference again
Mix 2h at 37 DEG C of the lower calcein of degree and 100 μ l of carboxylic acid Oseltamivir mixture, calcein and carboxylic acid Oseltamivir
0.25,0.5,1,2 and 4 times of the IC50 that concentration is selected as respectively relative to H7N9 wild types and saltant type neuraminidase is dense
The ratio of degree, the concentration for the calcein being added and the concentration of carboxylic acid Oseltamivir remains consistent, is the ratio of its IC50 value.
Later, incubation mixed liquor is separately added into 96 holes for having completed cell respectively, per 200 μ L of hole, each Incubating Solution does 3 multiple holes,
It is placed in incubator culture 2h, this is viruses adsorption course of infection.After 2h, the supernatant in each hole, the no blood of each hole mdck cell are discarded
Clear DMEM culture mediums clean 3 times, and 100 μ L viral dilutions are added per hole, sets and is cultivated in incubator for 24 hours, abandon supernatant, each hole later
It is cleaned 3 times with plasma-free DMEM medium, 100 μ L cell maintenance mediums is added per hole and (contain+1 μ g/ of DMEM culture solutions of 2% serum
ML pancreatin), after cultivating 3 days in incubator, the CPE in each hole is observed, detects each hole cytopathy degree in conjunction with mtt assay, this is simultaneously
Reflect the inhibition level of detection drug infected by influenza.15 μ L MTT will be added per hole in the 96 well culture plate holes for terminating observation
(5mg/mL) solution, gently mixing, sets after being incubated 4h in 37 DEG C of incubators, abandons supernatant, and 100 μ L DMSO are added per hole, gently mix
It is even, in reading the OD values at 570nm on enzyme-linked immunosorbent assay instrument in 30min, calculate the percentage of the vigor of cell, vigor percentage
The degree of protection of mdck cell damage is caused to virus than representing drug to a certain extent.
As a result as shown in Figure 3 and Figure 4, under the conditions of different disposal, calcein and carboxylic acid Oseltamivir mixed liquor is added
When with H7N9 wild types or saltant type influenza virus, with the increase of drug concentration, cell survival rate is consequently increased.Illustrate calcium
Yellowish green element can inhibit H7N9 wild types and saltant type influenza virus with Oseltamivir mixed liquor, protect mdck cell from virus
It destroys, and dose dependent is presented also with the increase of calcein and carboxylic acid Oseltamivir mixture concentration in protective effect.
Embodiment 4
In order to detect calcein and Oseltamivir mixture to infected H7N9 wild type influenza virus mouse whether
There are therapeutic effect, 18-20g female BABL/c mouse 50 only to be anaesthetized, then passed through with 0.2% 50 μ L/ of Nembutal sodium solution
The H7N9 wild type influenza virus dilutions of 10 times of LD50 concentration are instilled in mouse nasal cavity and establish mouse influenza by the mode of collunarium
Model, to instill the mouse of 50 μ L PBS buffer solution as a control group.50 mouse are randomly divided into 5 groups by us, respectively pair
According to group (PBS groups)；Viral group (10 times of LD50H7N9 wild type influenza virus)；Calcein group (10 times of LD50H7N9 wild types
Influenza virus+25mg/kg calceins)；Oseltamivir group (10 times of LD50H7N9 wild type influenza virus+25mg/kg Ao Sita
Wei)；Calcein and Oseltamivir mixture group (10 times of LD50H7N9 wild type influenza virus+25mg/kg calceins with
25mg/kg Oseltamivirs mixture).Every group 10.Start to be administered before 2 days viral to mouse inoculation, 2 times a day, continuous gavage
10 days, every mouse single administration 0.2mL.The weight and death condition of every group of mouse are recorded daily.At the 15th day, to mouse
After the jejunitas 8h that cuts off the water supply, dislocation execution is carried out, take out the lungs of mouse and is weighed.It is calculated according to formula：
The results are shown in Figure 5, can alleviate the mitigation of H7N9 wild type influenza virus infecting mouse weight, improve mouse
Survival condition makes mouse be gradually restored to the general level of the health.
The results are shown in table below, and it is small to illustrate that calcein with Oseltamivir mixed liquor can significantly reduce H7N9 wildtype influenzas
The Lung Exponent of mouse.
Result above illustrates that calcein mixes liquid energy with Oseltamivir and risen to the mouse for infecting H7N9 wildtype influenzas
To the effect for the treatment of, mouse is made to be gradually restored to the general level of the health.
Embodiment 5
In order to detect calcein and Oseltamivir mixture to infected H7N9 saltant type influenza viruses mouse whether
There are therapeutic effect, 18-20g female BABL/c mouse 50 only to be anaesthetized, then led to 0.2% 50 μ L/ of Nembutal sodium solution
Mouse stream will be established in the H7N9 saltant type influenza virus dilutions instillation mouse nasal cavity of 10 times of LD50 concentration by crossing the mode of collunarium
Model is felt, to instill the mouse of 50 μ L PBS buffer solution as a control group.50 mouse are randomly divided into 5 groups by us, respectively
Control group (PBS groups)；Viral group (10 times of LD50H7N9 saltant types influenza viruses)；(the 10 times of LD50H7N9 mutation of calcein group
Type influenza virus+25mg/kg calceins)；Oseltamivir group (10 times of departments of LD50H7N9 saltant type influenza virus+25mg/kg Austria
His Wei)；Calcein and Oseltamivir mixture group (10 times of LD50H7N9 saltant type influenza virus+25mg/kg calceins
With 25mg/kg Oseltamivirs mixture).Every group 10.Start to be administered before 2 days viral to mouse inoculation, it is 2 times a day, continuous to fill
Stomach 10 days, every mouse single administration 0.2mL.The weight and death condition of every group of mouse are recorded daily.At the 15th day, to small
After the jejunitas 8h that cuts off the water supply of mouse, dislocation execution is carried out, the lungs of mouse is taken out and weighs.It is carried out according to formula shown in embodiment 4
It calculates.
Shown in result figure 6, the mitigation of H7N9 saltant type influenza infection mouse weights can be alleviated, improve the life of mouse
State is deposited, mouse is made to be gradually restored to the general level of the health.
The results are shown in table below, and it is small to illustrate that calcein with Oseltamivir mixed liquor can significantly reduce H7N9 saltant type influenzas
The Lung Exponent of mouse.
Result above illustrates that calcein mixes liquid energy with Oseltamivir and risen to the mouse for infecting H7N9 saltant type influenzas
To the effect for the treatment of, mouse is made to be gradually restored to the general level of the health.
Claims (3)
1. calcein and Oseltamivir, which are combined, is preparing the application in preventing H7N9 type flu pharmaceuticals.
2. application according to claim 1, it is characterised in that：The calcein and the united administering mode of Oseltamivir
It is oral.
3. application according to claim 1, it is characterised in that：The calcein and the united administering mode of Oseltamivir
For injection.
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| CN105193785A (en) * | 2015-10-29 | 2015-12-30 | 王烨 | Application of calcein in drug for preventing and treating influenza A |
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Non-Patent Citations (2)
| Title |
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| Development and evaluation of a dry powder formulation of liposome-encapsulated oseltamivir phosphate for inhalation;Yue Tang et al.;《Drug Delivery》;20131204;第22卷(第5期);第608-618页 * |
| 抗病毒与免疫调节治疗H7N9禽流感研究进展;李丹等;《中国感染控制杂志》;20151031;第14卷(第10期);第717-720页，尤其是第717页左栏第1.1节、第718页右栏第1.3节 * |
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