CN109453155A - For assessing the construction method of the tree shrew model of anti-influenza virus medicament pharmacokinetics and pharmacodynamics - Google Patents
For assessing the construction method of the tree shrew model of anti-influenza virus medicament pharmacokinetics and pharmacodynamics Download PDFInfo
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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 1
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- 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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Abstract
The present invention provides a kind of for assessing the construction method of the tree shrew model of Oseltamivir pharmacokinetics and pharmacodynamics, this method and using tree shrew as platform, and evaluation Oseltamivir treatment is to the drug effect for infecting different influenza virus tree shrews.Present invention discover that tree shrew removes half-life period in plasma drug after oral Oseltamivir and peak time is close with mouse, Oseltamivir treatment can reduce the toxin expelling of H9N2 influenza virus and corresponding inflammatory reaction in tree shrew body, therefore tree shrew can be used as a kind of animal model of epidemic catarrh with drug therapy foresight.
Description
Technical field
The invention belongs to pharmaceutical technology fields, and in particular to one kind for assess anti-influenza virus medicament pharmacokinetics and
The construction method of the tree shrew model of pharmacodynamics.
Background technique
Influenza disease seriously threaten human health (Fischer WA 2nd, Gong M, Bhagwanjee S et al.,
Global burden of influenza as a cause of cardiopulmonary morbidity and
mortality.Glob Heart. 2014；9 (3): 325-336.), preparing a kind of can effectively replicate Human Influenza's genius morbi
It is commented with the animal model of good drug foresight for developing and screening the curative effect of new anti-anti-influenza virus medicament or vaccine
Antiviral therapy scheme is estimated with important value.The animal model of epidemic catarrh such as currently used mouse, ferret due to non-human species
Difference is excessive, and drug foresight is often poor, as mouse influenza pathogenic mechanism and the mankind there are larger difference (Darwish I,
Mubareka S,Liles W.Immunomodulatory therapy for severe influenza.Expert
review of anti-infective therapy.2011；9 (7): 807-822.), clinical simulation and drug foresight compared with
Difference, it is a variety of to verify effective anti-inflammatory agent through mouse model and stop at clinical test (Seok J, Warren HS, Cuenca AG etc.
People, Genomic responses in mouse models poorly mimic human inflammatory
diseases.Proc Natl Acad Sci U S A.2013；110(9):3507-3512.)；In addition the models such as mouse, ferret
Animal differs greatly compared with people in terms of anti-anti-influenza virus medicament pharmacokinetic parameter, as the mankind take orally Oseltamivir
Bioavilability is greater than 75% (Roche Laboratories Inc.Tamiflu (Oseltamivir Phosphate)
Capsules and for Oral Suspension,2008.Available online:http://www.fda.gov/
Downloads/Drugs/DrugSafety/ InformationbyDrugClass/UC M147992.pdf.), but mouse
Bioavilability with ferret be only 30% and 11% (Li W, Escarpe PA, Eisenberg EJ et al.,
Identification of GS 4104as an orally bioavailable prodrug of the influenza
virus neuraminidase inhibitor GS 4071[J].Antimicrob Agents Chemother.1998；42
(3):647-653.).Therefore a kind of novel experimental animal with preferable clinical simulation and drug foresight is developed and explores,
Objective reduction and anti-anti-influenza virus medicament Therapy study for influenza harmfulness are of great significance.
Tree shrew (Tupaia belangeri, family Tupaiidae) is a kind of small-sized mammalian for being similar to squirrel,
The areas such as Yunnan, Guizhou, Guangxi are distributed mainly in China, Chinese Academy of Sciences Kunming animal institute scientist has found tree shrew and spirit within 2013
Long class affiliation can be substituted for the large-scale spirit such as macaque more closely, having at many aspects than the conventional animals such as mouse model
Hereditary basis (Fan Y, Huang ZY, Cao CC et al., Genome of the Chinese tree of long class experimental animal
Shrew.Nature communications.2013,4:1426.), in a variety of disease models such as viral infectious diseases
(Zheng Yongtang, Yao Yonggang, Xu Lin etc., tree shrew fundamental biological knowledge and disease model, in October, 2014, Yunnan section is widely applied
Skill publishing house；Wang Xiaojuan, Yang Chun, Su Jianjia, new development of the tree shrew in medical experiment research, Chinese comparative medicine magazine,
2010；20 (2): 67-70), the eighties in last century tree shrew have been used for influenza virus research, this seminar further confirms and
Human influenza genius morbi is compared, and tree shrew is in flu symptom, virus replication, and respiratory tract receptor distribution etc. is compared with other common influenzas
Animal model has more simulation, however pharmacokinetics and pharmacodynamics of the anti-influenza virus medicament on tree shrew animal model are ground
Study carefully and but have not been reported, can tree shrew become a kind of animal model of epidemic catarrh with good drug therapy foresight, flow for people (fowl)
Feel disease and the effective detection platform of effective treatment method offer is provided, it is on the knees of the gods.
Summary of the invention
The purpose of the present invention is aiming at the above technical problems to be solved, provide one kind can effectively assess resisiting influenza virus
The construction method of drug pharmacokinetics and the tree shrew model of pharmacodynamics.
In order to achieve the goal above, the present invention provides one kind for assessing anti-influenza virus medicament pharmacokinetics and medicine
Imitate the construction method for the tree shrew model learned comprising following steps:
(1) tree shrew takes takes blood plasma for pharmacokinetic analysis after the anti-influenza virus medicament in different time, obtains
The anti-influenza virus medicament tree shrew pharmacokinetic parameter, and with the anti-influenza virus medicament in people, ferret, small
The intracorporal pharmacokinetic parameter of the animals such as mouse, rat compares；
(2) tree shrew takes the Tamiflu through rhinovaccination influenza virus, observes nasal cavity symptom, collects after infection the
1,3 and 5 days nose lavation row virus TCID50 are detected and to Nasal lavage fluid sedimentation cell differential counting, are examined after 21 days after infection
Survey serum neutralizing antibody titers.
Preferably, the anti-influenza virus medicament is Oseltamivir.
Preferably, the anti-influenza virus medicament is phosphate Oseltamivir.
Preferably, the tree shrew is middle remote tree shrew.It is preferred that the middle remote tree of the tree shrew of adult healthy, especially adult healthy
Shrew.
Preferably, described to take as oral stomach-filling oral way.
Preferably, in the step (1), after tree shrew fasting 12h, Oseltamivir 10mg/kg is given in oral stomach-filling, fills for the first time
Record administration time after stomach administration, respectively after administration 0.25,0.5,1,1.5,2,4,6,8 and 12 hour it is quiet through tail vein or stock
Arteries and veins blood sampling 0.5ml, collected blood are placed in centrifuge tube containing heparin, and 4 DEG C of centrifugation 10min of 1500g obtain tree shrew blood to be measured
Slurry；Appropriate Oseltamivir phosphate, carboxyl Oseltamivir and internal standard Peramivir are weighed, is made of 90%v/v methanol aqueous solution
1mg/mL stock solution, wherein Oseltamivir phosphate need to be converted to Oseltamivir concentration, then be diluted to gradient work step by step with methanol
Make solution；Internal standard Peramivir is made into the working solution of 1 μ g/mL with methanol；10 μ L Oseltamivir phosphate working solutions are taken respectively
With carboxyl Oseltamivir working solution, 50 μ L blank tree shrew blood plasma are added, the work for adding 130 μ L containing the internal standard Peramivirs is molten
Liquid, sample introduction is analyzed obtains Oseltamivir phosphate and carboxyl Oseltamivir gradient plasma concentration, system for vortex mixed centrifuging and taking supernatant
Standby standard curve, tree shrew blood plasma to be measured is by the processing of above-mentioned blank tree shrew blood plasma method and sample introduction is analyzed, linear fitting, acquisition
The Oseltamivir pharmacokinetic parameter of tree shrew blood plasma to be measured.
Preferably, in the step (2), tree shrew intranasal virus inoculation process is 3% Nembutal sodium solution of intraperitoneal injection
0.25ml influenza virus is perfused through nose after anesthesia.
Preferably, the influenza virus is human influenza virus H1N1 and/or avian influenza virus H9N2.
Preferably, it in the step (2), is inoculated with the oral stomach-filling in 2 hours before virus inoculation of the tree shrew of influenza virus and takes
Oseltamivir is administered twice a day, successive administration 5 days, daily to observe tree shrew sniffle, the 1st, 3 and 5 day tree shrew warp after infection
After the anesthesia of 3% Nembutal sodium solution is injected intraperitoneally, the 600 sterile PBS of μ L are drawn, are slowly dropped into from tree shrew side nostril, used
90mm2Plate collects Nasal lavage fluid, and Nasal lavage fluid is dispensed in right amount through 800g centrifuging and taking supernatant for detecting virus titer TCID50,
Nasal lavage fluid precipitating carries out classified counting of leucocyte.
Preferably, in the step (2), virus infection tree shrew Nasal lavage fluid virus titer TCID50 detection process includes nose
10 times of doubling dilutions of irrigating solution are at 10-1、10-2……10-7Gradient concentration is added in 96 orifice plates for covering with MDCK cell monolayers and trains
It supports 48 hours, observes cytopathogenic effect, and use blood coagulation tests verification result, viral half is calculated using Reed-Muench and is caused
Dead titre TCID50。
Method of the invention is being set afterwards by detection tree shrew oral anti-influenza virus medicament (especially Oseltamivir) by drug
Plasma changes in Shrew body, estimates its pharmacokinetic parameter, and with the Oseltamivir medicine of other animal models for power
It learns parameter to compare, provides foundation in the intracorporal rational use of medicines of tree shrew for anti-influenza virus medicament, on this basis, further study
Oral influence of the anti-influenza virus medicament to tree shrew nasal cavity toxin expelling and nosal inflammation lesion, can study it effective in tree shrew body
Inhibit influenza virus toxin expelling, so that demonstrating tree shrew is a kind of animal model of epidemic catarrh with good drug therapy foresight.
Detailed description of the invention
Fig. 1 is the average C-t curve of Oseltamivir medicine in blood plasma after tree shrew is administered orally.
Fig. 2 is the measured value C-t curve of Oseltamivir medicine in blood plasma after tree shrew is administered orally.
Specific embodiment
Combined with specific embodiments below, technical solution of the present invention is described in further detail, but the present invention is not limited to
Following embodiment.
As unspecified, related reagent can be bought by commercial sources below.For simplicity, part operation
The parameter, step and used instrument of operation are not described in detail, it should be understood that these are all well known to those skilled in the art and can
Repetition.
Embodiment 1: Oseltamivir pharmacokinetics and antiviral drug effect in tree shrew body
1. Oseltamivir pharmacokinetic
In 40 adult healthies after Burma tree shrew (male and female are unlimited) fasting 12h (free water), every group of 4 tree shrews are oral to fill
Stomach gives Oseltamivir 10mg/kg, records administration time after gastric infusion for the first time, respectively after administration 0.25,0.5,1,1.5,
2,4,6,8 and 12 hours through tail vein or femoral vein blood 0.5ml, and collected blood is respectively placed in centrifuge tube containing heparin
In, then blood plasma is transferred in EP pipe and sets -80 DEG C of profound hypothermia refrigerators preservations by 4 DEG C of centrifugation 10min of 1500g.
Appropriate Oseltamivir phosphate, carboxyl Oseltamivir and internal standard Peramivir are accurately weighed, it is water-soluble with 90%v/v methanol
About 1mg/mL stock solution is made in liquid, and wherein Oseltamivir phosphate need to be converted to Oseltamivir concentration, is then diluted step by step with methanol
At gradient working solution；Internal standard Peramivir is made into the working solution of 1 μ g/mL with methanol.10 μ L Oseltamivir phosphates are taken respectively
With carboxyl Oseltamivir working solution, 50 μ L blank tree shrew blood plasma are added, the work for adding 130 μ L containing the internal standard Peramivirs is molten
Liquid, sample introduction is analyzed obtains Oseltamivir phosphate and carboxyl Oseltamivir gradient plasma concentration, system for vortex mixed centrifuging and taking supernatant
Standby standard curve.Test plasma sample is by the processing of above-mentioned blank tree shrew blood plasma method and sample introduction is analyzed, linear fitting, obtains each
Blood sample measurement result and corresponding statistical moment parameter and Drug-time curve.
2. Oseltamivir treats the influence to tree shrew nasal cavity toxin expelling and cell count
Animal body quality is weighed, is calculated by Oseltamivir high dose (40mg/kg/d) and low dosage (4mg/kg/d) daily
Administration accumulated dose is administered in two times, and first administration is administered for 2 hours before virus inoculation, is administered twice a day, successive administration 5 days.
Daily observation tree shrew sniffle, the 1st, 3 and 5 day tree shrew is through 3% Nembutal sodium solution of intraperitoneal injection (penta bar of 3g ratio after infection
Appropriate sodium powder end is dissolved in 0.9% physiological saline of 100ml concentration (w/v)) about 300 μ L anesthesia after, draw the sterile PBS of 600 μ 0, from tree
The side Shrew nostril is slowly dropped into, and uses 90mm2Plate collects Nasal lavage fluid, and Nasal lavage fluid dispenses in right amount through 800g centrifuging and taking supernatant,
Nasal lavage fluid precipitating carries out classified counting of leucocyte, and the Nasal lavage fluid of collection detects virus titer TCID50。
3. serum antibody titer detects
By H1N1 and H9N2 infection group and viral infection group tree shrew (including viral group, Oseltamivir high dose (40mg/kg/d) administration
Group, Oseltamivir low dosage (4mg/kg/d) administration group) capable euthanasia is raised to the 21st day after virus infection, it is received after heart extracting blood
Collect serum, dispense into EP pipe, -80 DEG C save backup, and detect serum-virus neutralizing antibody titers by blood clotting Inhibition test.
Embodiment 2: Oseltamivir pharmacokinetics and antiviral pharmacodynamic result in tree shrew body
1. tree shrew is compared with other several animal model of epidemic catarrh pharmacokinetic parameters
Tree shrew is oral to give Oseltamivir phosphate 10mg/kg/d, and the Drug-time curve such as Fig. 1 and Fig. 2 after administration are (four in figure
Item difference curve respectively represents the respective C-t Drug-time curve of four tree shrews) shown in: it is dense by blood medicine after detecting oral Oseltamivir
Degree calculates it in the intracorporal main pharmacokinetic parameter of tree shrew, peak blood concentration (Cmax) it is 1.34 μ g/ml, peak time
(Tmax) it is 0.75h, serum removes half-life period (T1/2) it is 2.03h, lower area of blood concentration-time curve (AUC0-12) be
1.76mg*h/liter。
2. Oseltamivir infects H9N2 and H1N1 influenza virus pharmacodynamic study to tree shrew
The 1st day after the treatment, the administration of Oseltamivir high dose can obviously inhibit to infect the tree shrew nasal cavity toxin expelling of H9N2 virus,
Oseltamivir high dose administration group tree shrew is without toxin expelling when to the 3rd day, and virus group and Oseltamivir low dosage administration group nasal cavity
Toxin expelling significantly reduces and (is shown in Table 1)；But it is treated the 1st day and the 3rd day after tree shrew infection H1N1 virus, Oseltamivir drug treatment is to each
Group nasal cavity toxin expelling titre influences no significant difference, until Oseltamivir high dose administration group tree shrew nasal cavity is arranged when treating the 5th day
More viral group of poison is decreased obviously, and has 3/5 tree shrew not detect H1N1 influenza virus again, although virus group and Oseltamivir low dosage
Administration group nasal lavage fluid toxin expelling reduces earlier above, but all tree shrews still have toxin expelling (being shown in Table 1).
1 tree shrew of table infects different influenza strains and treats posterula toxin expelling titre through Oseltamivir
After influenza virus infection H9N2 and H1N1, clear yellow viscous secretion increases in the tree shrew nasal lavage fluid of part, is feeling
Cell count increases to peak in the 3rd day nasal lavage fluid after dye, and increased includes epithelial cell and lymphocyte is main inflammatory
Cell, wherein high dose Oseltamivir can obviously inhibit H9N2 infection group and viral infection group nasal cavity total number of cells, but to H1N1 virus sense
Each group nasal cavity total number of cells are contaminated without influence (being shown in Table 2).
2 tree shrew of table infects different influenza strain Nasal lavage fluid cell count (a 10 after Oseltamivir is treated4/ml)
3. Oseltamivir infects H9N2Y280-PB2-E627K and A/California/04/2009H1N1 influenza to tree shrew
Serum virus antibody influences
Pass through the corresponding virus antibody titer of tree shrew serum on the 21st after the detection infection of blood clotting Inhibition test, as the result is shown tree shrew sense
Corresponding antibodies, influenza virus A hominis H1N1 infection can be generated after dye influenza virus A hominis H1N1 and avian influenza virus H9N2
The antibody titer generated afterwards is between 640-2560, hence it is evident that higher than avian influenza virus H9N2 generate antibody titer (160-640),
The generation of Oseltamivir antiviral therapy infected by influenza antibody has no significant effect and (is shown in Table 3).
3 tree shrew influenza virus infection posterula toxin expelling titre of table and serum titer
Note: high dose dosage (40mg/kg/d)；Low dosage dosage (4mg/kg/d).
Claims (10)
1. a kind of for assessing the construction method of the tree shrew model of anti-influenza virus medicament pharmacokinetics and pharmacodynamics, feature
Be the following steps are included:
(1) tree shrew takes takes blood plasma for pharmacokinetic analysis after the anti-influenza virus medicament in different time, obtains institute
Anti-influenza virus medicament is stated in the pharmacokinetic parameter of tree shrew, and with the anti-influenza virus medicament people, ferret, mouse,
The intracorporal pharmacokinetic parameter of the animals such as rat compares；
(2) tree shrew takes the Tamiflu through rhinovaccination influenza virus, observes nasal cavity symptom, collects the 1st, 3 and after infection
5 days nose lavation row virus TCID50 are detected and to Nasal lavage fluid sedimentation cell differential counting, are detected serum after 21 days after infection
Neutralizing antibody titers.
2. the method according to claim 1, wherein the anti-influenza virus medicament is Oseltamivir.
3. the method according to claim 1, wherein the anti-influenza virus medicament is phosphate Oseltamivir.
4. the method according to claim 1, wherein the tree shrew is middle remote tree shrew.
5. the method according to claim 1, wherein described take as oral stomach-filling oral way.
6. the method according to claim 1, wherein in the step (1), after tree shrew fasting 12h, oral stomach-filling
Give Oseltamivir 10mg/kg, record administration time after gastric infusion for the first time, respectively after administration 0.25,0.5,1,1.5,2,
4, it is placed in centrifuge tube containing heparin through tail vein or femoral vein blood 0.5ml, collected blood within 6,8 and 12 hours, 1500g4
DEG C centrifugation 10min obtains tree shrew blood plasma to be measured；Appropriate Oseltamivir phosphate, carboxyl Oseltamivir and internal standard Peramivir are weighed,
1mg/mL stock solution is made with 90%v/v methanol aqueous solution, wherein Oseltamivir phosphate is converted to Oseltamivir concentration, then uses
Methanol is diluted to gradient working solution step by step；Internal standard Peramivir is made into the working solution of 1 μ g/mL with methanol；10 μ L are taken respectively
Oseltamivir phosphate working solution and carboxyl Oseltamivir working solution are added 50 μ L blank tree shrew blood plasma, add 130 μ L and contain
The working solution of internal standard Peramivir, sample introduction is analyzed obtains Oseltamivir phosphate and department, carboxyl Austria for vortex mixed centrifuging and taking supernatant
His Wei gradient plasma concentration prepares standard curve, and tree shrew blood plasma to be measured is by the processing of above-mentioned blank tree shrew blood plasma method and sample introduction point
Analysis, linear fitting, the Oseltamivir pharmacokinetic parameter of the tree shrew blood plasma to be measured of acquisition.
7. the method according to claim 1, wherein in the step (2), tree shrew intranasal virus inoculation process is
It is injected intraperitoneally after the anesthesia of 3% Nembutal sodium solution and 0.25ml influenza virus is perfused through nose.
8. the method according to claim 1, wherein the influenza virus is human influenza virus H1N1 and/or fowl
Influenza virus H9N2.
9. the method according to claim 1, wherein being inoculated with the tree shrew of influenza virus in disease in the step (2)
Oseltamivir is taken in poison inoculation oral stomach-filling in first 2 hours, is administered twice a day, successive administration 5 days, daily to observe tree shrew nose disease
Shape draws the 600 sterile PBS of μ L after the anesthesia of 3% Nembutal sodium solution is injected intraperitoneally in the 1st, 3 and 5 day tree shrew after infection, from
Tree shrew side nostril is slowly dropped into, and uses 90mm2Plate collects Nasal lavage fluid, and Nasal lavage fluid dispenses in right amount through 800g centrifuging and taking supernatant
For detecting virus titer TCID50, Nasal lavage fluid precipitating carries out classified counting of leucocyte.
10. the method according to claim 1, wherein in the step (2), virus infection tree shrew Nasal lavage fluid
Virus titer TCID50 detection process includes 10 times of doubling dilutions of Nasal lavage fluid at 10-1、10-2……10-7Gradient concentration is added
It covers in 96 orifice plates of MDCK cell monolayers and cultivates 48 hours, observe cytopathogenic effect, and use blood coagulation tests verification result, adopt
Viral median lethal titre TCID is calculated with Reed-Muench50。
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Citations (2)
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| EP1327687A1 (en) * | 2000-10-03 | 2003-07-16 | Tokyo Metropolitan Organization for Medical Research | Methods of infection with hepatitis c virus |
| WO2011123856A1 (en) * | 2010-04-02 | 2011-10-06 | Tsrl, Inc. | Neuraminidase inhibitors |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1327687A1 (en) * | 2000-10-03 | 2003-07-16 | Tokyo Metropolitan Organization for Medical Research | Methods of infection with hepatitis c virus |
| WO2011123856A1 (en) * | 2010-04-02 | 2011-10-06 | Tsrl, Inc. | Neuraminidase inhibitors |
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