口服药物的体外吸收模型.ppt

1、In Vitro Models for Oral Drug AbsorptionOutlinenPhysical and Biochemical Barrier for Oral Drug Absorption and In Vitro models for drug absorptionnEvaluation of the Method Caco-2 Cell SuitabilitynUse of Caco2 and Factor Affecting Permeation of NCEn Continuous Dissolution/Caco-2 System for Formulation

2、 Screening of NCEBLight micrograph of monkey small intestinal mucosa illustrating the three layers of the mucosa:the epithelium(E),the lamina propria(L),and the muscularis mucosa(M).(taken from J.L.Madera and J.S.Trier in Physiology of the Gastrointestinal Tract(L.R.Johnson,Ed.),2nd Edition,Raven Pr

3、ess,New York,1987,p.1210)LEMIntestinal Mucosa:A Physical and Biochemical Barrier for Oral Drug Absorption View of Intestinal Mucosa,Pre-1990ACurrent View of Intestinal Mucosa?BasolateralApical ParacellularDiffusionTranscellularDiffusionMetabolismA BEffluxTransportACDMultidrug Resistance Transporters

4、 and CYP3As Form a Concerted Barrier to Drug Absorption Multidrug Resistance Transporters (MDR1,MRP2,BCRP)CYP3AsApical sideBasolateral sideMetabolizedSubstrate drugDrug metaboliteMDR1(P-glycoprotein,P-gp)Inhibitors e.g.GF120918ABSubstratese.g.DigoxinExamples of Drugs That are Substrates for MDR1Canc

5、er drugsVinblastineVincristineTaxolDaunorubicinEtoposideImmunosuppressive DrugsCyclosporin AFK506SteroidsDexamethasoneProgesteroneCortisolHIV protease inhibitorsAmprenavirIndinavirCardiac drugsDigoxinQuinidineAnti-gout agentColchicineAntibioticErythromycinAnti-tuberculous agent RifampinSchematic Ill

6、ustration of MDR1,MRP1,MRP2,MRP3,MRP5 and BCRP in Transfected Polarized Epithelial CellsReference:P.Borst et al.,BBA 1461,347-357(1999)J.Jonker et al.,JNCI 92(20),1651-1656(2000)MDR1MRP1MRP2MRP3MRP5Apical sideBasolateral sideTight junctionBCRPModels Used to Determine the Intestinal Mucosal Permeatio

7、n Characteristics of a Drug CandidateOral dosing of animals or humansIntestinal perfusion studies in humansDosing of animals through a chronic cannula in the intestinal lumenIn Situ intestinal perfusion studies in animalsEfflux transporter-gene knockout miceIn Vitro permeation studies using excised

8、human or animal intestinal mucosal tissuesIn Vitro permeation studies using cell culture modelsPAMPA,IAMGastro PlusIntestinal Perfusion Studies in HumansCorrelation between Fraction Absorbed of d-Glucose and Phenazone in Human Regional Perfusion StudyDrug Absorption Study in situ and in Conscious Ra

9、tExtraction for LC/MS/MSAnalysisHPLCAnalysis0.2ml/min CentrifugationBlood FiltrationPerfusate In Situ Rat Intestinal Perfusion Mesenteric veinIleum Animals:MaleSprague-Dawley rats weighing 350-400g were used.Perfusion solution:NaH2PO4(57.9mM),Na2SO4(79.6mM),pH 7.5.PumpDonorBloodJugular veinPapp=DQDt

10、 A C0Basolateral(B)Apical(A)CellMonolayerBi-directional Transport Experiment Using Caco-2 Cell Monolayers and Calculation Substrate drug(e.g.Digoxin)Apically localized efflux transporter(e.g.MDR1)Q/t:The linear appearance rate of mass in the receiver solution.A:Cell monolayer surface area.C0:The ini

11、tial concentration in the donor solution.PB to APA to BRatio of PB to A/PA to B:An indicator of efflux activity of transporter in cell monolayerCorrelation between Oral Absorption in Humans and Membrane Permeability(Papp)in Caco-2 CellsP.Artursson et al.,Adv.Drug Del.Rev.46:27-43(2001)History of Cel

12、l Culture Models Used to Predict Drug Permeation across the Intestinal MucosaLate 1980s-Mid 1990s:Caco-2 cellsMid 1990s:Caco-2 cells vs.MDCK cellsLate 1990s:Caco-2 cells vs.MDCK cells vs.MDCK-MDR1 vs.MDCK-MRP2Caco-2 Cell Monolayer System Advantages Cells permit estimation of:both cellular uptake and

13、 drug transepithelial transport drug transport in the presence of metabolic reactions polarized efflux Cells are:of human origin(colon adenocarcinama)viable for long periods Disadvantages Relative long-period of time for culture(3 weeks)Underexpression of key drug-metabolizing enzymes(e.g.,CYP3A4)Va

14、riable expression levels of efflux transporters(e.g.,MDR1,MRP2)Papp values in MDCK cells correlated well with those in Caco-2 cells The correlation between MDCK Papp values and human intestinal absorption is similar to the results observed with Caco-2 MDCK grow faster than Caco-2(1 week vs 3 weeks)J

15、.D.Irvine et al.,JPS 88,28(1999)MDCK Cell Monolayer System Questions Concerning MDCK-MDR1/MDCK-MRP2 Cellsas the Model of the Intestinal MucosaMDR1/MRP2 cDNAMDCK cellsWhen compared with Caco-2 cells,do these cell lines:express the same protein?exhibit polarized efflux?exhibit the same kinetics and af

16、finity for substrates?MDCK-MDR1/MRP2 cellsExpression of MDR1 in Caco-2,MDCK-WT,and MDCK-MDR1 CellsM 1A 1B 2A 2B 3A 3B 4A 4BKD250148M:marker,A:5 g protein B:10 g protein.1:Caco-2,2:MDCK-WT,3:MDCK-MDR1(low passage),4:MDCK-MDR1(high passage).Ref.:Tang,F.,Horie,K,and Borchardt,R.T.(2002)Are MDCK cells transfected with human MDR1 gene a good model of the human intestinal mucosa?Pharm.Res.19 765-772 Tang,F.,Horie,K,and Borchardt,R.T.(2002)Are MDCK cells transfected with human MRP2 gene a good model of