Quantitative Evaluation of the Effect of P-Glycoprotein on Oral Drug Absorption

Transcription

1 Quantitative Evaluation of the Effect of P-Glycoprotein on Oral Drug Absorption -Assessment of Drug Permeability to Rat Small Intestine- College of Pharmacy, Setsunan University, Osaka, Japan Yoshiyuki Shirasaka

2 PURPOSE Transport Experiments by using P-gp Expressing Cell Monolayers Apical Basal Caco-2 cell, MDR1-MDCK cell Permeability ratio Transport Rate = f ( K m,, P passive ) Drug Concentration P-gp Expression Level Affinity to P-gp Passive Membrane Permeability Cellular Accumulation <Impact of P-gp on in vivo Oral Drug Absorption> Development of kinetic model that can predict the in vivo absorption of P-glycoprotein substrate drugs from in vitro data.

3 Effect of donor concentration on AP to BL and BL to AP transport of quinidine in Caco-2 monolayers P app ( 1-5 cm/sec) 3. AP to BL Donor concentration to Apical (µm) P app ( 1-5 cm/sec) BL to AP Donor concentration to Basal (µm) Permeability ratio (BL to AP / AP to BL) BL to AP / AP to BL Donor concentration (µm)

4 Various cell lines with different expression levels of P-gp 2 14 (kda) mole mass of standards 5., 1, 2, 5, Standard (µg) GAPDH cell line Protein (µg/cm 2 ) P-gp expression levels mrna ( 1-5 /GAPDH) 1 Non-cell (Blank).. 2 Normal Caco-2 cells P-gp-induced Caco-2 cells P-gp-highly induced Caco-2 cells MDR1-knockdown Caco-2 cells MDR1-MDCKII cells Protein levels were detected and quantified by Western blotting, loading total protein of 5µg for cells and standard. mrna levels were determined and quantified by Real-time quantitative PCR.

5 Effect of donor (apical) concentration on AP to BL transport of P-gp substrates in various cell monolayers P P app ( 1-5 app ( 1-5 cm/sec) cm/sec) 2. Quinidine Verapamil P app ( 1-5 cm/sec) P app ( 1-5 cm/sec).7 Normal Caco-2 P-gp induced Caco-2 P-gp highly induced Caco-2 P-gp knockdown Caco-2 MDR1-MDCK MDCKⅡ Inhibition of P-gp P ( Alprenolol ) Vinblastine Digoxin Donor concentration to Apical (µm) Donor concentration to Apical (µm).6

6 Analysis of P-gp function based on Sigmoid E max model C a C bind C b P-gp CL p-gp = / ( + C a ) (CLp) CLp max CLp passive = CLp max 1 (CLp max + CLp min ) 2 CLp = CLp max CL P-gp CLp min (C a ) Sigmoid E max model Flux rate = CLp max C a C a r r + C a r To add the flexibility, Hill Coefficient (r) was introduced to Sigmoid E max model

7 Estimation of, and hill coefficient of three P-gp substrates by fitting CLp to Sigmoid E max model Cell line P-gp expression level MDR1-kd. Caco-2 Normal Caco-2 P-gp id. Caco-2 P-gp highly id. Caco-2 MDR1- MDCKⅡ Verapamil Quinidine (µm) ( 1-7 µmol/min/cm 2 ) r (Hill coefficient) (µm) ( 1-7 µmol/min/cm 2 ) r (Hill coefficient) Vinblastine (µm) ( 1-7 µmol/min/cm 2 ) r (Hill coefficient)

8 Why does value fluctuate depending on the expression level of P-gp in cells Lower expression level P-gp High Conc. Drug concentration at drug bindingsite of P-gp increase easily when the apical concentration become high. Higher expression level Binding-site concentration P-gp expression Low Conc. Drug concentration at drug bindingsite of P-gp is kept low even when the apical concentration become high.

9 Correlation between P-gp expression level and value of three P-gp substrates Quinidine Verapamil Vinblastine ( 1-5 µmol/min/cm 2 ) versus Protein R 2 = R 2 =.995 R 2 = Protein level (µg/cm 2 ) versus mrna R 2 = R 2 =.863 R 2 = mrna level ( 1-5 /GAPDH)

10 Correlation between P-gp expression level and value of three P-gp substrates Quinidine Verapamil Vinblastine versus Protein versus mrna 4 4 (µm) 3 R 2 = R 2 =.98 2 R 2 = Protein level (µg/cm 2 ) R 2 = R 2 =.896 R 2 = mrna level ( 1-5 /GAPDH) 12

11 How to estimate P-gp-mediated efflux in human intestine? How to predict the permeability in human intestine? V or K max m(app) P-gp expression level in human intestine Protein or mrna level of P-gp (1) Passive permeability : Possible to be predicted from Caco-2 data by using P-gp inhibitor. (2) P-gp mediated efflux : CL p-gp = / ( + C a ) Permeability In vivo permeability Passive permeability Human intestine Luminal concentration

12 Regional difference in P-gp expression levels in rat small intestine (proximal and distal) 2 14 (kda) mole mass of standards 5., 1, 2, 5, 1 Standard (µg) GAPDH Rat # / Region P-gp expression levels Protein (µg/cm 2 ) 1 Blank. Rat #1 Rat #2 Rat # Protein (µg/cm 2 ) Ave Protein levels were detected and quantified by Western blotting, loading 1µg for rat BBM; 5µg for standard. 77.7

13 Estimation of and values of quinidine in rat small intestine (proximal and distal) ( 1-5 µmol/min/cm 2 ) Y = 3.61x1-8 X (R 2 =.995) P-gp () 77.7 µg/cm 2 Protein level (µg/cm 2 ) P-gp () µg/cm 2 Quinidine ( 1-5 µmol/min/cm 2 ) (µm) Rat intestine

14 Passive permeability P app of quinidine in rat small intestine (proximal and distal) Quinidine Cell monolayers Passive P app ( 1-5 cm/sec) 1.41 Ratio (Rat/Caco-2) - Rat intestine P app of quinidine to rat small intestine was measured by in situ singlepass perfusion experiments.

15 Simulation of concentration-dependent permeability of quinidine in rat small intestine (proximal and distal) P app ( 1-5 cm/sec) Experimental Predicted P app ( 1-5 cm/sec) Experimental Predicted Luminal concentration (µm) Luminal concentration (µm) (x1-5 µmol/min/1cm gut) (µm) / (x1-5 L/min /1cm gut) (x1-5 µmol/min/1cm gut) (µm) / (x1-5 L/min /1cm gut) Predicted Experimental

16 Simulation of concentration-dependent permeability of verapamil in rat small intestine (proximal and distal) P app ( 1-5 cm/sec) 1 5. Predicted Experimental P app ( 1-5 cm/sec) 1 5. Predicted Experimental Luminal concentration (µm) Luminal concentration (µm) ( 1-5 µmol/min/1cm gut) (µm) / ( 1-5 L/min /1cm gut) ( 1-5 µmol/min/1cm gut) (µm) / ( 1-5 L/min /1cm gut) Predicted Experimental

17 Simulation of concentration-dependent permeability of vinblastine in rat small intestine (proximal and distal) 1 1 P app ( 1-5 cm/sec) Experimental Predicted P app ( 1-5 cm/sec) Experimental Predicted Luminal concentration (µm) Luminal concentration (µm) ( 1-5 µmol/min/1cm gut) (µm) / ( 1-5 L/min /1cm gut) ( 1-5 µmol/min/1cm gut) (µm) / ( 1-5 L/min /1cm gut) Predicted Experimental

18 CONCLUSIONS Sigmoid E max model for permeability-concentration curve of P-gp substrate drugs can provide the fundamental parameters of P-gp-mediated transport, and. This study clearly demonstrated the possibility to estimate and values in human intestine from P-gp expression level and to predict concentration-dependent permeability of P-gp substrate drugs on human intestinal absorption. P-gp expression level In vitro experiment In vivo permeability Permeability, Passive permeability Permeability Human intestine Donor concentration Luminal concentration

19 In vivo permeability SIGNIFICANCE Permeability-concentration curve of P-gp substrate drugs can predict how P-gp affect the pharmacokinetic profiles of its drugs and how much permeability of its drugs will be changed by dose adjustment and drug-drug interactions in clinical treatment. Permeability Human intestine Luminal concentration Drug interactions Intestine (Absorption) Brain (Distribution) Liver (Metabolism) Kidney (Excretion) Therapeutic dose

20 ACKNOWLEDGMENTS - Setsunan University - Prof. S. Yamashita Dr. S. Sakuma Dr. M. Kataoka Ms. Y. Masaoka Ms. Y. Nagai Mr. N. Kitamura - Shujitsu University - Dr. T. Sakane - University of Tokyo - Prof. Y. Sugiyama Dr. R. Onuki Ms. T. Watanabe - Kobe University - Prof. K. Okumura Dr. T. Sakaeda Ms. Y. Moriya Mr. H. Omatsu - University of Mainz - Prof. P. Langguth - University of Washington - Prof. K. E. Thummel