Drug Transport in the Brain: Models of BBB and Brain Parenchyma

Transcription

1 Drug Transport in the Brain: Models of BBB and Brain Parenchyma Reina Bendayan, Pharm.D. Professor and Career Scientist, MHO Department of Pharmaceutical Sciences Leslie Dan Faculty of Pharmacy University of Toronto 10 th International ISSX Conference, Toronto, September 2013

2 Leslie Dan Faculty of Pharmacy University of Toronto Toronto, ON Canada Patrick Ronaldson Shannon Dallas Tamima Ashraf Tozammel Hoque Wenlei Jiang

3 Blood-Brain Barrier (BBB) Microvessel Lumen Endothelium Pericytes Human brain microvascular network (Zlokovic and Apuzzo Neurosurgery 43: ) Human cortical blood vessels (Rodriguez-Baeza et al The Anatomical Record 273A: ) Basement Membranes Electron microscopy of human brain microvasculature (Bendayan R. et al Journal of Histochemistry & Cytochemistry. 54(10): ) Brain microvessels/capillaries Microvessels per adult brain: 100 Billion Total length: 650 km (TO Ottawa Montréal) Surface Area: m 2 Electron microscopy of rat brain microvasculature (Bendayan et al Microscopy Research and Technique. 57(5): )

4 From: Bendayan R. et al. (2002). Microsc Res Tech. 57:

5 Structure of the BBB: Neurovascular Unit Adapted from Abbott et al Neurobiology of Disease 37 :13 25 Differences compared to the periphery: Joined by tight junctions, non-fenestrated, highly-polarized and low pinocytotic activity Endothelial interactions: astrocytes foot processes, pericytes, neuronal axons and basal lamina

6 Mechanisms of Drug Transport Solute Carrier (SLC) Family ATP-Binding Cassette (ABC) Family PASSIVE Diffusion Drug Uptake Drug Efflux OATPs OCTs OATs Pgp MRPs BCRP Drug Uptake Drug Efflux

7 From: Simionescu M. et al. (2002). Microsc Res Tech. 57:

8 Endocytosis and Transcytosis From: Simionescu M. et al. (2002). Microsc Res Tech. 57:

9 Abluminal Luminal Basolateral Apical MRP1 Mrp2 MRP4 MRP5 BCRP Mrp4 Brain Blood MRP4 MRP1 Mrp5 CSF BBB CP Oat3 Oatp1c1 Oatp2a1 Oatp1a4 OATP1A2 OCT1 OCT2 Oat3 CNT3 Oatp1c1 Oatp1a4 OATP3A1.V1 BCRP MRP4 Oatp1c1 Oatp1a4 OATP1A2 Oat3 OAT1 Oatp1a1 Oatp1a5 Oatp2b1 Oatp5a1 OATP3A1.V2 P-gp P-gp P-gp ENT2 MRP1 OCT3 OCT2 OCTN2 OCTN2

10 Mrp5 Mrp6 CNT2 OCT3 Oatp3a1 Oatp2a1 Pept2 CNT3 Mrp6 Mrp5 P-gp BCRP MRP1 MRP4 P-gp BCRP MRP1 Mrp4 OCTN2 ENT1 ENT2 OCT3 Oatp1a5 MRP1 Mrp3 MRP4 MRP5 Mrp6 MRP8 Neurons P-gp OATP3A1.V2 Oat1 OCT2 Oatp2b1 Astrocytes Microglia

11 Methods for the Study of Drug Transport In Vitro In Situ In Vivo

12 In Vitro BBB Models Isolated Brain capillaries In Vitro Cell-based BBB Models Cell-free Models Static models 1- or 2- dimensional Dynamic models 3-dimensional Primary cultured cells Immortalized brain microvessel endothelial cells Cell culture on hollow fibres exposed to blood flow

13 Immunocytochemical Localization of P-gp in Rodent BBB

14 Double Labeling of P-gp and GFAP Bendayan et al (2006). J Histochem Cytochem.

15 Immortalized Rat Brain Microvessel Endothelial Cell Line (RBE4) From: Bendayan R. et al. (2002). Microsc Res Tech. 57:

16 P-gp Localization in RBE4 Cells

17 Immunolocalization of P-gp in Human Brain Tissue

18 Immunolocalization of P-gp in Human Brain Microvessel Endothelial Cells

19 Zastre J, Chan GNY, et al JNR. 87(4): Light Contrast Image of hcmec/d3 cells Weksler et al., FASEB Journal 19(13): Morphology & biochemically similar to primary cultures of brain microvessel endothelial cells Microvessel endothelial markers and tight junction proteins ABC transporters (MRP4, MRP5, MDR1, BCRP, MRP1, MRP3 and MRP2) Restricted permeability to paracellular diffusion markers & small molecules

20 Immunocytochemical localization of P-gp and hpxr in hcmec/d3 cells Zastre et al. JNR, 2009

21 P-gp Inhibitor P-gp Inhibitor P-gp Inhibitor MRPs Inhibitor BCRP Inhibitor Functional expression of P-gp in hcmec/d3 cells P-gp substrate (Rhodamine6G, R-6G) cellular accumulation studies: The presence P-gp-mediated transport in hcmec/d3 cells hcmec/d3 cells P-gp overexpressing cells P-gp selectivity of R-6G Zastre J, Chan GNY, et al JNR. 87(4): h, n=4 1 h, n=4 Chan GNY Unpublished data.

22 P-gp protein induction following treatment with hpxr or hcar agonist in hcmec/d3 cells Immunoblots of P-gp hpxr antagonist hpxr agonist hcar antagonist hcar agonist Chan GNY, et al JNC. 118(2): * p < 0.05,n = 4

23 Immunocytochemical localization of hpxr in hpxr agonist-treated hcmec/d3 cells Confocal Microscopy Chan GNY, et al JNC. 118(2): p < Cells / treatment from 3 passages

24 P-gp function in hcmec/d3 cells following treatment with antiretroviral drugs that are ligands of hpxr and hcar R6G Cellular Accumulation Chan GNY. et al. Antimicrob Agents Chemotherapy min, * p < 0.05,n = 4

25 P-gp Function at the BBB A key efflux mechanism to restrict brain entry of xenobiotics Systemic Circulation Endothelial Cells of Brain Vasculature Brain Parenchyma Xenobiotics P-gp Tight Junctions and Adhesion Molecules P-gp P-gp Brain Microvessel Endothelial Cells

26 Isolated Mouse Brain Capillary

27 A Primary Cultures of Rat Astrocytes B From: Ronaldson P.T. et al. (2004). J Neurochem. 89:

28 P-gp Localization in Astrocyte Cultures Ronaldson et al. (2004) J Neurochem. 89:

29 Primary Cultures of Rat Microglia

30 P-gp Localization in Microglia Cultures From: Lee G. et al. (2001). J Pharmacol Exp Ther. 299:

31 Pgp/actin ratio Comparative western blot for Pgp in various brain cell culture systems RBE4 MLS9 Primary astrocytes Primary microglia hcmec

32 P-gp Activity in Cultured Glial Cells Astrocytes MLS-9 Cells (Microglia) Intracellular Digoxin Accumulation ( pmol/mg protein ) 0.6 Control PSC ** ** ** ** Time ( min ) ** p < 0.01

33 HIV-1 gp120 decreases expression of P-gp in astrocytes 250 kda Untreated gp120 (1nM) MDCK- MDR1 160 kda 50 kda 38 kda P-gp Actin *p< H-Digoxin Accumulation (pmol/mg protein) * * * * Control 1.0 nm Gp120 * * gp120 * * Untreated Time (minutes) * p < 0.05 * Ashraf T, Ronaldson P, Persidsky Y, Bendayan R. Journal Neuroscience Research, 2011

34 Ronaldson PT and Bendayan R. J Neurochem (2008). Functional Studies in Astrocytes Mrp1 Intracellular BCECF Retention (pmol/mg protein) * ** * Time (minutes) ** Untreated control 1.0 nm Gp120 * p < 0.05 ** p < 0.01 *

35 Functional activity of MRP1 in Astrocytes in the presence of TNF-α Ronaldson, Ashraf, Bendayan, Mol Pharmacol, 2010

36 Microglia P - gp MRP1 MRP3 MRP4 MRP5 Astrocyte foot process MRP1 MRP3 MRP4 MRP5 P-gp Brain P-gp MRP5 MRP4? P-gp MRP2 ABCG2 MRP1 Blood

37 In Situ BBB Techniques In Situ Brain Perfusion -Carotid artery perfusion of test substrate (very short time periods) -Allows for complex kinetic analysis and transport characterization -Avoids substrate metabolism -Reflects events at the luminal BBB

38 In Vivo Techniques Brain Distribution PK Method (Use of KO models) Intraventricular Administration Intracerebral Microdialysis Technique.Direct sampling of ECF (use of a probe)

39 Atazanavir Brain : Plasma Concentration Ratios in KO Model Brain : Plasma C brain /C plasma * * * * AUC Brain /AUC plasma Time (hours) From: Robillard et al., Submitted Time (hours) mean ± SEM, n=4-5 animals per time point, *(p<0.05)

40 Intracerebroventricular (ICV) injection of gp120 in Wistar rats Stereotaxic surgery: Implantation of cannula Administration gp120 Research plan: Characterization of cytokine secretion Regulation of ABC transporters

41 Rodent Brain Microdialysis

42 Intracerebral microdialysis: In vivo P-gp induction at the mouse BBB affects brain ECF concentrations of the P-gp substrate, quinidine C Brain ECF > C Dialysate Exchange between ECF & blood Diffusion from ECF into perfusate Brain ECF Sawchuk and Elmquist Advanced Drug Delivery Reviews 45: , De Lange Advanced Drug Delivery Reviews,

43 Ratio of [Quinidine] brain ECF / [unbound Quinidine] plasma Chan GNY. et al. J. Neurochem, 2013 * p < 0.05 n = 5

44 Drug efflux transporters in neurovascular unit MRP1 Mrp5 Mrp4 P-gp Bcrp MRP1 P-gp Mrp3 MRP1 MRP4 Mrp2 MRP5 Mrp4 MRP4 Mrp6 P-gp MRP5 P-gp BCRP Bcrp P-gp MRP1 Mrp3 MRP4 MRP5 Mrp6 MRP8 Astrocyte Neurovascular Unit Mrp6 Microglia Neuron Blood Endothelium Brain Adapted from book chapter: Ashraf et al., Drug Transporters at Brain Barriers. In Biology and Regulation of Blood-Tissue Barriers by Landes Bioscience.

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46 Acknowledgment Collaborators: Dr. Lyanne Schlichter, U. Health Network, Toronto Dr. Moise Bendayan, University of Montreal Dr. Alex Prat, University of Montreal Dr. Yuri Persidsky, Temple University, Philadelphia Drs Jean-Michel Sherrmann, Pierre-Olivier Couraud, University Paris Descartes, France Dr. David Miller, NIEHS, NIH, Research Triangle, NC Dr. Ines de Lannoy, InterVivoSolutions, Toronto, ON Dr. Carolyn Cummins, University of Toronto Funding Agencies: CIHR, OHTN (MHO), CANFAR

47 Funding Agencies